AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a ...AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.展开更多
Objective： To investigate the expressions and correlations of Pin1, β-catenin and cyclin D1 in elderly lung carcinomas. Methods： The expressions of Pin1, β-catenin and cyclin D1 were examined in the specimens of 9...Objective： To investigate the expressions and correlations of Pin1, β-catenin and cyclin D1 in elderly lung carcinomas. Methods： The expressions of Pin1, β-catenin and cyclin D1 were examined in the specimens of 92 elderly lung carcinomas and 10 normal lung tissues by immunohistochemistry and explored the relationship between the expression levels and clinicopathological factors. Results： （1） The overexpression of Pin1 and cyclin D1 in lung carcinomas was 46 （50%） cases and 60 （65.22%） cases respectively and 56 （60.82%） cases showed positive immunoreactivity for 13-catenin in the nuclear and （or） cytoplasmic fraction in tumor tissues. In normal tissue, the expressions of Pin1 and cyclin D1 were negative, the expression of β-catenin was lied in cell membrane. （2） In lung carcinomas the expressions of Pin1, β-catenin and cyclin D1 correlated with tumor differentiation （P 〈 0.05）. The pesitive expression rate and intensity of Pin1 correlated with tumor stage （P = 0.032） and lymph node positive disease （P = 0.041）. The expression of β-catenin correlated with lymph node positive disease （P = 0.012）. （3） High expression levels of Pin1 correlated with aberrant I]-catenin expression （P = 0.000） but did not show a correlation with cyclin D1 （P = 0.157）. Conclusion： In elderly lung carcinomas, the positive expression of Pin1 causes abnormal accumulation of β-catenin and actives its target gene, however, this target gene was not cyclin DI. The detection of Pin1 expression had some clinical significance in estimating prognosis of elderly patient with lung carcinomas.展开更多
AIM:To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-RNA (HCV-RNA) amount and to analyze whether this effect was assoc...AIM:To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-RNA (HCV-RNA) amount and to analyze whether this effect was associated with clinical response to IFN.METHODS:Twenty-seven patients with histologically proven chronic hepatitis C were given intravenous administration of 6 million units (MU) IFN-β daily for 6 weeks followed by three times weekly for 20 weeks. PBMC collected before IFN therapy were incubated with IFN-β and HCV-RNA in PMBC was semi-quantitatively determined.RESULTS: Twenty-five patients completed IFN therapy.Eight patients (32%) had sustained loss of serum HCV-RNA with normal serum ALT levels after IFN therapy (complete responders).HCV-RNA in PBMC was detected in all patients,whereas it was not detected in PBMC from healthy subjects.In vitro administration of IFN-β decreased the amount of HCV-RNA in PMBC in 18 patients (72%). Eight of these patients obtained complete response. On the other hand,none of the patients whose HCV-RNA in PBMC did not decrease by IFN-β was complete responders. Multiple logistic regression analysis revealed that the decrease of HCV-RNA amount in PBMC by IFN-β was the only independent predictor for complete response (P<0.05).CONCLUSION:The effect of in vitro IFN-β on HCV in PBMC reflects clinical response and would be taken into account as a predictive marker of IFN therapy for chronic hepatitis C.展开更多
To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector...To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.展开更多
基金the National Natural Science Foundation of China,No.81503406the China Postdoctoral Science Foundation,No.2017M6232+1 种基金the Special Fund for Traditional Chinese Medicine by the Health Department of Guangxi Zhuang Autonomous Region,No.GZBZ16-07,GZLC16-23the Natural Science Foundation of Guangxi Zhuang Autonomous Region,No.2014GXNSFAA118177~~
基金the"Hundred Talents"Program of Shanghai Municipal Government,No.98BR018
文摘AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.
文摘Objective： To investigate the expressions and correlations of Pin1, β-catenin and cyclin D1 in elderly lung carcinomas. Methods： The expressions of Pin1, β-catenin and cyclin D1 were examined in the specimens of 92 elderly lung carcinomas and 10 normal lung tissues by immunohistochemistry and explored the relationship between the expression levels and clinicopathological factors. Results： （1） The overexpression of Pin1 and cyclin D1 in lung carcinomas was 46 （50%） cases and 60 （65.22%） cases respectively and 56 （60.82%） cases showed positive immunoreactivity for 13-catenin in the nuclear and （or） cytoplasmic fraction in tumor tissues. In normal tissue, the expressions of Pin1 and cyclin D1 were negative, the expression of β-catenin was lied in cell membrane. （2） In lung carcinomas the expressions of Pin1, β-catenin and cyclin D1 correlated with tumor differentiation （P 〈 0.05）. The pesitive expression rate and intensity of Pin1 correlated with tumor stage （P = 0.032） and lymph node positive disease （P = 0.041）. The expression of β-catenin correlated with lymph node positive disease （P = 0.012）. （3） High expression levels of Pin1 correlated with aberrant I]-catenin expression （P = 0.000） but did not show a correlation with cyclin D1 （P = 0.157）. Conclusion： In elderly lung carcinomas, the positive expression of Pin1 causes abnormal accumulation of β-catenin and actives its target gene, however, this target gene was not cyclin DI. The detection of Pin1 expression had some clinical significance in estimating prognosis of elderly patient with lung carcinomas.
文摘AIM:To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-RNA (HCV-RNA) amount and to analyze whether this effect was associated with clinical response to IFN.METHODS:Twenty-seven patients with histologically proven chronic hepatitis C were given intravenous administration of 6 million units (MU) IFN-β daily for 6 weeks followed by three times weekly for 20 weeks. PBMC collected before IFN therapy were incubated with IFN-β and HCV-RNA in PMBC was semi-quantitatively determined.RESULTS: Twenty-five patients completed IFN therapy.Eight patients (32%) had sustained loss of serum HCV-RNA with normal serum ALT levels after IFN therapy (complete responders).HCV-RNA in PBMC was detected in all patients,whereas it was not detected in PBMC from healthy subjects.In vitro administration of IFN-β decreased the amount of HCV-RNA in PMBC in 18 patients (72%). Eight of these patients obtained complete response. On the other hand,none of the patients whose HCV-RNA in PBMC did not decrease by IFN-β was complete responders. Multiple logistic regression analysis revealed that the decrease of HCV-RNA amount in PBMC by IFN-β was the only independent predictor for complete response (P<0.05).CONCLUSION:The effect of in vitro IFN-β on HCV in PBMC reflects clinical response and would be taken into account as a predictive marker of IFN therapy for chronic hepatitis C.
文摘To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.
