To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we establishe...To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.展开更多
Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-...Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hlGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochernistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghlGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. Results Recombinant retroviral plasmid vector pLghlGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening, hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast （P〈 0.05）. The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast （P〈 0.05）. Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myobiast group （P〈 0.05）. Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.展开更多
AIM: To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas,and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro.METHODS: The expre...AIM: To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas,and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro.METHODS: The expression of DPC4 was determined in 40 pancreatic adenocarcinoma and 36 non-malignant pancreatic specimens by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohisto-chemistry.Furthermore,we constructed retroviral vectors containing DPC4,which then infected the pancreatic carcinoma cell line BxPC-3.Cell growth in vitro after being infected was observed,and the vascular endothelial growth factor (VEGF) mRNA level in the daughter cells was determined by semi-quantitative PCR assay.RESULTS: The RT-PCR assay showed a positive rate of DPC4 mRNA in 100% (36/36) of normal specimens,compared to 40% (16/40) in adenocarcinoma specimens.The regional and intense positive cases of DPC4 expression in adenocarcinoma detected by immunohistochemistry were 10 and four,whereas it was all positive expression in normal tissues.There was a significant difference of DPC4 expression between them.The stable expression of DPC4 in the pancreatic carcinoma cells BxPC-3 could be resumed by retroviral vector pLXSN transfection,and could inhibit cell growth in vitro.Rather,DPC4 could decrease VEGF mRNA transcription levels.CONCLUSION: The deletion of DPC4 expression in pancreatic carcinoma suggests that loss of DPC4 may be involved in the development of pancreatic carcinoma.The retroviral vector pLXSN containing DPC4 can inhibit the proliferation of pancreatic carcinoma cells,and down-regulate the level of VEGF.展开更多
This paper describes the first human gene therapy trial for hemophilia B. Retroviruses were used to introduce human factor Ⅸ into autologous, primary human skin fibroblasts from the patients. Recombinant retroviral v...This paper describes the first human gene therapy trial for hemophilia B. Retroviruses were used to introduce human factor Ⅸ into autologous, primary human skin fibroblasts from the patients. Recombinant retroviral vector containing human FIX cDNA driven by viral LTR promoter (XL-Ⅸ) and double-copy retroviral vector driven by human cytomegalovirus enhancer-promoter (N2CMV-Ⅸ)were constructed. After the safety assessment, including soft-agar test, cell morphology observation, analysis of endotoxin, chromosome karyotype, allergic reaction test, nude mice test, routine pathological test, electromicroscopic analysis, and virus detection by PCR, etc., the engineered cells were pooled and embedded in collagen mixture, autologously injected into the patients respectively. The concentration of human FIX protein of Patient 1 increased from 71 ng/ml to 220 ng/ml, witha maximum level of 245 ng/ml. The expression of FIX has lasted for 6 months at the time of writing. The clotting activity also increased from 2.9% to 6.3%, his clinical symptoms have been alleviated obviously. The secretion rate of FIX for Patient 2 increased from 130 to 250 ng/ml, maintained at the level of 220 ng/ml for 5.5 months at the time of writing, but the clotting activity has not been increased steadily. There is no deleterious effect to be found in the two patients since the ex-vivo cells were implanted. The two patients are now under follow-up investigation. We suggested that retrovirus-mediated transfer of genes into skin fibroblasts, to be embedded in collagen and subcutaneously injected into patients, is a simple and effective approach for the gene therapy for hemophilia B.展开更多
基金This work was kindly supported by Na-tional Natural Science Foundation of China(No.39670308)
文摘To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.
基金This work was supported by a grant from the National Natural Science Foundation of China (No.30470457).
文摘Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hlGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochernistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghlGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. Results Recombinant retroviral plasmid vector pLghlGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening, hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast （P〈 0.05）. The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast （P〈 0.05）. Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myobiast group （P〈 0.05）. Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.
文摘AIM: To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas,and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro.METHODS: The expression of DPC4 was determined in 40 pancreatic adenocarcinoma and 36 non-malignant pancreatic specimens by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohisto-chemistry.Furthermore,we constructed retroviral vectors containing DPC4,which then infected the pancreatic carcinoma cell line BxPC-3.Cell growth in vitro after being infected was observed,and the vascular endothelial growth factor (VEGF) mRNA level in the daughter cells was determined by semi-quantitative PCR assay.RESULTS: The RT-PCR assay showed a positive rate of DPC4 mRNA in 100% (36/36) of normal specimens,compared to 40% (16/40) in adenocarcinoma specimens.The regional and intense positive cases of DPC4 expression in adenocarcinoma detected by immunohistochemistry were 10 and four,whereas it was all positive expression in normal tissues.There was a significant difference of DPC4 expression between them.The stable expression of DPC4 in the pancreatic carcinoma cells BxPC-3 could be resumed by retroviral vector pLXSN transfection,and could inhibit cell growth in vitro.Rather,DPC4 could decrease VEGF mRNA transcription levels.CONCLUSION: The deletion of DPC4 expression in pancreatic carcinoma suggests that loss of DPC4 may be involved in the development of pancreatic carcinoma.The retroviral vector pLXSN containing DPC4 can inhibit the proliferation of pancreatic carcinoma cells,and down-regulate the level of VEGF.
文摘This paper describes the first human gene therapy trial for hemophilia B. Retroviruses were used to introduce human factor Ⅸ into autologous, primary human skin fibroblasts from the patients. Recombinant retroviral vector containing human FIX cDNA driven by viral LTR promoter (XL-Ⅸ) and double-copy retroviral vector driven by human cytomegalovirus enhancer-promoter (N2CMV-Ⅸ)were constructed. After the safety assessment, including soft-agar test, cell morphology observation, analysis of endotoxin, chromosome karyotype, allergic reaction test, nude mice test, routine pathological test, electromicroscopic analysis, and virus detection by PCR, etc., the engineered cells were pooled and embedded in collagen mixture, autologously injected into the patients respectively. The concentration of human FIX protein of Patient 1 increased from 71 ng/ml to 220 ng/ml, witha maximum level of 245 ng/ml. The expression of FIX has lasted for 6 months at the time of writing. The clotting activity also increased from 2.9% to 6.3%, his clinical symptoms have been alleviated obviously. The secretion rate of FIX for Patient 2 increased from 130 to 250 ng/ml, maintained at the level of 220 ng/ml for 5.5 months at the time of writing, but the clotting activity has not been increased steadily. There is no deleterious effect to be found in the two patients since the ex-vivo cells were implanted. The two patients are now under follow-up investigation. We suggested that retrovirus-mediated transfer of genes into skin fibroblasts, to be embedded in collagen and subcutaneously injected into patients, is a simple and effective approach for the gene therapy for hemophilia B.
