【作者】 丁艳华；

【导师】 牛俊奇；

【作者基本信息】 吉林大学 ， 内科学， 2007， 博士

【摘要】 全世界目前己有1.7亿HCV感染者,我国普通人群抗-HCV阳性率为3.2%。急性HCV感染者中,大约80%可发展为慢性持续感染,其中20%的患者经历20-30年将发展为肝硬化,1%-5%的患者可进展为肝细胞癌。HCV所导致的终末期肝病是重要的死亡原因之一,也是肝移植的原因。目前慢性丙型肝炎的标准治疗是干扰素联合利巴韦林,但有效率仅仅40%-80%左右,人们试图开发新型、高效的治疗药物来解决这一问题。自RNAi技术发现以来,已广泛应用于分子生物学领域的研究,并且已经证实其在细胞水平能有效抑制HIV、脊髓灰质炎病毒、HBV及HCV的复制和表达。本文构建了三个靶向(targeting to)HCV 5’NCR和C区的小发夹RNA质粒表达载体,与含有HCV 5’NCR和C区部分基因及荧光素酶报告基因的质粒共转染人肝细胞HL-7702。通过检测荧光素酶报告基因和Western blot来检测目的蛋白表达。发现靶向HCV 5’NCR NCR和靶向C区的shRNA能明显抑制HCV 5’NCR和C区的表达。目前小干涉RNA进行体内实验的主要障碍是缺乏安全有效的转基因载体。我们应用了一种非病毒类载体古菌组蛋白(HPhA)作为转基因载体。在实验中发现HPhA能与DNA结合,毒性很小,能在血清存在的条件下转染小干涉RNA表达质粒进入细胞抑制HCV 5’NCR和C区的表达。所以HPhA有潜力发展为适合体内实验的转基因载体。本文首次应用HPhA作为转基因载体,为小干涉RNA抗病毒基因治疗的体内实验提供了重要的理论基础、技术路线和实验方法。更多还原

【Abstract】 Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Effective therapies against viral infection are not available at present. There are some problems involved in the available techniques of HCV gene therapies, such as antioligonuclitide and ribozyme and deoxyribozyme. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.RNA interference (RNAi) is a process during which double stranded RNA (dsRNA) induces the sequence-specific degradation of homologous single-stranded RNA. As a result, the expression of the target gene is suppressed. RNAi can therefore be used to develop specific dsRNA based gene therapeutics by knocking down specific genes such as the gene related to viral infection, carcinoma, and the others diseases.In cultured mammalian cells, the sequence-specific gene silencing can be accomplished by introducing small interfering RNA (siRNA) that is cleaved from dsRNA. Only RNA molecules<30 bases in length can be used to exclusively induce RNAi in mammalian cells because longer molecules also activate the nonspecific double-stranded RNA-dependent IFN response. With the advance of producing siRNA, several laboratories made progress in using RNAi to control viral infection and showed that siRNA can inhibit infection by polio, HIV, HCV and influenza virus. Recently it was reported that RNAi could also reduced HCV replication and expression in the post-transcriptional level.The likelihood of HCV developing escape mutations illustrates the importance of careful siRNA target sequence selection during the development of treatment strategies. Several conserved regions within the HCV5’noncoding region (5’-NCR) have been found to be viable targets for RNA interference and have potential in therapeutic RNA interference strategies. For example, the 5′UTR acts as an internal ribosome entry sequence, and its activity is determined by RNA structural characteristics. As such, the sequence does not tolerate nucleotide changes and is highly conserved between different HCV genotypes. Therefore, siRNA target sequences based on the 5′UTR region offer promise for siRNA-based treatments.We construct HCV-specific shRNA expression plasmid targeted 5’NCR and C region (Psilencirle A, B and D), luciferase-specific siRNA expression plasmid (Psilencirle E) and scrambled siRNA expression plasmid(Psilencirle C) by SilenCircleTM RNAi Transcription Kit. Then we transfect above plasmids and plasmids pCMV/T7-NCRC?-luc to HL-7702 cells, pCMV/T7-NCRC?-luc containing HCV 5’NCR, partial core region and luciferase sequences. The luciferase gene is utilized as a reporter gene, its expression can reflect the synthesis of HCV 5’NCR and core protein. The results show that Psilencirle A,B and D can inhibit the expression of HCV 5’NCR and core protein in HL-7702 cells, the inhibiting rate are 38%, 49%, 55% respectedly.An obvious challenge in the use of siRNA as a therapeutic agent is the development of suitable delivery methods. There have been recent advances in delivery of siRNA using peptide and polymeric vehicles and in vivo application through injection of a large volume of liquid siRNA solutions into the tail veins of mice. The latter treatment could induce RNA interference in the mouse liver and prevent Fas-mediated apoptosis. In our study we introduced the new transgene carrier HPhA (gene engineering production of jilin university) to transfect siRNA expression plasmid into HL-7702 cells.Gel electrophoresis mobility shift assays demonstrate that the purified HphA has high affinity to DNA. The HphA can increase plasmid electrophoresis mobilities with proper HphA/DNA mass ratios.We determined the toxicty of HphA to cells by MTT methods, and compared with LipofectamineTM 2000. The results shows that HphA almost don’t affect the growth of cells in relative low concentration(20ug/ml), but LipofectamineTM 2000 cause relative cell viability obviously come down. Cells will die only when the concentration of HphA is higher than 100ug/ml . The toxicty of 1000ug/ml HphA to HL-7702 cells is similar with that of 40ug/ml LipofectamineTM 2000. Considering of the maximum amount of HphA per well is only 9ug when transfection, we can conclude that HphA is nontoxic to cells.HCV-specific siRNA expression plasmid mediated by HphA can inhibit the expression of HCV 5’NCR and core protein, its efficiency is similar with LipofectamineTM2000. The most important is that when medium containe10% fetal calf serum (FCS), the efficiency of HphA mediated transfection is similar with that without fetal calf serum, and LipofectamineTM2000 can’t transfec plasmids into HL-7702 cells to express HCV 5’NCR and core protein ,when medium containe10% fetal calf serum (FCS). So we concluded that HphA is suitable to transfection in vivo, and it is a safe and effective transgene carrier and will play an import role in RNAi-based genetherapy.更多还原