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IL-10对稳定转染HBV X基因的HL-7702细胞炎症相关因子的影响

发布时间:2018-03-14 06:35

  本文选题:乙肝病毒X蛋白 切入点:乙肝病毒X基因 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】乙型肝炎病毒(hepatitis B virus,HBV)感染是引起病毒性肝炎的主要病原体之一,其中X基因编码的X蛋白具有多种生物学功能,,可与宿主细胞的多种蛋白相互作用,调控宿主细胞基因表达,进而影响宿主细胞的信号转导、增殖与分化等。HBV感染可激活多种信号通路并影响炎症相关基因的表达,如IL-6、环氧化酶-2(cyclooxygenase-2,COX-2)和PGE2等。本研究拟探讨炎症相关因子在稳定转染HBV X基因的肝细胞中的表达情况,观察IL-10对肝细胞炎症相关因子表达的影响,为进一步阐明乙肝病毒X蛋白在HBV致病过程中的作用机制提供一些实验依据。 【方法】应用免疫细胞化学S-P法检测HL-7702、HL-7702空载、HL-7702X三种细胞中COX-2蛋白的表达情况;以不同浓度的白介素10(IL-10)(0、40、60、80ng/ml)处理体外培养的肝细胞,了解在不同时间内(24、48h)各组细胞中COX-2蛋白的表达情况。采用半定量RT-PCR方法检测各组细胞中COX-2mRNA的表达。体外培养肝细胞,以不同浓度IL-10(0、40、60、80ng/ml)处理后收集细胞上清液,采用ELISA法检测上清液中IL-6、TNF-α、PGE2的含量。 【结果】稳定转染HBx基因的HL-7702细胞中COX-2蛋白和mRNA的表达较对照组及空载组明显升高(P0.05;P0.05),不同时间段变化无明显差异(P0.05)。在加入40、60、80ng/ml的IL-10处理24h及48h后,COX-2蛋白及mRNA的表达均明显下调,且各组间比较均有统计学意义(P0.05)。细胞上清中均有IL-6、TNF-α、PGE2的表达,但稳定转染HBx基因的HL-7702细胞中因子的表达无明显差异(P0.05),加入IL-10干预后各组炎症因子的表达无明显改变。 【结论】稳定转染HBx基因的肝细胞COX-2的表达均明显上调。外源性的IL-10可以抑制肝细胞中COX-2蛋白和mRNA的表达,从而减少肝细胞炎症反应,但细胞上清中IL-6、TNF-α、PGE2的表达未见明显异常,IL-10的影响不明显。
[Abstract]:[objective] Hepatitis B virus (HBV) infection is one of the main pathogens of viral hepatitis. X protein encoded by X gene has many biological functions and can interact with many proteins in host cells. HBV infection can activate many signaling pathways and affect the expression of inflammatory genes, which can affect the signal transduction, proliferation and differentiation of host cells. For example, IL-6, cyclooxygenase-2cyclooxygenase-2 (COX-2) and PGE2. This study was to investigate the expression of inflammatory related factors in hepatocytes transfected with HBV X gene stably, and to observe the effect of IL-10 on the expression of inflammatory related factors in hepatocytes. To further clarify the role of hepatitis B virus X protein in the pathogenesis of HBV provides some experimental evidence. [methods] Immunocytochemistry S-P method was used to detect the expression of COX-2 protein in HL-7702HL-7702 empty loaded HL-7702X cells. To investigate the expression of COX-2 protein in the cells of each group at different time. The expression of COX-2mRNA was detected by semi-quantitative RT-PCR method. Hepatocytes were cultured in vitro, and the supernatants were collected after treatment with different concentrations of IL-10, 40,60,60,80ng / ml. The content of IL-6 TNF- 伪 PGE2 in supernatant was determined by ELISA. [results] the expression of COX-2 protein and mRNA in HL-7702 cells with stable transfection of HBx gene was significantly higher than that in control group and no-load group, and there was no significant difference in the changes of P0.05 protein and mRNA in different time periods. The expression of COX-2 protein and mRNA in HL-7702 cells treated with 4060 mg / ml IL-10 for 24 h and 48 h were significantly down-regulated. The expression of IL-6 TNF- 伪 pGE2 was found in the supernatant of the cells, but there was no significant difference in the expression of cytokines in the HL-7702 cells transfected with HBx gene stably. The expression of inflammatory factors did not change after the intervention of IL-10. [conclusion] the expression of COX-2 in hepatocytes transfected with HBx gene was significantly up-regulated, and exogenous IL-10 could inhibit the expression of COX-2 protein and mRNA in hepatocytes, thus reducing the inflammatory response of hepatocytes. However, the expression of IL-6, TNF- 伪 and PGE2 in the supernatant was not significantly abnormal, and the effect of IL-10 was not obvious.
【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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