TLR7信号通路的激活对Hela细胞增殖及细胞因子表达的影响

发布时间：2018-01-21 22:43

本文关键词： Hela细胞细胞因子 Gardiquimod增殖信号通路周期蛋白　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的（1）探讨TLR7在Hela细胞中的表达（2）TLR7信号通路激活后对Hela细胞相关细胞因子、增殖、细胞周期的影响。方法（1）培养Hela细胞，采用荧光定量PCR（Real-time PCR）及Western Blot的方法分析TLR7在Hela细胞中的表达,同时用正常人外周血单个核细胞（peripheral blood mononuclear cell, PBMC）作为阳性对照。（2）Western-Blot分析Gardiquimod对Hela细胞丝裂原蛋白激活激酶-胞外信号调节激酶（MAPKs-ERK1/2）及磷脂酰肌醇激酶-丝氨酸/苏氨酸激酶（PI3K-AKT）蛋白磷酸化水平的影响,以及MAPKs-ERK1/2特异性阻断剂PD98059，PI3K-AKT特异性阻断剂LY294002阻断这两个信号通路，其磷酸化蛋白水平的变化。（3）通过Real-time PCR分析不同时间点Gardiquimod对Hela细胞下游VEGF，TIMP1，MMP2，IL-6，IL-15表达变化的影响，并通过特异性的信号通路阻断剂PD98059，LY294002分析下游的VEGF，TIMP1等表达的变化是否依赖上述MAPKs-ERK1/2及PI3K-AKT这两条信号通路的蛋白磷酸化水平的变化（。4）使用不同浓度的TLR7激动剂Gardiquimod经过不同的时间刺激Hela细胞，采用MTT比色法分析其对Hela细胞增殖的影响。（5）流式细胞技术检测Gardiquimod作用于Hela细胞后其细胞周期的变化。（6）Western-Blot分析Hela细胞中TLR7激活后,细胞周期蛋白B1（CyclinB1）及细胞周期蛋白E（CyclinE）表达的变化。结果（1）Real-time PCR及Western-Blot结果显示，与PBMC相比，TLR7在Hela细胞呈现组成性弱表达。（2）Western-Blot结果表明，Gardiquimod激活TLR7后可以显著增加Hela细胞中ERK1/2和AKT的蛋白磷酸化水平，特异性的阻断剂PD98059，LY294002研究显示，Gardiquimod依赖MAPK-ERK1/2和PI3K-AKT途径发挥其促进Hela细胞增殖的作用。（3）Real-time PCR分析，Gardiquimod经不同时间处理Hela细胞后，其下游VEGF,TIMP1，MMP2，IL-6，，IL-15的表达都有不同程度的增加，并用上述特异性的信号阻断剂后，其下游VEGF等因子的表达呈现不同程度的降低。（4）MTT结果显示，TLR7激动剂Gardiquimod可促进Hela细胞的增殖，且呈时间及剂量效应。（5）流式细胞检测结果表明，TLR7配体促进Hela细胞的增殖。（6）Western-Blot结果显示，Gardiquimod可上调Hela细胞中CyclinB1和CyclinE蛋白的表达。结论Gardiquimod通过MAPKs-ERK1/2和PI3K-AKT信号通路诱导Hela细胞下游VEGF等因子的表达，并促进Hela细胞增殖。促增殖作用可能通过增加CyclinB1,CyclinE蛋白的表达。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2

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