BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provid...BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.展开更多
BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno- associated ...BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno- associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS: The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase- polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantita- tive PCR was used to measure the expression of IL-2, IFN-γ, IL-4 and IL-10 in the allografts. RESULTS: The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-γ, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62±0.09, 1.52±0.11, 1.50± 0.07 and 1.43±0.07 versus 1.29±0.09, 1.32±0.07, 1.34±0.06 and 1.35±0.04, respectively).CONCLUSIONS: pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological findings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.展开更多
Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or ...Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.展开更多
Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous sys- tem...Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous sys- tem. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plas- mids were encapsidated as recombinant virions. PCl2 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus or AAV vectors by South- ern blot and transcript situation or foreign genes by dot blot. Results The hybridization tests showed that AAV in- troduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PCl2 cells. Conclusion AAV vectors can serve as high efficiency vectors or target genes in neuronal PC12 cells.展开更多
Adeno-associated virus(AAV) is a small,non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprote...Adeno-associated virus(AAV) is a small,non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms,especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation,column chromatography,or a combination,have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road,such as crossspecies variation in vector tissue tropism and gene transfer efficiency,pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients,the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants,and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.展开更多
Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells ...Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.展开更多
Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases.Recombinant rabies viral vectors allow for th...Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases.Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing,making these vectors powerful candidates for the dissection of synaptic inputs.Although several attenuated rabies viral vectors have been developed,their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors.To overcome these limitations,we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus.Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably:(1)N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro,and the efficiency was six times higher than that of rAAV9-Retro;(2)the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2–3 times higher than that of oG-mediated SAD-B19-ΔG;(3)CVS-N2c-ΔG could delivery modified genes for neural activity monitoring,and the time window during which this was maintained was 3 weeks;and(4)CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination.These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.展开更多
In recent years,significant breakthroughs have been made in the field of gene ther-apy.Adeno-associated virus(AAV)is one of the most promising gene therapy vectors and a powerful tool for delivering the gene of intere...In recent years,significant breakthroughs have been made in the field of gene ther-apy.Adeno-associated virus(AAV)is one of the most promising gene therapy vectors and a powerful tool for delivering the gene of interest.Among the AAV vectors,AAV serotype 8(AAv8)has attracted much attention for its efficient and stable gene transfection into specific tissues.Currently,recombinant AAv8 has been widely used in gene therapy research on a va-riety of diseases,including genetic diseases,cancers,autoimmune diseases,and viral diseases.展开更多
H9N2 avian influenza virus(AIV) infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin(HA) gene sequences of H9N2 AIV of ...H9N2 avian influenza virus(AIV) infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin(HA) gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.展开更多
BACKGROUND: Data on the mechanical ventilation(MV) characteristics and radiologic features for the cases with H7 N9-induced ARDS were still lacking.METHODS: We describe the MV characteristics and radiologic features o...BACKGROUND: Data on the mechanical ventilation(MV) characteristics and radiologic features for the cases with H7 N9-induced ARDS were still lacking.METHODS: We describe the MV characteristics and radiologic features of adult patients with ARDS due to microbiologically confirmed H7 N9 admitted to our ICU over a 3-month period.RESULTS: Eight patients(mean age 57.38±16.75; 5 male) were diagnosed with H7 N9 in the first quarter of 2014. All developed respiratory failure complicated by acute respiratory distress syndrome(ARDS), which required MV in ICU. The baseline APACHE II and SOFA score was 11.77±6.32 and 7.71±3.12. The overall CT scores of the patients was 247.68±34.28 and the range of CT scores was 196.3–294.7. The average MV days was 14.63±6.14, and 4 patients required additional rescue therapies for refractory hypoxemia. Despite these measures, 3 patients died.CONCLUSION: In H7 N9-infected patients with ARDS, low tidal volume strategy was the conventional mode. RM as one of rescue therapies to refractory hypoxemia in these patients with serious architectural distortion and high CT scores, which could cause further lung damage, may induce bad outcomes and requires serious consideration. Prone ventilation may improve mortality, and should be performed at the early stage of the disease, not as a rescue therapy.展开更多
Objective: To evaluate the expression of matrix metalloproteinase-9 (MMP9) in nasopharyngeal carcinoma and the association between MMP9 and Epstein-Barr virus infection. Methods: The MMP9 expression was studied by imm...Objective: To evaluate the expression of matrix metalloproteinase-9 (MMP9) in nasopharyngeal carcinoma and the association between MMP9 and Epstein-Barr virus infection. Methods: The MMP9 expression was studied by immunohistochemical analysis; and Epstein-Barr virus encoded small nuclear mRNA-1 (EBER-1) produced by in situ hybridization were examined in 41 nasopharyngeal carcinoma sections, and the relation between them, and the associations of MMP9 with clinical features were statistically analyzed. Results: Positive expression rate of MMP9 was 73.17%. The expression of MMP9 showed significant positive correlation with the expression of EBER-1(γ=0.483, P=0.001 ). There was significant association of MMP9 expression with lymph nodes metastasis and clinical stage (P<0.001), non-significant association with age, gender, pathological classification and T classification. Conclusions: The highly pronounced expression of MMP9 is associated with cervical lymph nodes metastasis. Epstein-Barr virus can enhance NPC metastasis by up-regulating the expression of MMP9.展开更多
Objective: To delineate the H9N2 influenza virus circulation within Iran and its neighboring countries, the potential source of the epidemic in these countries, and its date of origin.Methods: We obtained all hemagglu...Objective: To delineate the H9N2 influenza virus circulation within Iran and its neighboring countries, the potential source of the epidemic in these countries, and its date of origin.Methods: We obtained all hemagglutinin(HA) and neuraminidase(NA) nucleotide sequences of influenza H9N2 available up to December 25, 2020 from Iran and its neighboring countries(i.e., Pakistan, Afghanistan, Turkmenistan, Armenia, Azerbaijan, Turkey, and Iraq). We also performed a Bayesian Markov chain Monte Carlo method to infer the evolutionary dynamic and the most recent common ancestor for the HA and NA sequences.Results: H9N2 epidemic may have started in Iran and Pakistan much earlier than the other investigated countries in the region, and an ongoing bidirectional dispersion of the virus between the investigated countries was also observed. The mean time of the most recent common ancestor of H9N2 viruses was 1988 for HA, and 1992 for NA.Conclusions: Strains from investigated countries rooted in Pakistan and Iran. Regular surveillance of H9N2 viruses, especially in the live bird markets, enhancing the biosecurity of poultry industry and screening newly arriving immigrants and tourists from neighboring countries at border should be considered to control spread of the virus. Furthermore, surveillance of viral molecular evolution should be initiated for effective prevention of epidemic and pandemic spreads.展开更多
CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9...CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.展开更多
A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of...A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.展开更多
Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheles...Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.展开更多
The majority of hepatocellular carcinoma(HCC)cases are associated with the hepatitis B virus(HBV)infection.Autophagy related protein 9A(ATG9A)is a transmembrane protein required for autophagosome formation.In order to...The majority of hepatocellular carcinoma(HCC)cases are associated with the hepatitis B virus(HBV)infection.Autophagy related protein 9A(ATG9A)is a transmembrane protein required for autophagosome formation.In order to investigate the role of ATG9A in HBV-associated HCC,ATG9A protein expression was determined in tumor liver tissues and compared with adjacent nontumor tissues from HCC patients with or without HBV infection.In HBVassociated HCC tissues,ATG9A protein level was increased in tumor liver tissues,but not in cases of non-HBV HCC.Our findings suggested that ATG9A might be involved in HBV and cancer cell survival.Therefore,we aimed to analyze the function of ATG9A in HBV replication using RNA interference to evaluate the HBV DNA level using real-time PCR.In the present study,there were no significant differences between shATG9A-transfected HepG2.2.15 cells and the mock control.However,we found that silencing ATG9A affected apoptosis in HepG2.2.15 and HepG2 cell lines.Our results indicated that ATG9A might be partly involved in the survival of HCC.Thus,the inhibition of ATG9A together with other targets might be a potential drug target for HCC treatment.展开更多
Background: One of potentially dangerous problems for a human organism is the new strain of a virus of bird flu-A/H7N9. As it is regular mutation of bird flu virus, it obvious, that of antibacterial preparations is no...Background: One of potentially dangerous problems for a human organism is the new strain of a virus of bird flu-A/H7N9. As it is regular mutation of bird flu virus, it obvious, that of antibacterial preparations is not efficient. Efficiency decreases when the number of agents with multiple stability to antimicrobic remedy vastly increases, the part of associate infections enlarges, and aggression of opportunistic pathogenic flora rises. This reduces the role of the preparations in prevention of epidemics. Therefore, the optimization of only etiotropic therapies does not fully solve the problem. In this connection natural preparations seem extremely promising which strengthen the functional condition of immune system and, thereby, activate protective forces of macroorganism. Objectives: One of such preparations is BAE Synergy Liquid, a natural mineral water which was underwent subtle energetic changes at the natural energetic deposit. Design: An estimation of protective efficiency of naturally modified mineral BAE SL water was performed on white outbred mice-males in models of H7N9 virus. The animals were monitored during 16 days after infection, and survived and fallen mice were counted daily. Results: The results revealed significant effect of the investigated preparation as possible prophylactic care and medical remedy to the mentioned virus. This means that one can be considered as potential effective remedy for human. Conclusions: As significant effect of the immune system resistance was revealed, the experimental model with studied naturally modified mineral water is potentially generalizable.展开更多
A novel influenza virus of the H7N9 subtype has infected more than 350 people in China since 19 February 2013. Evolutionary analysis indicates that the virus is a reassortant originated from H7, N9 and H9N2 avian infl...A novel influenza virus of the H7N9 subtype has infected more than 350 people in China since 19 February 2013. Evolutionary analysis indicates that the virus is a reassortant originated from H7, N9 and H9N2 avian influenza viruses, and bears some amino acids associated with mammalian receptor binding, raising concern over the possibility of a new influenza pandemic. Besides HA and NA, the mutation of the polymerase is known to have an important role in virulence, host adaptation and transmissibility in mammalians. In this article, the annotation of the polymerase protein domain associated with molecular function has been highlighted, suggesting the combination of RNA polymerase of H7N9 viruses is still not stable for host adaptation. In addition, the mutation hallmarks in polymerase gene of H7N9 are compared, providing the potential determinants of the evolution in the H7N9 influenza A virus.展开更多
目的:探究单纯疱疹病毒(HSV)感染后脑脊液(CSF)中S100B、Cys-C、MMP-9水平对自身免疫性脑炎(AE)的预测价值。方法:选取2016年1月至2021年3月河北中石油中心医院收治的200例HSV感染患者为研究对象,根据是否继发AE分为研究组(继发AE,35例...目的:探究单纯疱疹病毒(HSV)感染后脑脊液(CSF)中S100B、Cys-C、MMP-9水平对自身免疫性脑炎(AE)的预测价值。方法:选取2016年1月至2021年3月河北中石油中心医院收治的200例HSV感染患者为研究对象,根据是否继发AE分为研究组(继发AE,35例)和对照组(未继发AE,165例)。多因素Logistic回归分析HSV感染患者继发AE的独立影响因素。Spearman法分析脑脊液中Cys-C、MMP-9与S100B水平的相关性。受试者工作特征(ROC)曲线分析S100B、Cys-C、MMP-9对AE的预测价值。构建风险预测模型并进行评价。结果:多因素Logistic回归分析显示,MRI异常、脑脊液S100B、MMP-9升高、EEG异常是HSV感染患者继发AE的独立危险因素,脑脊液Cys-C是其保护因素(P<0.05)。Spearman分析显示,HSV感染患者Cys-C浓度与S100B水平呈负相关(r=-0.83,P<0.05),MMP-9浓度与S100B水平呈正相关(r=0.88,P<0.05)。构建的联合预测因子pre1诊断HSV患者继发AE的AUC明显大于S100B、Cys-C、MMP-9单独预测的AUC(0.876 vs 0.827、0.787、0.750)。构建的风险预测模型具有良好的区分度和一致性。结论:脑脊液中S100B、Cys-C、MMP-9水平均可对HSV感染患者诱发AE的可能性进行有效预测,且三项指标联合预测价值最大,其次是S100B蛋白、Cys-C、MMP-9。展开更多
基金Supported by the National Natural Science Foundation of China,No.82172297Natural Science Foundation of Jiangsu Province of China,No.BK20211346 and No.BK20201011+1 种基金Natural Science Foundation of Jiangsu Higher Education Institutions of China,No.22KJA310007Xuzhou Science and Technology Project,No.KC22055.
文摘BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.
文摘BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno- associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS: The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase- polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantita- tive PCR was used to measure the expression of IL-2, IFN-γ, IL-4 and IL-10 in the allografts. RESULTS: The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-γ, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62±0.09, 1.52±0.11, 1.50± 0.07 and 1.43±0.07 versus 1.29±0.09, 1.32±0.07, 1.34±0.06 and 1.35±0.04, respectively).CONCLUSIONS: pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological findings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.
文摘Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.
文摘Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous sys- tem. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plas- mids were encapsidated as recombinant virions. PCl2 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus or AAV vectors by South- ern blot and transcript situation or foreign genes by dot blot. Results The hybridization tests showed that AAV in- troduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PCl2 cells. Conclusion AAV vectors can serve as high efficiency vectors or target genes in neuronal PC12 cells.
文摘Adeno-associated virus(AAV) is a small,non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms,especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation,column chromatography,or a combination,have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road,such as crossspecies variation in vector tissue tropism and gene transfer efficiency,pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients,the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants,and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.
文摘Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.
基金supported by the National Natural Science Foundation of China,Nos.32100899(to KZL),31830035(to FQX),31771156(to FQX),21921004(to FQX)the National Science and Technology Innovation 2030,No.2021ZD0201003(to FQX)+2 种基金the Key-Area Research and Development Program of Guangdong Province,No.2018B030331001(to FQX)the Strategic Priority Research Program of the Chinese Academy of Sciences,No.XDB32030200(to FQX)the Shenzhen Key Laboratory of Viral Vectors for Biomedicine,No.ZDSYS20200811142401005(to FQX)。
文摘Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases.Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing,making these vectors powerful candidates for the dissection of synaptic inputs.Although several attenuated rabies viral vectors have been developed,their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors.To overcome these limitations,we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus.Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably:(1)N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro,and the efficiency was six times higher than that of rAAV9-Retro;(2)the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2–3 times higher than that of oG-mediated SAD-B19-ΔG;(3)CVS-N2c-ΔG could delivery modified genes for neural activity monitoring,and the time window during which this was maintained was 3 weeks;and(4)CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination.These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.
文摘In recent years,significant breakthroughs have been made in the field of gene ther-apy.Adeno-associated virus(AAV)is one of the most promising gene therapy vectors and a powerful tool for delivering the gene of interest.Among the AAV vectors,AAV serotype 8(AAv8)has attracted much attention for its efficient and stable gene transfection into specific tissues.Currently,recombinant AAv8 has been widely used in gene therapy research on a va-riety of diseases,including genetic diseases,cancers,autoimmune diseases,and viral diseases.
基金supported by the National Modern Agricultural Industry Technology System Project of China(CARS-41)the Science and Technology Plan Project of Guangdong Province,China(2012B020306002 and 2012B091100078)
文摘H9N2 avian influenza virus(AIV) infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin(HA) gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.
基金supported by the National Natural Science Foundation of China(grant number 81501654)Natural Science Foundation of Shanghai(grant number 14ZR1433700)
文摘BACKGROUND: Data on the mechanical ventilation(MV) characteristics and radiologic features for the cases with H7 N9-induced ARDS were still lacking.METHODS: We describe the MV characteristics and radiologic features of adult patients with ARDS due to microbiologically confirmed H7 N9 admitted to our ICU over a 3-month period.RESULTS: Eight patients(mean age 57.38±16.75; 5 male) were diagnosed with H7 N9 in the first quarter of 2014. All developed respiratory failure complicated by acute respiratory distress syndrome(ARDS), which required MV in ICU. The baseline APACHE II and SOFA score was 11.77±6.32 and 7.71±3.12. The overall CT scores of the patients was 247.68±34.28 and the range of CT scores was 196.3–294.7. The average MV days was 14.63±6.14, and 4 patients required additional rescue therapies for refractory hypoxemia. Despite these measures, 3 patients died.CONCLUSION: In H7 N9-infected patients with ARDS, low tidal volume strategy was the conventional mode. RM as one of rescue therapies to refractory hypoxemia in these patients with serious architectural distortion and high CT scores, which could cause further lung damage, may induce bad outcomes and requires serious consideration. Prone ventilation may improve mortality, and should be performed at the early stage of the disease, not as a rescue therapy.
文摘Objective: To evaluate the expression of matrix metalloproteinase-9 (MMP9) in nasopharyngeal carcinoma and the association between MMP9 and Epstein-Barr virus infection. Methods: The MMP9 expression was studied by immunohistochemical analysis; and Epstein-Barr virus encoded small nuclear mRNA-1 (EBER-1) produced by in situ hybridization were examined in 41 nasopharyngeal carcinoma sections, and the relation between them, and the associations of MMP9 with clinical features were statistically analyzed. Results: Positive expression rate of MMP9 was 73.17%. The expression of MMP9 showed significant positive correlation with the expression of EBER-1(γ=0.483, P=0.001 ). There was significant association of MMP9 expression with lymph nodes metastasis and clinical stage (P<0.001), non-significant association with age, gender, pathological classification and T classification. Conclusions: The highly pronounced expression of MMP9 is associated with cervical lymph nodes metastasis. Epstein-Barr virus can enhance NPC metastasis by up-regulating the expression of MMP9.
文摘Objective: To delineate the H9N2 influenza virus circulation within Iran and its neighboring countries, the potential source of the epidemic in these countries, and its date of origin.Methods: We obtained all hemagglutinin(HA) and neuraminidase(NA) nucleotide sequences of influenza H9N2 available up to December 25, 2020 from Iran and its neighboring countries(i.e., Pakistan, Afghanistan, Turkmenistan, Armenia, Azerbaijan, Turkey, and Iraq). We also performed a Bayesian Markov chain Monte Carlo method to infer the evolutionary dynamic and the most recent common ancestor for the HA and NA sequences.Results: H9N2 epidemic may have started in Iran and Pakistan much earlier than the other investigated countries in the region, and an ongoing bidirectional dispersion of the virus between the investigated countries was also observed. The mean time of the most recent common ancestor of H9N2 viruses was 1988 for HA, and 1992 for NA.Conclusions: Strains from investigated countries rooted in Pakistan and Iran. Regular surveillance of H9N2 viruses, especially in the live bird markets, enhancing the biosecurity of poultry industry and screening newly arriving immigrants and tourists from neighboring countries at border should be considered to control spread of the virus. Furthermore, surveillance of viral molecular evolution should be initiated for effective prevention of epidemic and pandemic spreads.
基金financially supported by the National Key Research and Development Program of China(No.2017YFD0500103)the Beijing Natural Science Foundation(No.5152023)+4 种基金the National Natural Science Foundation of China(No.31772747 and31272385)the Jilin Province Science and Technology Development Projects(20150204077NY)the Graduate Innovation Fund of Jilin Universitythe Program for Chang jiang Scholarsthe University Innovative Research Team(No.IRT1248)
文摘CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.
文摘A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.
基金sponsored by grants from the National Natural Science Foundation of China(No.82030117)the Wenzhou major scientific and technological innovation project(No.ZY2022001).
文摘Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.
文摘The majority of hepatocellular carcinoma(HCC)cases are associated with the hepatitis B virus(HBV)infection.Autophagy related protein 9A(ATG9A)is a transmembrane protein required for autophagosome formation.In order to investigate the role of ATG9A in HBV-associated HCC,ATG9A protein expression was determined in tumor liver tissues and compared with adjacent nontumor tissues from HCC patients with or without HBV infection.In HBVassociated HCC tissues,ATG9A protein level was increased in tumor liver tissues,but not in cases of non-HBV HCC.Our findings suggested that ATG9A might be involved in HBV and cancer cell survival.Therefore,we aimed to analyze the function of ATG9A in HBV replication using RNA interference to evaluate the HBV DNA level using real-time PCR.In the present study,there were no significant differences between shATG9A-transfected HepG2.2.15 cells and the mock control.However,we found that silencing ATG9A affected apoptosis in HepG2.2.15 and HepG2 cell lines.Our results indicated that ATG9A might be partly involved in the survival of HCC.Thus,the inhibition of ATG9A together with other targets might be a potential drug target for HCC treatment.
文摘Background: One of potentially dangerous problems for a human organism is the new strain of a virus of bird flu-A/H7N9. As it is regular mutation of bird flu virus, it obvious, that of antibacterial preparations is not efficient. Efficiency decreases when the number of agents with multiple stability to antimicrobic remedy vastly increases, the part of associate infections enlarges, and aggression of opportunistic pathogenic flora rises. This reduces the role of the preparations in prevention of epidemics. Therefore, the optimization of only etiotropic therapies does not fully solve the problem. In this connection natural preparations seem extremely promising which strengthen the functional condition of immune system and, thereby, activate protective forces of macroorganism. Objectives: One of such preparations is BAE Synergy Liquid, a natural mineral water which was underwent subtle energetic changes at the natural energetic deposit. Design: An estimation of protective efficiency of naturally modified mineral BAE SL water was performed on white outbred mice-males in models of H7N9 virus. The animals were monitored during 16 days after infection, and survived and fallen mice were counted daily. Results: The results revealed significant effect of the investigated preparation as possible prophylactic care and medical remedy to the mentioned virus. This means that one can be considered as potential effective remedy for human. Conclusions: As significant effect of the immune system resistance was revealed, the experimental model with studied naturally modified mineral water is potentially generalizable.
文摘A novel influenza virus of the H7N9 subtype has infected more than 350 people in China since 19 February 2013. Evolutionary analysis indicates that the virus is a reassortant originated from H7, N9 and H9N2 avian influenza viruses, and bears some amino acids associated with mammalian receptor binding, raising concern over the possibility of a new influenza pandemic. Besides HA and NA, the mutation of the polymerase is known to have an important role in virulence, host adaptation and transmissibility in mammalians. In this article, the annotation of the polymerase protein domain associated with molecular function has been highlighted, suggesting the combination of RNA polymerase of H7N9 viruses is still not stable for host adaptation. In addition, the mutation hallmarks in polymerase gene of H7N9 are compared, providing the potential determinants of the evolution in the H7N9 influenza A virus.
文摘目的:探究单纯疱疹病毒(HSV)感染后脑脊液(CSF)中S100B、Cys-C、MMP-9水平对自身免疫性脑炎(AE)的预测价值。方法:选取2016年1月至2021年3月河北中石油中心医院收治的200例HSV感染患者为研究对象,根据是否继发AE分为研究组(继发AE,35例)和对照组(未继发AE,165例)。多因素Logistic回归分析HSV感染患者继发AE的独立影响因素。Spearman法分析脑脊液中Cys-C、MMP-9与S100B水平的相关性。受试者工作特征(ROC)曲线分析S100B、Cys-C、MMP-9对AE的预测价值。构建风险预测模型并进行评价。结果:多因素Logistic回归分析显示,MRI异常、脑脊液S100B、MMP-9升高、EEG异常是HSV感染患者继发AE的独立危险因素,脑脊液Cys-C是其保护因素(P<0.05)。Spearman分析显示,HSV感染患者Cys-C浓度与S100B水平呈负相关(r=-0.83,P<0.05),MMP-9浓度与S100B水平呈正相关(r=0.88,P<0.05)。构建的联合预测因子pre1诊断HSV患者继发AE的AUC明显大于S100B、Cys-C、MMP-9单独预测的AUC(0.876 vs 0.827、0.787、0.750)。构建的风险预测模型具有良好的区分度和一致性。结论:脑脊液中S100B、Cys-C、MMP-9水平均可对HSV感染患者诱发AE的可能性进行有效预测,且三项指标联合预测价值最大,其次是S100B蛋白、Cys-C、MMP-9。
