干扰素在2’-5’OAS启动子调控的重组caspase-3质粒诱导肝细胞凋亡中的作用
发布时间：2019-03-17 08:02
【摘要】：背景与目的：世界卫生组织统计,全世界约有3%的人感染丙型肝炎病毒(HCV),约1.7亿的慢性携带者有发展成肝硬化和肝细胞癌(HCC)的危险性。丙型肝炎的防治是目前全世界面临的一项重大问题,由于HCV极易发生变异,使得其防治更为困难。至今尚无理想的抗HCV药物和疫苗防治HCV感染,特别是感染细胞内的HCV难以清除,成为丙型肝炎复发的病毒来源。本课题组前期体内外实验表明：丙型肝炎病毒的核心蛋白可以特异性的结合并激活2’-5’寡腺苷酸合成酶(OAS)启动子调控的重组caspase-3质粒系统,从而诱导HCV感染的肝细胞凋亡。干扰素是目前治疗丙型肝炎的常用药物之一,日本学者Kerr等发现用干扰素作用细胞,可在其细胞提取液中找到2’-5’寡腺苷酸合成酶。本研究旨在明确干扰素a-2b能否特异性的激活OAS启动子调控的重组caspase-3质粒,为OAS-re-caspase-3治疗系统进 一步应用提供实验依据。 方法：(1)构建重组质粒GV230/EGFP-OAS-re-caspase-3,PCR扩增、琼脂糖凝胶电泳及扩增产物测序验证；(2)建立稳定转染细胞系:HL7702/EGFP-OAS-re-caspase-3和HepG2/EGFP-OAS-re-caspase-3(3)用流式细胞仪和MTT检测比较HL7702/OAS-re-caspase-3和HL7702/EGFP细胞在不同浓度干扰素作用下的凋亡情况；结晶紫染色及TUNEL检测比较HepG2/EGFP-OAS-re-caspase-3和HepG2两组细胞在不同浓度干扰素作用下的凋亡指数；(4)裸鼠成瘤：将稳定转染的HepG2/EGFP-OAS-re-caspase-3、HepG2细胞接种裸鼠皮下,待肿瘤形成后,瘤内注射治疗剂量干扰素,48小时后处死动物,TUNEL显色法检测比较两组细胞的凋亡指数。 结果：(1)PCR及凝胶电泳和测序结果表明成功构建了重组质粒EGFP-OAS-re-caspase-3；(2)转染24h后,在荧光显微镜下观察计数,EGFP-OAS-re-caspase-3、EGFP质粒转染HL7702、HepG2细胞,转染效率达70%以上；G418筛选2周左右可见抗性克隆,维持筛选出稳定转染细胞株；(3)流式细胞仪、MTT、结晶紫染色及TUNEL体外实验表明：干扰素α-2b可特异激活OAS-re-caspase-3系统,引起转染GV230/EGFP-OAS-re-caspase-3质粒的细胞凋亡,且随着干扰素浓度的递增,凋亡指数依次递增,呈现剂量依赖关系(P0.05)；而转染EGFP的对照组细胞,在不同浓度干扰素作用下,细胞凋亡指数差别不明显(P0.05);(4)裸鼠成瘤实验结果：成功构建裸鼠皮下肝癌移植瘤模型,成瘤率100%,但HepG2/EGFP-OAS-re-caspase-3组肿瘤体积明显小于HepG2组(P0.05):TUNEL技术检测比较HepG2/EGFP-OAS-re-caspase-3组和HepG2组细胞的凋亡指数,经统计分析得出HepG2/EGFP-OAS-re-caspase-3组的凋亡指数较HepG2组明显增高(P0.05) 结论：1.干扰素a-2b在体内外均能特异性地诱导转染EGFP-OAS-re-caspase-3质粒的肝细胞凋亡；2.在一定范围内随着干扰素浓度的增加,转染EGFP-OAS-re-caspase-3质粒的肝细胞凋亡率依次递增,呈现剂量依赖关系；3.干扰素有可能增强OAS-re-caspase-3系统治疗丙型肝炎的性能。
[Abstract]:Background & objective: according to the World Health Organization (WHO), about 3% of chronic carriers infected with hepatitis C virus (HCV),) and about 170 million of chronic carriers worldwide are at risk of developing (HCC) in liver cirrhosis and hepatocellular carcinoma (HCC). The prevention and treatment of hepatitis C is one of the most important problems in the world at present. Because HCV is easy to mutate, it is more difficult to prevent and cure hepatitis C. Up to now, there is no ideal anti-HCV drug and vaccine to prevent and cure HCV infection, especially HCV in infected cells is difficult to be cleared, so it is the source of HCV recurrence. Previous experiments in vivo and in vitro showed that the core protein of hepatitis C virus (HCV) could specifically bind to and activate the recombinant caspase-3 plasmid system regulated by the promoter of 2'5 'oligoadenylate synthase (OAS). Thus, apoptosis of hepatocytes infected with HCV was induced. Interferon (IFN) is one of the commonly used drugs in the treatment of hepatitis C. Japanese scholar Kerr et al found that cells treated with interferon can find 2'5 'oligoadenylate synthetase in its cell extract. The aim of this study was to determine whether interferon a 伪-2b could specifically activate the recombinant caspase-3 plasmid regulated by OAS promoter, and to provide experimental basis for further application of OAS-re-caspase-3 therapy system. Methods: (1) the recombinant plasmid GV230/EGFP-OAS-re-caspase-3,PCR was constructed and confirmed by agarose gel electrophoresis and sequencing. (2) Establishment of stable transfected cell lines: HL7702/EGFP-OAS-re-caspase-3 and HepG2/EGFP-OAS-re-caspase-3 (3) HL7702/OAS-re-caspase-3 and HL7702/EGFP cells in different concentrations were detected by flow cytometry and MTT. Degree interferon induced apoptosis; Crystal violet staining and TUNEL were used to detect the apoptosis index of HepG2/EGFP-OAS-re-caspase-3 and HepG2 cells treated with different concentrations of interferon. (4) Nude mice tumorigenesis: the stably transfected HepG2/EGFP-OAS-re-caspase-3,HepG2 cells were inoculated subcutaneously into nude mice. After tumor formation, interferon was injected into the tumor and the animals were killed 48 hours later. The apoptosis index of the two groups was measured by TUNEL colorimetric assay. Results: (1) the recombinant plasmid EGFP-OAS-re-caspase-3; was successfully constructed by PCR, gel electrophoresis and sequencing. (2) 24 hours after transfection, HL7702,HepG2 cells were transfected with EGFP-OAS-re-caspase-3,EGFP plasmid, and the transfection efficiency was more than 70%. Resistant clones could be seen in G418 screening for about 2 weeks, and stable transfected cell lines were selected after G418 selection. (3) flow cytometry, MTT, crystal violet staining and TUNEL assay in vitro showed that interferon 伪-2b could specifically activate OAS-re-caspase-3 system and induce apoptosis of transfected GV230/EGFP-OAS-re-caspase-3 plasmid. And with the increase of interferon concentration, the apoptosis index increased in turn, showing a dose-dependent relationship (P0.05). However, in the control group transfected with EGFP, there was no significant difference in apoptosis index under different concentrations of interferon (P0.05). (4) the results of nude mice tumorigenesis experiment: the transplanted tumor model of subcutaneously liver cancer in nude mice was successfully constructed, and the tumor formation rate was 100%. However, the tumor volume of HepG2/EGFP-OAS-re-caspase-3 group was significantly smaller than that of HepG2 group (P0.05): TUNEL), and the apoptosis index of HepG2/EGFP-OAS-re-caspase-3 group and HepG2 group was compared with that of HepG2 group. The results of statistical analysis showed that the apoptosis index of HepG2/EGFP-OAS-re-caspase-3 group was significantly higher than that of HepG2 group (P0.05) conclusion: 1. Interferon-a-伪-2b can specifically induce apoptosis of hepatocytes transfected with EGFP-OAS-re-caspase-3 plasmid in vitro and in vivo. In a certain range, with the increase of interferon concentration, the apoptosis rate of hepatocytes transfected with EGFP-OAS-re-caspase-3 plasmid increased in turn, showing a dose-dependent relationship; 3. Interferon may enhance the performance of the OAS-re-caspase-3 system in the treatment of hepatitis C.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.63
本文编号：2442090
[Abstract]:Background & objective: according to the World Health Organization (WHO), about 3% of chronic carriers infected with hepatitis C virus (HCV),) and about 170 million of chronic carriers worldwide are at risk of developing (HCC) in liver cirrhosis and hepatocellular carcinoma (HCC). The prevention and treatment of hepatitis C is one of the most important problems in the world at present. Because HCV is easy to mutate, it is more difficult to prevent and cure hepatitis C. Up to now, there is no ideal anti-HCV drug and vaccine to prevent and cure HCV infection, especially HCV in infected cells is difficult to be cleared, so it is the source of HCV recurrence. Previous experiments in vivo and in vitro showed that the core protein of hepatitis C virus (HCV) could specifically bind to and activate the recombinant caspase-3 plasmid system regulated by the promoter of 2'5 'oligoadenylate synthase (OAS). Thus, apoptosis of hepatocytes infected with HCV was induced. Interferon (IFN) is one of the commonly used drugs in the treatment of hepatitis C. Japanese scholar Kerr et al found that cells treated with interferon can find 2'5 'oligoadenylate synthetase in its cell extract. The aim of this study was to determine whether interferon a 伪-2b could specifically activate the recombinant caspase-3 plasmid regulated by OAS promoter, and to provide experimental basis for further application of OAS-re-caspase-3 therapy system. Methods: (1) the recombinant plasmid GV230/EGFP-OAS-re-caspase-3,PCR was constructed and confirmed by agarose gel electrophoresis and sequencing. (2) Establishment of stable transfected cell lines: HL7702/EGFP-OAS-re-caspase-3 and HepG2/EGFP-OAS-re-caspase-3 (3) HL7702/OAS-re-caspase-3 and HL7702/EGFP cells in different concentrations were detected by flow cytometry and MTT. Degree interferon induced apoptosis; Crystal violet staining and TUNEL were used to detect the apoptosis index of HepG2/EGFP-OAS-re-caspase-3 and HepG2 cells treated with different concentrations of interferon. (4) Nude mice tumorigenesis: the stably transfected HepG2/EGFP-OAS-re-caspase-3,HepG2 cells were inoculated subcutaneously into nude mice. After tumor formation, interferon was injected into the tumor and the animals were killed 48 hours later. The apoptosis index of the two groups was measured by TUNEL colorimetric assay. Results: (1) the recombinant plasmid EGFP-OAS-re-caspase-3; was successfully constructed by PCR, gel electrophoresis and sequencing. (2) 24 hours after transfection, HL7702,HepG2 cells were transfected with EGFP-OAS-re-caspase-3,EGFP plasmid, and the transfection efficiency was more than 70%. Resistant clones could be seen in G418 screening for about 2 weeks, and stable transfected cell lines were selected after G418 selection. (3) flow cytometry, MTT, crystal violet staining and TUNEL assay in vitro showed that interferon 伪-2b could specifically activate OAS-re-caspase-3 system and induce apoptosis of transfected GV230/EGFP-OAS-re-caspase-3 plasmid. And with the increase of interferon concentration, the apoptosis index increased in turn, showing a dose-dependent relationship (P0.05). However, in the control group transfected with EGFP, there was no significant difference in apoptosis index under different concentrations of interferon (P0.05). (4) the results of nude mice tumorigenesis experiment: the transplanted tumor model of subcutaneously liver cancer in nude mice was successfully constructed, and the tumor formation rate was 100%. However, the tumor volume of HepG2/EGFP-OAS-re-caspase-3 group was significantly smaller than that of HepG2 group (P0.05): TUNEL), and the apoptosis index of HepG2/EGFP-OAS-re-caspase-3 group and HepG2 group was compared with that of HepG2 group. The results of statistical analysis showed that the apoptosis index of HepG2/EGFP-OAS-re-caspase-3 group was significantly higher than that of HepG2 group (P0.05) conclusion: 1. Interferon-a-伪-2b can specifically induce apoptosis of hepatocytes transfected with EGFP-OAS-re-caspase-3 plasmid in vitro and in vivo. In a certain range, with the increase of interferon concentration, the apoptosis rate of hepatocytes transfected with EGFP-OAS-re-caspase-3 plasmid increased in turn, showing a dose-dependent relationship; 3. Interferon may enhance the performance of the OAS-re-caspase-3 system in the treatment of hepatitis C.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.63
【参考文献】
中国期刊全文数据库 前2条
1 Takumi Kawaguchi;Michio Sata;;Importance of hepatitis C virus-associated insulin resistance:Therapeutic strategies for insulin sensitization[J];World Journal of Gastroenterology;2010年16期
2 Chao-Hung Hung;Jing-Houng Wang;Tsung-Hui Hu;Chien-Hung Chen;Kuo-Chin Chang;Yi-Hao Yen;YuanHung Kuo;Ming-Chao Tsai;Chuan-Mo Lee;;Insulin resistance is associated with hepatocellular carcinoma in chronic hepatitis C infection[J];World Journal of Gastroenterology;2010年18期
，本文编号：2442090
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