本文選題：乙肝病毒X蛋白　切入點(diǎn)：乙肝病毒X基因　出處：《福建醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【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體之一，其中X基因編碼的X蛋白具有多種生物學(xué)功能，，可與宿主細(xì)胞的多種蛋白相互作用，調(diào)控宿主細(xì)胞基因表達(dá)，進(jìn)而影響宿主細(xì)胞的信號轉(zhuǎn)導(dǎo)、增殖與分化等。HBV感染可激活多種信號通路并影響炎癥相關(guān)基因的表達(dá)，如IL-6、環(huán)氧化酶-2(cyclooxygenase-2，COX-2)和PGE2等。本研究擬探討炎癥相關(guān)因子在穩(wěn)定轉(zhuǎn)染HBV X基因的肝細(xì)胞中的表達(dá)情況，觀察IL-10對肝細(xì)胞炎癥相關(guān)因子表達(dá)的影響，為進(jìn)一步闡明乙肝病毒X蛋白在HBV致病過程中的作用機(jī)制提供一些實(shí)驗(yàn)依據(jù)。 【方法】應(yīng)用免疫細(xì)胞化學(xué)S-P法檢測HL-7702、HL-7702空載、HL-7702X三種細(xì)胞中COX-2蛋白的表達(dá)情況；以不同濃度的白介素10（IL-10）(0、40、60、80ng/ml)處理體外培養(yǎng)的肝細(xì)胞，了解在不同時(shí)間內(nèi)(24、48h)各組細(xì)胞中COX-2蛋白的表達(dá)情況。采用半定量RT-PCR方法檢測各組細(xì)胞中COX-2mRNA的表達(dá)。體外培養(yǎng)肝細(xì)胞，以不同濃度IL-10(0、40、60、80ng/ml)處理后收集細(xì)胞上清液，采用ELISA法檢測上清液中IL-6、TNF-α、PGE2的含量。 【結(jié)果】穩(wěn)定轉(zhuǎn)染HBx基因的HL-7702細(xì)胞中COX-2蛋白和mRNA的表達(dá)較對照組及空載組明顯升高（P0.05;P0.05），不同時(shí)間段變化無明顯差異（P0.05）。在加入40、60、80ng/ml的IL-10處理24h及48h后，COX-2蛋白及mRNA的表達(dá)均明顯下調(diào)，且各組間比較均有統(tǒng)計(jì)學(xué)意義（P0.05）。細(xì)胞上清中均有IL-6、TNF-α、PGE2的表達(dá)，但穩(wěn)定轉(zhuǎn)染HBx基因的HL-7702細(xì)胞中因子的表達(dá)無明顯差異（P0.05），加入IL-10干預(yù)后各組炎癥因子的表達(dá)無明顯改變。 【結(jié)論】穩(wěn)定轉(zhuǎn)染HBx基因的肝細(xì)胞COX-2的表達(dá)均明顯上調(diào)。外源性的IL-10可以抑制肝細(xì)胞中COX-2蛋白和mRNA的表達(dá)，從而減少肝細(xì)胞炎癥反應(yīng)，但細(xì)胞上清中IL-6、TNF-α、PGE2的表達(dá)未見明顯異常，IL-10的影響不明顯。
[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- 偽 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- 偽 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- 偽 and PGE2 in the supernatant was not significantly abnormal, and the effect of IL-10 was not obvious.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.62

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