本文關(guān)鍵詞： Hela細胞 細胞因子 Gardiquimod增殖 信號通路 周期蛋白　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的（1）探討TLR7在Hela細胞中的表達（2）TLR7信號通路激活后對Hela細胞相關(guān)細胞因子、增殖、細胞周期的影響。 方法（1）培養(yǎng)Hela細胞，采用熒光定量PCR（Real-time PCR）及Western Blot的方法分析TLR7在Hela細胞中的表達,同時用正常人外周血單個核細胞（peripheral blood mononuclear cell, PBMC）作為陽性對照。（2）Western-Blot分析Gardiquimod對Hela細胞絲裂原蛋白激活激酶-胞外信號調(diào)節(jié)激酶（MAPKs-ERK1/2）及磷脂酰肌醇激酶-絲氨酸/蘇氨酸激酶（PI3K-AKT）蛋白磷酸化水平的影響,以及MAPKs-ERK1/2特異性阻斷劑PD98059，PI3K-AKT特異性阻斷劑LY294002阻斷這兩個信號通路，其磷酸化蛋白水平的變化。（3）通過Real-time PCR分析不同時間點Gardiquimod對Hela細胞下游VEGF，TIMP1，MMP2，IL-6，IL-15表達變化的影響，并通過特異性的信號通路阻斷劑PD98059，LY294002分析下游的VEGF，TIMP1等表達的變化是否依賴上述MAPKs-ERK1/2及PI3K-AKT這兩條信號通路的蛋白磷酸化水平的變化（。4）使用不同濃度的TLR7激動劑Gardiquimod經(jīng)過不同的時間刺激Hela細胞，采用MTT比色法分析其對Hela細胞增殖的影響。（5）流式細胞技術(shù)檢測Gardiquimod作用于Hela細胞后其細胞周期的變化。（6）Western-Blot分析Hela細胞中TLR7激活后,細胞周期蛋白B1（CyclinB1）及細胞周期蛋白E（CyclinE）表達的變化。 結(jié)果（1）Real-time PCR及Western-Blot結(jié)果顯示，與PBMC相比，TLR7在Hela細胞呈現(xiàn)組成性弱表達。（2）Western-Blot結(jié)果表明，Gardiquimod激活TLR7后可以顯著增加Hela細胞中ERK1/2和AKT的蛋白磷酸化水平，特異性的阻斷劑PD98059，LY294002研究顯示，Gardiquimod依賴MAPK-ERK1/2和PI3K-AKT途徑發(fā)揮其促進Hela細胞增殖的作用。（3）Real-time PCR分析，Gardiquimod經(jīng)不同時間處理Hela細胞后，其下游VEGF,TIMP1，MMP2，IL-6，，IL-15的表達都有不同程度的增加，并用上述特異性的信號阻斷劑后，其下游VEGF等因子的表達呈現(xiàn)不同程度的降低。（4）MTT結(jié)果顯示，TLR7激動劑Gardiquimod可促進Hela細胞的增殖，且呈時間及劑量效應(yīng)。（5）流式細胞檢測結(jié)果表明，TLR7配體促進Hela細胞的增殖。（6）Western-Blot結(jié)果顯示，Gardiquimod可上調(diào)Hela細胞中CyclinB1和CyclinE蛋白的表達。 結(jié)論Gardiquimod通過MAPKs-ERK1/2和PI3K-AKT信號通路誘導(dǎo)Hela細胞下游VEGF等因子的表達，并促進Hela細胞增殖。促增殖作用可能通過增加CyclinB1,CyclinE蛋白的表達。
[Abstract]:Objective 1) to investigate the effect of TLR7 expression in Hela cells after activation of TLR7 signaling pathway on cytokines, proliferation and cell cycle of Hela cells. Methods 1) Hela cells were cultured. Fluorescence quantitative PCR(Real-time PCR and Western Blot were used to analyze the expression of TLR7 in Hela cells. Peripheral blood mononuclear cell was also used in normal human peripheral blood mononuclear cells. Western blot analysis of mitogen-activated kinase-extracellular signal-regulated kinase (ECK) in Hela cells by Gardiquimod as a positive control. Effects of MAPKs-ERK1 / 2) and phosphatidylinositol kinase-serine / threonine kinase (PI3K-AKT) on phosphorylation levels. The two signaling pathways were blocked by MAPKs-ERK1/2 specific blocker PD98059 and PI3K-AKT specific blocker LY294002. The changes of phosphorylated protein level in Hela cells were analyzed by Real-time PCR. The effect of MMP2 on the expression of IL-15 was observed and the downstream VEGF was analyzed by PD98059 and LY294002, a specific signal pathway blocker. Whether the changes in expression of TIMP1 et al depend on the changes in protein phosphorylation level of the two signaling pathways, MAPKs-ERK1/2 and PI3K-AKT, mentioned above? Different concentrations of TLR7 agonist Gardiquimod were used to stimulate Hela cells for different time. The effect of Gardiquimod on the proliferation of Hela cells was analyzed by MTT colorimetry. Flow cytometry (FCM) was used to detect the cell cycle changes of Hela cells treated with Gardiquimod. Western-Blot was used to detect the activation of TLR7 in Hela cells. The expression of cyclin B _ (1) and cyclin E (E). Results Real-time PCR and Western-Blot showed that compared with PBMC. The results of Western-Blot showed that TLR7 was constitutively weakly expressed in Hela cells. Gardiquimod activation of TLR7 could significantly increase the level of protein phosphorylation of ERK1/2 and AKT in Hela cells and the specific blocker PD98059. LY294002 study showed. Gardiquimod depends on the MAPK-ERK1/2 and PI3K-AKT pathway to play its role in promoting the proliferation of Hela cells. Real-time PCR analysis. After Gardiquimod was treated with Hela cells at different time, the expression of IL-6 IL-15 was increased in the downstream of Hela cells. After the above specific signal blockers, the expression of VEGF and other factors in the downstream of the cells decreased in varying degrees. TLR7 agonist Gardiquimod could promote the proliferation of Hela cells in a time-and dose-dependent manner. The results of Western-Blot showed that TLR7 ligand promoted the proliferation of Hela cells. Gardiquimod can up-regulate the expression of CyclinB1 and CyclinE in Hela cells. Conclusion Gardiquimod induces the expression of VEGF and other factors downstream of Hela cells through MAPKs-ERK1/2 and PI3K-AKT signaling pathway. The proliferation of Hela cells may be enhanced by increasing the expression of cyclin E protein.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R329.2

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