干擾素在2’-5’OAS啟動子調控的重組caspase-3質粒誘導肝細胞凋亡中的作用
發(fā)布時間：2019-03-17 08:02
【摘要】：背景與目的：世界衛(wèi)生組織統(tǒng)計,全世界約有3%的人感染丙型肝炎病毒(HCV),約1.7億的慢性攜帶者有發(fā)展成肝硬化和肝細胞癌(HCC)的危險性。丙型肝炎的防治是目前全世界面臨的一項重大問題,由于HCV極易發(fā)生變異,使得其防治更為困難。至今尚無理想的抗HCV藥物和疫苗防治HCV感染,特別是感染細胞內的HCV難以清除,成為丙型肝炎復發(fā)的病毒來源。本課題組前期體內外實驗表明：丙型肝炎病毒的核心蛋白可以特異性的結合并激活2’-5’寡腺苷酸合成酶(OAS)啟動子調控的重組caspase-3質粒系統(tǒng),從而誘導HCV感染的肝細胞凋亡。干擾素是目前治療丙型肝炎的常用藥物之一,日本學者Kerr等發(fā)現(xiàn)用干擾素作用細胞,可在其細胞提取液中找到2’-5’寡腺苷酸合成酶。本研究旨在明確干擾素a-2b能否特異性的激活OAS啟動子調控的重組caspase-3質粒,為OAS-re-caspase-3治療系統(tǒng)進 一步應用提供實驗依據(jù)。 方法：(1)構建重組質粒GV230/EGFP-OAS-re-caspase-3,PCR擴增、瓊脂糖凝膠電泳及擴增產(chǎn)物測序驗證；(2)建立穩(wěn)定轉染細胞系:HL7702/EGFP-OAS-re-caspase-3和HepG2/EGFP-OAS-re-caspase-3(3)用流式細胞儀和MTT檢測比較HL7702/OAS-re-caspase-3和HL7702/EGFP細胞在不同濃度干擾素作用下的凋亡情況；結晶紫染色及TUNEL檢測比較HepG2/EGFP-OAS-re-caspase-3和HepG2兩組細胞在不同濃度干擾素作用下的凋亡指數(shù)；(4)裸鼠成瘤：將穩(wěn)定轉染的HepG2/EGFP-OAS-re-caspase-3、HepG2細胞接種裸鼠皮下,待腫瘤形成后,瘤內注射治療劑量干擾素,48小時后處死動物,TUNEL顯色法檢測比較兩組細胞的凋亡指數(shù)。 結果：(1)PCR及凝膠電泳和測序結果表明成功構建了重組質粒EGFP-OAS-re-caspase-3；(2)轉染24h后,在熒光顯微鏡下觀察計數(shù),EGFP-OAS-re-caspase-3、EGFP質粒轉染HL7702、HepG2細胞,轉染效率達70%以上；G418篩選2周左右可見抗性克隆,維持篩選出穩(wěn)定轉染細胞株；(3)流式細胞儀、MTT、結晶紫染色及TUNEL體外實驗表明：干擾素α-2b可特異激活OAS-re-caspase-3系統(tǒng),引起轉染GV230/EGFP-OAS-re-caspase-3質粒的細胞凋亡,且隨著干擾素濃度的遞增,凋亡指數(shù)依次遞增,呈現(xiàn)劑量依賴關系(P0.05)；而轉染EGFP的對照組細胞,在不同濃度干擾素作用下,細胞凋亡指數(shù)差別不明顯(P0.05);(4)裸鼠成瘤實驗結果：成功構建裸鼠皮下肝癌移植瘤模型,成瘤率100%,但HepG2/EGFP-OAS-re-caspase-3組腫瘤體積明顯小于HepG2組(P0.05):TUNEL技術檢測比較HepG2/EGFP-OAS-re-caspase-3組和HepG2組細胞的凋亡指數(shù),經(jīng)統(tǒng)計分析得出HepG2/EGFP-OAS-re-caspase-3組的凋亡指數(shù)較HepG2組明顯增高(P0.05) 結論：1.干擾素a-2b在體內外均能特異性地誘導轉染EGFP-OAS-re-caspase-3質粒的肝細胞凋亡；2.在一定范圍內隨著干擾素濃度的增加,轉染EGFP-OAS-re-caspase-3質粒的肝細胞凋亡率依次遞增,呈現(xiàn)劑量依賴關系；3.干擾素有可能增強OAS-re-caspase-3系統(tǒng)治療丙型肝炎的性能。
[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 偽-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 偽-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-偽-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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.63
本文編號：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 偽-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 偽-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-偽-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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.63
【參考文獻】
中國期刊全文數(shù)據(jù)庫 前2條
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
，本文編號：2442090
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