乙型肝炎病毒X蛋白对HL7702肝细胞增殖的影响及机制

发布时间：2018-02-15 06:59

本文关键词： 乙肝病毒X蛋白（HBx）乙型肝炎病毒（HBV）肝细胞癌（HCC）细胞增殖环氧合酶-2（COX-2）　出处：《福建医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：【目的】： 1.探讨HBx蛋白的稳定表达对HL7702肝细胞增殖以及细胞中COX-2表达的影响。 2.观察COX-2选择性抑制剂NS-398对HL7702/HBx细胞的影响，探讨COX-2 在HBx蛋白促进肝细胞增殖中的作用。【方法】： HL7702/HBx细胞、HL7702/Mock细胞、HL7702细胞分别复苏和培养，CCK-8法和克隆形成实验观察和检测上述三种细胞增殖能力；以β-actin为内参，运用RT-PCR检测上述三种细胞中COX-2在mRNA水平的表达情况，Western blot检测COX-2在蛋白水平的表达情况；相同浓度COX-2选择性抑制剂NS398作用体外培养的各组细胞，并以DMSO组为空白对照，采用CCK-8法检测经处理24h、48h、72h后各组细胞的增殖活性；在50umol/L NS-398作用72h后，以DMSO作为对照组，Western blot检测各组细胞中COX-2蛋白的表达水平。【结果】：细胞增殖实验及克隆形成实验显示：与HL7702/Mock和HL7702两组细胞相比较，HL7702/HBx增殖能力更强（P0.05），克隆形成率也更高（P0.05）。通过对各组细胞COX-2mRNA的半定量RT-PCR检测发现，，相对于HL7702/Mock细胞、HL7702细胞，转染HBV X基因的HL7702细胞内COX-2的mRNA相对表达量明显增高（P0.05）。Western blot检测示：HL7702/HBx细胞中的COX-2蛋白相对表达水平较高（P0.05），而HL7702/Mock及HL7702之间则无明显差异。NS-398能以时间依赖的方式抑制各组细胞的增殖能力，而在HL7702/HBx细胞中增殖抑制率在3个时间点均高于其他两组细胞（P0.05）。经50μmol/LNS-398处理后，各组细胞的COX-2蛋白水平显著降低，而在HL7702/HBx细胞中更加明显（P0.05）。【结论】： 1. HBx蛋白可促进HL7702细胞分裂增殖、促进细胞生长；HBx蛋白可显著升高肝细胞中COX-2的mRNA及蛋白表达水平；HBx蛋白能通过调控COX-2的表达而促进肝细胞增殖，诱导肝细胞恶性转化。 2. COX-2选择性抑制剂NS-398能通过降低COX-2的表达，抑制高表达的COX-2的HL7702/HBx细胞的恶性增殖，阻止肝细胞在肝癌早期阶段的癌变转化。
[Abstract]:[purpose]:. 1. To investigate the effect of stable expression of HBx protein on the proliferation and COX-2 expression of HL7702 hepatocytes. 2. To observe the effect of NS-398, a selective inhibitor of COX-2, on HL7702/HBx cells and to explore COX-2. The role of HBx protein in promoting hepatocyte proliferation. [methods]:. Resuscitation and culture of HL-7702 / Mock cells from HL7702/HBx cells by CCK-8 method and clone formation assay were used to observe and test the proliferative ability of these three cells, 尾 -actin was used as the internal reference, and 尾 -actin was used as the internal reference, and 尾 -actin was used as the internal reference. RT-PCR was used to detect the expression of COX-2 at the mRNA level in the three kinds of cells, and Western blot was used to detect the expression of COX-2 at the protein level, and the same concentration of NS398, a selective inhibitor of COX-2, was used to treat the cultured cells in vitro, and DMSO group was used as the blank control. The proliferative activity of each group was detected by CCK-8 method after treatment for 24 h or 48 h or 72 h, and the expression level of COX-2 protein was detected with DMSO as control group after treatment with 50 mmol / L NS-398 for 72 h. [outcome]:. Cell proliferation assay and clone formation assay showed that HL7702 / HBx had stronger proliferative ability and higher clone formation rate than those of HL7702/Mock and HL7702 groups. By semi-quantitative RT-PCR detection of COX-2mRNA in each group, it was found that compared with HL7702/Mock cells, HL7702 cells were higher than HL7702 cells. The relative expression of COX-2 in HL7702 cells transfected with HBV X gene was significantly higher than that in HL7702 cells transfected with HBV X gene. Western blot detection showed that the relative expression level of COX-2 protein was higher in the cell line of 10 HL7702 / HBX, but there was no significant difference between HL7702/Mock and HL7702. NS-398 could inhibit the expression of COX-2 protein in a time-dependent manner. The proliferative ability of the group, The inhibitory rate of proliferation in HL7702/HBx cells at three time points was higher than that in the other two groups. After 50 渭 mol/LNS-398 treatment, the COX-2 protein level of the cells in each group was significantly decreased, and that in HL7702/HBx cells was more obvious. [conclusion]:. 1. HBx protein can promote the division and proliferation of HL7702 cells, and promote cell growth. HBX protein can significantly increase the level of mRNA and protein expression of COX-2 in hepatocytes. HBX protein can promote the proliferation of hepatocytes and induce malignant transformation of hepatocytes by regulating the expression of COX-2. 2. NS-398, a selective inhibitor of COX-2, can inhibit the malignant proliferation of HL7702/HBx cells with high expression of COX-2 by reducing the expression of COX-2, thus preventing the transformation of hepatocytes from cancerous transformation in the early stage of HCC.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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