SP600125抑制UVA激活皮肤成纤维细胞JNK通路的最佳浓度

发布时间：2018-04-20 10:34

  本文选题：SP + c-Jun氨基末端激酶通路　； 参考：《中山大学学报(医学科学版)》2014年02期

【摘要】：【目的】研究抑制UVA激活的皮肤成纤维细胞JNK通路的最佳SP600125浓度,为研究JNK信号通路在光老化中的作用机制奠定基础。【方法】原代培养取自儿童包皮的皮肤成纤维细胞。用CCK-8法结合荧光倒置显微镜研究不同剂量UVA和不同浓度SP600125及两者联合对皮肤成纤维细胞活性和形态的影响,以选择非细胞毒性的UVA剂量和SP600125浓度。再用Western-blot检测UVA辐照后不同时间点皮肤成纤维细胞中磷酸化c-Jun(P-c-Jun)表达以确定其被诱导表达的时间点。最后用Western-blot检测不同浓度SP600125对UVA上调的P-c-Jun表达的抑制。【结果】UVA和SP600125分别辐照细胞和孵育细胞时,UVA≤10 J/cm2和SP600125≤2μmol/L均能保持细胞活性在90%以上。10 J/cm2的UVA辐照不同浓度SP600125孵育的细胞时,≤800 nmol/L的SP600125孵育的细胞活性在90%以上;1μmol/L的SP600125使细胞活性明显下降至67.1%,镜下见部分细胞皱缩、碎裂、死亡;2μmol/L的SP600125使细胞活性进一步下降至7.6%,镜下见绝大部分细胞变形、碎裂、死亡。Western-blot结果显示P-c-Jun在UVA辐照后0.75 h、1.5 h均较0 h升高,3 h后恢复至0 h水平。200~800 nmol/L SP600125呈剂量依赖性地抑制UVA上调的P-c-Jun表达,差异有统计学意义。【结论】低于常用浓度的200~800 nmol/L SP600125即可抑制UVA激活的皮肤成纤维细胞的JNK通路。JNK通路可能在UVA辐照的皮肤成纤维细胞中起抗凋亡作用。
[Abstract]:[objective] to study the optimal SP600125 concentration of JNK pathway of skin fibroblasts activated by UVA, and to lay a foundation for studying the mechanism of JNK signaling pathway in photoaging. [methods] Primary cultured skin fibroblasts from prepuce of children were cultured. The effects of different doses of UVA, different concentrations of SP600125 and their combination on the activity and morphology of skin fibroblasts were studied by CCK-8 method and fluorescence inverted microscope, in order to select the non-cytotoxic UVA dose and SP600125 concentration. Then Western-blot was used to detect the phosphorylated c-Jun-P-c-Jun-Jun expression in skin fibroblasts at different time points after UVA irradiation to determine the time point at which it was induced to express. Finally, Western-blot was used to detect the inhibitory effect of different concentrations of SP600125 on the up-regulated P-c-Jun expression of UVA. [results] UVA and SP600125 irradiated cells and incubated cells with UVA 鈮，

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