Gene transfer in primary cultures of human hepatocytes

Summary Using liposomes as the mediator of DNA transfer, we were successful in the transfection of human hepatocytes isolated from surgical samples with an E. coli β-galactosidase gene (β-gal). A comparison of transfection efficiency showed that of the four promoters used, cytomegalovirus (CMV) promoter yielded higher transfection efficiencies than Rous sarcoma virus (RSV), Simian virus-40 (SV-40) and human alpha-l antitrypsin (AAT) promoters. These studies represent the first report on the successful transfection of primary cultures of human hepatocytes.     

Safety and efficacy of adeno-associated viral vector-mediated insulin gene transfer via portal vein to the livers of streptozotocin-induced diabetic Sprague-Dawley rats

BACKGROUND: Previous studies demonstrating the efficacy of insulin gene therapy have mostly involved use of adenoviral vectors or naked DNA to deliver the insulin gene. However, this procedure may not guarantee long-term insulin production. To improve the performance, we prepared recombinant adeno-associated viral vectors (rAAV) harboring the gene encoding a furin-modified human insulin under the cytomegalovirus (CMV) promoter [rAAV-hPPI(F12)]. METHODS: Streptozotocin (STZ)-induced diabetic Sprague-Dawley rats were used as a diabetic animal model. The levels of blood glucose, insulin, and HbA1c were measured to test the effect. An intraperitoneal glucose tolerance test was performed to test the capability of blood glucose disposal. Immunohistochemical staining and Northern blot analyses were performed to survey the expression pattern of the therapeutic insulin gene. RESULTS: STZ-induced diabetic Sprague-Dawley rats infused via the portal vein with rAAV-hPPI(F12) produced human insulin and after a 6-h fast were normoglycemic for over 90 days post-treatment, whereas diabetic rats treated with recombinant adenoviral vector harboring the hPPI(F12) gene [rAV-hPPI(F12)] were normoglycemic only for days 3 to 13 post-treatment. Insulin mRNA was detected mainly in the liver of the rAAV-hPPI(F12)-treated diabetic rats. The glucose tolerance capability of the rAAV-hPPI(F12)-treated diabetic rats was comparable to that of non-diabetic rats, even without injection of recombinant insulin. Furthermore, blood HbA1c concentrations in rAAV-hPPI(F12)-treated diabetic rats were reduced to almost the normal level. Importantly, studies of rAV or rAAV vector-dependent side effects on the targeted liver strongly suggested that only rAAV treatment caused no side effects. CONCLUSIONS: These results demonstrate that our rAAV-mediated in vivo insulin gene therapy provides safer maintenance of the insulin gene expression required for long-term and thus more effective blood glycemic control.     

Inhibition of corneal neovascularization with endostatin delivered by adeno-associated viral (AAV) vector in a mouse corneal injury model

The use of a recombinant adeno-associated viral (rAAV) vector carrying endostatin gene as an anti-angiogenesis strategy to treat corneal neovascularization in a mouse model was evaluated. Subconjunctival injection of recombinant endostatin-AAV was used to examine the inhibition of corneal neovascularization induced by silver nitrate cauterization in mice. The results showed that gene expression in corneal tissue was observed as early as 4 days after gene transfer and stably lasted for over 8 months with minimal immune reaction. Subconjunctival injection of a high-titer rAAV-endostatin successfully inhibited neovascularization. Immunohistchemistry staining of CD 31 and endostatin showed that the treatment significantly inhibits angiogenesis in cornea. We concluded that the rAAV was capable of directly delivering genes to the ocular surface epithelium by way of subconjunctival injection and was able to deliver sustained, high levels of gene expression in vivo to inhibit angiogenesis.     

昆虫细胞制备AAV-ITR基因表达微载体

AAV-ITR基因表达微载体是只含有腺相关病毒(Adeno-associated virus,AAV)倒置末端重复序列(Inverted terminal repeats,ITR)、基因表达顺式元件和目的基因,而不含有其他外源DNA序列的双链或单链DNA。本研究利用杆状病毒表达系统,制备得到两种重组杆状病毒Bac-ITR-EGFP和Bac-inrep,并将二者的P3代病毒共同感染昆虫细胞Spodoptera frugiperda(Sf9),抽提小分子量DNA,获得AAV-ITR-EGFP基因表达微载体,2×107的Sf9细胞抽提可以得到100μg AAV-ITR-EGFP基因表达微载体,核酸电泳显示AAV-ITR-EGFP基因表达微载体主要以单体和二聚体的形式存在。将AAV-ITR-EGFP基因表达微载体通过polyethylenimine(PEI)转染HEK 293T细胞,24 h后荧光显微镜观察有EGFP表达,48 h后达到高峰,转化效率达到65%。     

microRNA调控的靶向性病毒载体在基因治疗中的应用

安全、有效、具有靶向性的病毒载体是基因治疗药物在临床上得以应用的关键。microRNA是一类单链、内源性的转录后调控小分子,它的发现为开发具有靶向性调控能力的病毒载体提供了新的研究方法。以下在介绍microRNA调节病毒载体靶向性原理的基础上,着重介绍microRNA在清除复制能力病毒的污染、消除转基因特异性免疫、增强肿瘤靶向性基因治疗、开发活体疫苗等领域的应用。     

A simplified model of hypoxic injury in primary cultured rat hepatocytes

Summary The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + β-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/β-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.     

Effects of vanadate on the expression of genes involved in fuel homeostasis in animal models of Type I and Type II diabetes

Vanadium is a trace element that has raised increasing interest in diabetology since the discovery of its insulin-like propertiesin vitro andin vivo. This brief article reviews the most recent data concerning the beneficial effects of vanadium compounds on fuel homeostasis in animal models of insulinopenic (Type I) or insulin-resistant (Type II) diabetes. These studies open obvious therapeutic possibilities in diabetes, and more particularly, in states of insulin resistance.     

Selective gene therapy for human lung adenocarcinoma by tumor-specific expression of herpes simplex virus thymidine kinase gene

According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious "bystander effect" in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice