IL-6干预下HBx基因对L02细胞增殖影响的研究

发布时间：2018-08-01 13:26

【摘要】：目的(1)构建出能够稳定表达HBx基因的L02-HBx细胞模型,以期进一步探索HBx蛋白所具备的理化性质和生物学特性及其与原发性肝癌(HCC)发生和转归存在的内在关系。(2)观察外源性IL-6共培养后感染了HBx的L-02细胞生长趋势以及细胞周期的改变情况,以期在探索IL-6能否促进肝脏再生或改善肝硬化方面取得新进展,也期望能为乙肝病毒感染患者提供新的细胞因子治疗靶点和理论依据。方法(1) 设计以嘌呤霉素为研究目的的剂量——反应分析实验,摸索嘌呤霉素对于L-02的最低致死浓度。(2)通过慢病毒载体的方法建立稳定表达HBx的L02细胞株,利用摸索得到的嘌呤霉素的最低致死浓度筛选L02-HBx细胞以获取阳性克隆,扩增培养L02-HBx细胞,并以L02-con和L02细胞作为对照,运用普通PCR实验、荧光定量RT-PCR的方法以及蛋白印记Western blot技术手段检测HBxDNA的表达、HBx mRNA的转录以及HBx蛋白的翻译情况,从而确保被HBx感染的L02细胞模型最终构建成功。(3)运用IL-6共培养L02-HBx细胞,不加IL-6同步培养同一传代数的L02-HBx细胞,以此作为L02-HBx细胞的阴性对照。利用高配置的倒置相差显微镜观察各组当中L02-HBx细胞在形态学方面的显著特点,并且运用CCK8法检测并且观察对比各组中细胞的增殖情况,同时运用流式细胞仪进一步检测各组细胞的细胞周期以及处于各个细胞周期细胞占细胞总数的比例,以便对各组细胞的增殖情况和细胞周期的异同进行客观分析和比较。结果(1)嘌呤霉素剂量-反应分析实验结果表明,浓度为400-1000ng/ml的嘌呤霉素将培养的L02细胞全部杀死,而400ng/ml以下均有细胞存活,故400ng/ml为嘌呤霉素筛选L02细胞的最低致死浓度。(2) 400ng/ml的嘌呤霉素筛选转染HBx的L02细胞5-7天后,可见阳性细胞的克隆,以L02-con和L02细胞作为对照组进行扩大培养以后,经普通PCR、RT-PCR技术手段检测发现均分别有HBx DNA的表达、HBx mRNA的转录,Western blot检测有HBx蛋白的翻译。(3)倒置相差显微镜下观察可见使用工L-6共培养的L02-HBx细胞与对照组细胞相比生长方式独特,以团状、叠层的方式生长,边界不太清楚。CCK8检测结果进一步显示,经过与IL-6共培养后,L02-HBx细胞生长受到抑制。与对照组相比,L02-HBx细胞中处于S期的细胞所占比例显著降低,而处于G2/M期的细胞所占比例显著增高。结论(1)稳定表达HBx基因的L02-HBx细胞模型构建成功,为进一步探索HBx蛋白所具备的理化性质和生物学特性及其与原发性肝癌HCC发生发展和转化的内在关系奠定了深厚的基础。(2)在外源性IL-6共培养的环境下,L02-HBx细胞的生长明显受到抑制,细胞周期出现G2/M阻滞,细胞很可能因此发生突变变异进而最终导致恶变而致癌。并且揭示了最重要的一点,HBx可能在其所处的特定环境下发挥特定的生物学效应。
[Abstract]:Objective (1) to construct a L02-HBx cell model with stable expression of HBx gene. The aim of this study was to explore the physicochemical and biological properties of HBx protein and its relationship with the occurrence and outcome of (HCC) in hepatocellular carcinoma. (2) to observe the growth trend of L-02 cells infected with HBx after co-culture of exogenous IL-6. Cell cycle changes, In order to explore whether IL-6 can promote liver regeneration or improve liver cirrhosis, we hope to provide new target and theoretical basis of cytokine therapy for patients with hepatitis B virus infection. Methods (1) A dose-response assay was designed to investigate the lowest lethal concentration of purine mycin against L-02. (2) L02 cell line expressing HBx stably was established by lentivirus vector method. L02-HBx cells were screened with the lowest lethal concentration of purine mycin to obtain positive clones, L02-HBx cells were amplified and cultured, and L02-con and L02 cells were used as controls. Fluorescence quantitative RT-PCR and protein imprinting Western blot technique were used to detect the transcription of HBxDNA and the translation of HBx protein, so as to ensure that the L02 cell model infected by HBx was successfully constructed. (3) L02-HBx cells were co-cultured with IL-6. L02-HBx cells of the same transmission algebra were cultured without IL-6 synchronously as negative control of L02-HBx cells. The morphological characteristics of L02-HBx cells in each group were observed by inverted phase-contrast microscope with high configuration, and the proliferation of L02-HBx cells in each group was detected and compared by CCK8 method. At the same time, flow cytometry was used to detect the cell cycle and the proportion of the cells in each cell cycle to the total number of cells, so as to analyze and compare the cell proliferation and the similarities and differences of cell cycle in each group. Results (1) the results of dose-response analysis of purine mycin showed that purine mycin at concentration of 400-1000ng/ml killed all of the cultured L02 cells, while cells survived below 400ng/ml. Therefore, 400ng/ml was the lowest lethal concentration of purine mycin to screen L02 cells. (2) after 5-7 days of 400ng/ml purine mycin screening and transfection of HBx L02 cells, the positive cells were cloned, and L02-con and L02 cells were used as control group. The expression of HBx DNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and the translation of HBx protein was detected by Western blot. (3) under inverted phase contrast microscope, the growth pattern of L02-HBx cells co-cultured with engineering L-6 was different from that of control cells. The boundary of CCK8 was not clear. The results of CCK8 further showed that the growth of L02-HBx cells was inhibited after co-culture with IL-6. Compared with the control group, the percentage of S phase cells in L02-HBx cells was significantly decreased, while that in G 2 / M phase was significantly increased. Conclusion (1) the L02-HBx cell model with stable expression of HBx gene was successfully constructed. It lays a solid foundation for further exploring the physicochemical and biological properties of HBx protein and its relationship with the development and transformation of HCC in primary liver cancer. (2) the generation of L02-HBx cells in exogenous IL-6 co-culture environment. The length is obviously suppressed, G 2 / M arrest occurs in cell cycle, which may lead to mutation and mutagenesis, which eventually leads to malignant change and carcinogenesis. It also reveals that the most important point is that HBX may play a specific biological effect in its specific environment.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R512.62

【相似文献】