TOLL样受体7（TLR7）激活对HaCaT细胞增殖和分化的影响
发布时间：2019-05-11 18:13
【摘要】：目的探讨TLR7的激活对HaCaT细胞增殖与分化的影响及其可能的机制。 方法培养HaCaT细胞，，以不同剂量的TLR7配体Gardiquimod经不同的时间体外刺激HaCaT细胞，MTT及流式细胞术分析TLR7的激活对HaCaT细胞增殖的影响。以不同剂量的TLR7配体Gardiquimod经不同的时间体外刺激HaCaT细胞，加入氯化钙诱导HaCaT细胞分化，Western-Blot分析HaCaT细胞的分化Markers（颗粒层：Keratin1，基底层：Keratin5和棘层：Involucrin）并以此分析TLR7的激活对氯化钙诱导HaCaT细胞分化的影响。 Western-blotting分析TLR7在HaCaT细胞中激活的信号通路PI3K-AKT和RAS-MAPK等。在TLR7配体Gardiquimod处理HaCaT细胞前1h，分别加入特异性阻断剂(PD98059及LY2940002)阻断TLR7配体Gardiquimod激活的相关信号通路，然后分析阻断剂对TLR7配体Gardiquimod调控HaCaT细胞增殖及分化影响，从而探讨PI3K-AKT和RAS-MAPK信号通路在TLR7配体Gardiquimod对HaCaT细胞增殖及分化调控中的作用。 结果MTT及流式细胞分析结果显示：TLR7配体Gardiquimod促进HaCaT细胞增殖，且具有时间及剂量依赖性；TLR7配体Gardiquimod能够抑制氯化钙诱导的HaCaT细胞分化markers（Keratin1及Involucrin）的表达，存在时间效应及剂量效应；信号通路分析揭示TLR7配体Gardiquimod能够增加ERK1/2和MAPK的水平；阻断剂的研究发现TLR7配体Gardiquimod部分依赖PI3K-AKT和RAS-MAPK途径发挥其促进增殖及抑制分化作用。 结论TLR7配体Gardiquimod部分依赖PI3K-AKT和RAS-MAPK途径发挥其促进增殖及抑制分化作用。
[Abstract]:Objective to investigate the effect of TLR7 activation on proliferation and differentiation of HaCaT cells and its possible mechanism. Methods HaCaT cells were cultured and HaCaT cells were stimulated with different doses of TLR7 ligand Gardiquimod at different time. The effect of TLR7 activation on the proliferation of HaCaT cells was analyzed by MTT and flow cytometry. HaCaT cells were stimulated with different doses of TLR7 ligand Gardiquimod at different time. HaCaT cells were induced to differentiate by adding calcium chloride. The differentiation Markers (granule layer: Keratin1,) of HaCaT cells was analyzed by Western-Blot. Basal layer: Keratin5 and spinous layer: Involucrin) were used to analyze the effect of TLR7 activation on the differentiation of HaCaT cells induced by calcium chloride. The signal pathways PI3K-AKT and RAS-MAPK activated by TLR7 in HaCaT cells were analyzed by Western-blotting. One hour before HaCaT cells were treated with TLR7 ligand Gardiquimod, specific blockers (PD98059 and LY2940002) were added to block the related signaling pathway of TLR7 ligand Gardiquimod activation, and then the effects of TLR7 ligand Gardiquimod on the proliferation and differentiation of HaCaT cells were analyzed. To investigate the role of PI3K-AKT and RAS-MAPK signaling pathways in the regulation of TLR7 ligand Gardiquimod on the proliferation and differentiation of HaCaT cells. Results the results of MTT and flow cytometry showed that TLR7 ligand Gardiquimod promoted the proliferation of HaCaT cells in a time-and dose-dependent manner. TLR7 ligand Gardiquimod could inhibit the expression of markers (Keratin1 and Involucrin) induced by calcium chloride in HaCaT cells with time-effect and dose-effect. Signal pathway analysis showed that TLR7 ligand Gardiquimod could increase the levels of ERK1/2 and MAPK. It was found that TLR7 ligand Gardiquimod partly relied on PI3K-AKT and RAS-MAPK pathways to promote proliferation and inhibit differentiation. Conclusion TLR7 ligand Gardiquimod partly depends on PI3K-AKT and RAS-MAPK pathways to promote proliferation and inhibit differentiation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341
本文编号：2474730
[Abstract]:Objective to investigate the effect of TLR7 activation on proliferation and differentiation of HaCaT cells and its possible mechanism. Methods HaCaT cells were cultured and HaCaT cells were stimulated with different doses of TLR7 ligand Gardiquimod at different time. The effect of TLR7 activation on the proliferation of HaCaT cells was analyzed by MTT and flow cytometry. HaCaT cells were stimulated with different doses of TLR7 ligand Gardiquimod at different time. HaCaT cells were induced to differentiate by adding calcium chloride. The differentiation Markers (granule layer: Keratin1,) of HaCaT cells was analyzed by Western-Blot. Basal layer: Keratin5 and spinous layer: Involucrin) were used to analyze the effect of TLR7 activation on the differentiation of HaCaT cells induced by calcium chloride. The signal pathways PI3K-AKT and RAS-MAPK activated by TLR7 in HaCaT cells were analyzed by Western-blotting. One hour before HaCaT cells were treated with TLR7 ligand Gardiquimod, specific blockers (PD98059 and LY2940002) were added to block the related signaling pathway of TLR7 ligand Gardiquimod activation, and then the effects of TLR7 ligand Gardiquimod on the proliferation and differentiation of HaCaT cells were analyzed. To investigate the role of PI3K-AKT and RAS-MAPK signaling pathways in the regulation of TLR7 ligand Gardiquimod on the proliferation and differentiation of HaCaT cells. Results the results of MTT and flow cytometry showed that TLR7 ligand Gardiquimod promoted the proliferation of HaCaT cells in a time-and dose-dependent manner. TLR7 ligand Gardiquimod could inhibit the expression of markers (Keratin1 and Involucrin) induced by calcium chloride in HaCaT cells with time-effect and dose-effect. Signal pathway analysis showed that TLR7 ligand Gardiquimod could increase the levels of ERK1/2 and MAPK. It was found that TLR7 ligand Gardiquimod partly relied on PI3K-AKT and RAS-MAPK pathways to promote proliferation and inhibit differentiation. Conclusion TLR7 ligand Gardiquimod partly depends on PI3K-AKT and RAS-MAPK pathways to promote proliferation and inhibit differentiation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341
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