【摘要】：目的探討TLR7的激活對HaCaT細胞增殖與分化的影響及其可能的機制。 方法培養(yǎng)HaCaT細胞，，以不同劑量的TLR7配體Gardiquimod經(jīng)不同的時間體外刺激HaCaT細胞，MTT及流式細胞術分析TLR7的激活對HaCaT細胞增殖的影響。以不同劑量的TLR7配體Gardiquimod經(jīng)不同的時間體外刺激HaCaT細胞，加入氯化鈣誘導HaCaT細胞分化，Western-Blot分析HaCaT細胞的分化Markers（顆粒層：Keratin1，基底層：Keratin5和棘層：Involucrin）并以此分析TLR7的激活對氯化鈣誘導HaCaT細胞分化的影響。 Western-blotting分析TLR7在HaCaT細胞中激活的信號通路PI3K-AKT和RAS-MAPK等。在TLR7配體Gardiquimod處理HaCaT細胞前1h，分別加入特異性阻斷劑(PD98059及LY2940002)阻斷TLR7配體Gardiquimod激活的相關信號通路，然后分析阻斷劑對TLR7配體Gardiquimod調(diào)控HaCaT細胞增殖及分化影響，從而探討PI3K-AKT和RAS-MAPK信號通路在TLR7配體Gardiquimod對HaCaT細胞增殖及分化調(diào)控中的作用。 結果MTT及流式細胞分析結果顯示：TLR7配體Gardiquimod促進HaCaT細胞增殖，且具有時間及劑量依賴性；TLR7配體Gardiquimod能夠抑制氯化鈣誘導的HaCaT細胞分化markers（Keratin1及Involucrin）的表達，存在時間效應及劑量效應；信號通路分析揭示TLR7配體Gardiquimod能夠增加ERK1/2和MAPK的水平；阻斷劑的研究發(fā)現(xiàn)TLR7配體Gardiquimod部分依賴PI3K-AKT和RAS-MAPK途徑發(fā)揮其促進增殖及抑制分化作用。 結論TLR7配體Gardiquimod部分依賴PI3K-AKT和RAS-MAPK途徑發(fā)揮其促進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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R341

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