AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor(PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization(CNV), and investigates the mechani...AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor(PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization(CNV), and investigates the mechanism by which PEDF inhibits CNV in rats.METHODS: Brown Norway(BN) rats(n =204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein(GFP) or lentivirus-control GFP(free fluorescent protein). Following induction and treatment,the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography(FFA), optical coherence tomography(OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction(PCR) and Western blot.RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated PEDG gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28 d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size,thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28 d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.展开更多
Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVEC...Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVECs.Methods We constructed recombinant lentivirus vectors of NT4-alphastatin fusion gene containing neurotrophin-4 signal peptide,pro-region sequences and alphastatin,then transfected the recombinant lentivirus vectors into HUVECs to obtain secretory protein alphastatin and test its anti-angiogenic activities in vitro.Results Our data showed that recombinant self-inactivating lentivirus vectors of NT4-alphastatin were successfully constructed,and stable NT4-alphastatin transduced HUVECs were capable of sustainably secreting alphastatin which significantly suppressed HUVECs migration and differentiation but not VEGF-induced proliferation.Conclusion This report represents the first time on the use of lentivirus-based vectors to deliver alphastatin,the endogenous angiogenesis inhibitor,and reveals the potential utility of anti-angiogenic gene therapy with lentivirus vectors for treating cancer.展开更多
AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HI...AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P >0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.展开更多
Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm(TE) lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lin...Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm(TE) lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA(shRNA) and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.展开更多
Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse...Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lenti-viruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.展开更多
Spermatogonial stem cells(SSCs)transmit genetic information to the next progeny in males.Thus,SSCs are a potential target for germ I i ne modifications to gen erate tran sgenic an imals.In this study,we report a techn...Spermatogonial stem cells(SSCs)transmit genetic information to the next progeny in males.Thus,SSCs are a potential target for germ I i ne modifications to gen erate tran sgenic an imals.In this study,we report a technique for the gen erati on of tran sgenic rats by in vivo manipulation of SSCs with a high success rate.SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harbori ng enhan ced green fluoresce nt protei n and mated with wild-type females to create foun der rats.These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%.Subsequent generations of progeny from the founder rats both expressed and passed on the transgene.Thus,direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline.This technology could be adapted to larger animals,in which existing methods for gene modificatio n are in adequate or in applicable,resulti ng in the gen eration of tran sge nic an imals in a variety of species.展开更多
AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The re...AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.01).Moreover,the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability(CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%,P < 0.01).However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.CONCLUSION:miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.展开更多
Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system.In brain research,a series of powerful and novel gene editing technologi...Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system.In brain research,a series of powerful and novel gene editing technologies are based on these systems.Although many viral vectors are used in rodents,their full application has been limited in non-human primates.To identify viral vectors that can stably and effectively express exogenous genes within nonhuman primates,eleven commonly used recombinant adeno-associated viral and lentiviral vectors,each carrying a gene to express green or red fluorescence,were injected into the parietal cortex of four rhesus monkeys.The expression of fluorescent cells was used to quantify transfection efficiency.Histological results revealed that recombinant adeno-associated viral vectors,especially the serotype 2/9 coupled with the cytomegalovirus,human synapsin I,or Ca2+/calmodulin-dependentproteinkinaseII promoters,and lentiviral vector coupled with the human ubiquitin C promoter,induced higher expression of fluorescent cells,representing high transfection efficiency.This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates(NHPs).These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.展开更多
AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem c...AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.展开更多
BACKGROUND: Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture(CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a(miR-34a) lentivirus regulating Notc...BACKGROUND: Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture(CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a(miR-34a) lentivirus regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide(LPS)-induced human umbilical vein endothelial cells(HUVEC).METHODS: HUVEC were divided into four groups as the following: they were infected with negative control lentivirus(NC group) or miR-34a lentivirus(OE group); LPS(1 g/mL) was added on the third day on the basis of NC group and OE group for 24 hours(NC+LPS group or OE+LPS group). The levels of TNF-α, IL-1β, IL-6, and IL-10 in the cell supernatants, and the mRNA and protein expression of Notch-1 and NF-κB in the HUVEC were evaluated.RESULTS: After 24 hours, the levels of TNF-α, IL-1β, IL-6 in the cell supernatants and the protein expression of NF-κB from NC+LPS group were significantly higher than those of NC group, but IL-10 level and the protein expression of Notch-1 in NC+LPS group were the opposite. After intervention of miR-34a lentivirus, the cell supernatants TNF-α and the protein expression of NF-κB in OE+LPS group after 24 hours markedly decreased compared to NC+LPS group. While the cell supernatants IL-1β and IL-6 and the mRNA expression of NF-κB slightly decreased in OE+LPS group, IL-10 and the mRNA and protein expression of Notch-1 were the opposite.CONCLUSION: miR-34a regulating Notch-1/NF-κB signaling pathway can reduce the HUVEC damage caused by LPS stimulation.展开更多
AIM:To investigate the effects of lentivirus(LV)mediated integrin-linked kinase(ILK)RNA interference(RNAi)on biological behaviors of human lens epithelial cells(LECs).·METHODS:Human cataract LECs and immortalized...AIM:To investigate the effects of lentivirus(LV)mediated integrin-linked kinase(ILK)RNA interference(RNAi)on biological behaviors of human lens epithelial cells(LECs).·METHODS:Human cataract LECs and immortalized human LEC line,human lens epithelial(HLE)B-3 cells were transfected by lentiviral vector expressing ILKspecific short hairpin RNA(sh RNA)and then stimulated by transforming growth factor-β(TGF-β),the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot methods;biological behaviors including cell cycle and apoptosis,cell morphology,α-smooth muscle actin(SMA)stress fiber formation and cell migration were examined.·RESULTS:Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-sh RNA vector;flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells.Lessα-SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs.·CONCLUSION:The present study demonstrated that ILK was an important regulator for LECs proliferation and migration.LV mediated ILK RNAi is an effective way todecrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis,as well as,to prevent cell migration by inhibiting TGF-βinducedα-SMA stress fiber formation.Thus,LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.展开更多
BACKGROUND Invasion and migration are the irreversible stages of colorectal cancer(CRC).The key is to find a sensitive,reliable molecular marker that can predict the migration of CRC at an early stage.N-myc downstream...BACKGROUND Invasion and migration are the irreversible stages of colorectal cancer(CRC).The key is to find a sensitive,reliable molecular marker that can predict the migration of CRC at an early stage.N-myc downstream regulated gene 1(NDRG1)is a multifunctional gene that has been tentatively reported to have a strong relationship with tumor invasion and migration,however the current molecular role of NDRG1 in CRC remains unknown.AIM To explore the role of NDRG1 in the development of CRC.METHODS NDRG1 stably over-expressed Caco2 cell line was established by lentiviral infection and NDRG1 knock-out Caco2 cell line was established by CRISPR/Cas9.Furthermore,the mRNA and protein levels of NDRG1 in Caco2 cells after NDRG1 over-expression and knockout were detected by real-time polymerase chain reaction and western blot.The cell proliferation rate was measured by the cell counting kit-8 method;cell cycle and apoptosis were detected by flow cytometry;invasion and migration ability were detected by the 24-transwell method.RESULTS NDRG1 over-expression inhibited Caco2 proliferation and the cell cycle could be arrested at the G1/S phase when NDRG1 was over-expressed,while the number of cells in the G2 phase was significantly increased when NDRG1 was knocked out.This suggests that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase.Our data also demonstrated that NDRG1 promotes early cell apoptosis.Invasion and migration of cells were extensively inhibited when NDRG1 was over-expressed.CONCLUSION NDRG1 inhibits tumor progression in Caco2 cells which may represent a potential novel therapeutic strategy for the treatment of CRC.展开更多
In the last decade, RNA interference(RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased ...In the last decade, RNA interference(RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased inhibitors against the chronic infection with human immunodeficiency virus type 1(HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules(sh RNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 sh RNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three sh RNAs. These sh RNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome,exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial sh RNA vector will soon move forward to the first clinical studies.展开更多
基金Supported by the National Natural Science Foundation of China(No.81070735)
文摘AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor(PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization(CNV), and investigates the mechanism by which PEDF inhibits CNV in rats.METHODS: Brown Norway(BN) rats(n =204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein(GFP) or lentivirus-control GFP(free fluorescent protein). Following induction and treatment,the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography(FFA), optical coherence tomography(OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction(PCR) and Western blot.RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated PEDG gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28 d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size,thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28 d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.
基金supported by the National Natural Science Foundation of China(No.30672162)
文摘Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVECs.Methods We constructed recombinant lentivirus vectors of NT4-alphastatin fusion gene containing neurotrophin-4 signal peptide,pro-region sequences and alphastatin,then transfected the recombinant lentivirus vectors into HUVECs to obtain secretory protein alphastatin and test its anti-angiogenic activities in vitro.Results Our data showed that recombinant self-inactivating lentivirus vectors of NT4-alphastatin were successfully constructed,and stable NT4-alphastatin transduced HUVECs were capable of sustainably secreting alphastatin which significantly suppressed HUVECs migration and differentiation but not VEGF-induced proliferation.Conclusion This report represents the first time on the use of lentivirus-based vectors to deliver alphastatin,the endogenous angiogenesis inhibitor,and reveals the potential utility of anti-angiogenic gene therapy with lentivirus vectors for treating cancer.
基金Supported by Natural Science Foundation of China(No.30901395)the Doctoral Fund of Ministry of Education of China(No.20090142120012,20110142120021)
文摘AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P >0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.
基金Supported by the Scientifi c Research Fund of Heilongjiang Provincial Education Department(11551039)
文摘Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm(TE) lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA(shRNA) and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.
文摘Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lenti-viruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.
文摘Spermatogonial stem cells(SSCs)transmit genetic information to the next progeny in males.Thus,SSCs are a potential target for germ I i ne modifications to gen erate tran sgenic an imals.In this study,we report a technique for the gen erati on of tran sgenic rats by in vivo manipulation of SSCs with a high success rate.SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harbori ng enhan ced green fluoresce nt protei n and mated with wild-type females to create foun der rats.These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%.Subsequent generations of progeny from the founder rats both expressed and passed on the transgene.Thus,direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline.This technology could be adapted to larger animals,in which existing methods for gene modificatio n are in adequate or in applicable,resulti ng in the gen eration of tran sge nic an imals in a variety of species.
基金Supported by National Natural Science Foundation of China,No.81101896
文摘AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.01).Moreover,the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability(CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%,P < 0.01).However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.CONCLUSION:miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.
基金supported by the National Program on Key Basic Research Project(973 Programs 2015CB755605)the National Natural Science Foundation of China(81471312)
文摘Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system.In brain research,a series of powerful and novel gene editing technologies are based on these systems.Although many viral vectors are used in rodents,their full application has been limited in non-human primates.To identify viral vectors that can stably and effectively express exogenous genes within nonhuman primates,eleven commonly used recombinant adeno-associated viral and lentiviral vectors,each carrying a gene to express green or red fluorescence,were injected into the parietal cortex of four rhesus monkeys.The expression of fluorescent cells was used to quantify transfection efficiency.Histological results revealed that recombinant adeno-associated viral vectors,especially the serotype 2/9 coupled with the cytomegalovirus,human synapsin I,or Ca2+/calmodulin-dependentproteinkinaseII promoters,and lentiviral vector coupled with the human ubiquitin C promoter,induced higher expression of fluorescent cells,representing high transfection efficiency.This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates(NHPs).These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.
基金Supported by National Institute of Genetic Engineering and Biotechnology,Ministry of Science Research and Technology,Tehran,Iran
文摘AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.
基金supported by a grant from Natural Science Foundation of Zhejiang Province of China(LY14H150003)
文摘BACKGROUND: Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture(CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a(miR-34a) lentivirus regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide(LPS)-induced human umbilical vein endothelial cells(HUVEC).METHODS: HUVEC were divided into four groups as the following: they were infected with negative control lentivirus(NC group) or miR-34a lentivirus(OE group); LPS(1 g/mL) was added on the third day on the basis of NC group and OE group for 24 hours(NC+LPS group or OE+LPS group). The levels of TNF-α, IL-1β, IL-6, and IL-10 in the cell supernatants, and the mRNA and protein expression of Notch-1 and NF-κB in the HUVEC were evaluated.RESULTS: After 24 hours, the levels of TNF-α, IL-1β, IL-6 in the cell supernatants and the protein expression of NF-κB from NC+LPS group were significantly higher than those of NC group, but IL-10 level and the protein expression of Notch-1 in NC+LPS group were the opposite. After intervention of miR-34a lentivirus, the cell supernatants TNF-α and the protein expression of NF-κB in OE+LPS group after 24 hours markedly decreased compared to NC+LPS group. While the cell supernatants IL-1β and IL-6 and the mRNA expression of NF-κB slightly decreased in OE+LPS group, IL-10 and the mRNA and protein expression of Notch-1 were the opposite.CONCLUSION: miR-34a regulating Notch-1/NF-κB signaling pathway can reduce the HUVEC damage caused by LPS stimulation.
基金Supported by the National Natural Science Foundation of China(No.81273605,No.30901655)
文摘AIM:To investigate the effects of lentivirus(LV)mediated integrin-linked kinase(ILK)RNA interference(RNAi)on biological behaviors of human lens epithelial cells(LECs).·METHODS:Human cataract LECs and immortalized human LEC line,human lens epithelial(HLE)B-3 cells were transfected by lentiviral vector expressing ILKspecific short hairpin RNA(sh RNA)and then stimulated by transforming growth factor-β(TGF-β),the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot methods;biological behaviors including cell cycle and apoptosis,cell morphology,α-smooth muscle actin(SMA)stress fiber formation and cell migration were examined.·RESULTS:Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-sh RNA vector;flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells.Lessα-SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs.·CONCLUSION:The present study demonstrated that ILK was an important regulator for LECs proliferation and migration.LV mediated ILK RNAi is an effective way todecrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis,as well as,to prevent cell migration by inhibiting TGF-βinducedα-SMA stress fiber formation.Thus,LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.
基金the National Natural Science Foundation of China,No.81260361Incubation Project of Mianyang Central Hospital,No.2020FH05.
文摘BACKGROUND Invasion and migration are the irreversible stages of colorectal cancer(CRC).The key is to find a sensitive,reliable molecular marker that can predict the migration of CRC at an early stage.N-myc downstream regulated gene 1(NDRG1)is a multifunctional gene that has been tentatively reported to have a strong relationship with tumor invasion and migration,however the current molecular role of NDRG1 in CRC remains unknown.AIM To explore the role of NDRG1 in the development of CRC.METHODS NDRG1 stably over-expressed Caco2 cell line was established by lentiviral infection and NDRG1 knock-out Caco2 cell line was established by CRISPR/Cas9.Furthermore,the mRNA and protein levels of NDRG1 in Caco2 cells after NDRG1 over-expression and knockout were detected by real-time polymerase chain reaction and western blot.The cell proliferation rate was measured by the cell counting kit-8 method;cell cycle and apoptosis were detected by flow cytometry;invasion and migration ability were detected by the 24-transwell method.RESULTS NDRG1 over-expression inhibited Caco2 proliferation and the cell cycle could be arrested at the G1/S phase when NDRG1 was over-expressed,while the number of cells in the G2 phase was significantly increased when NDRG1 was knocked out.This suggests that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase.Our data also demonstrated that NDRG1 promotes early cell apoptosis.Invasion and migration of cells were extensively inhibited when NDRG1 was over-expressed.CONCLUSION NDRG1 inhibits tumor progression in Caco2 cells which may represent a potential novel therapeutic strategy for the treatment of CRC.
基金Supported by The NWO-CW(Chemical Sciences),Zon Mw(Medical Sciences),the Dutch AIDS Fund(project 2006006)the DAAD(German Academic Exchange Service)the FRM(Fondation pour la Recherche Medicale)
文摘In the last decade, RNA interference(RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased inhibitors against the chronic infection with human immunodeficiency virus type 1(HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules(sh RNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 sh RNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three sh RNAs. These sh RNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome,exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial sh RNA vector will soon move forward to the first clinical studies.
