绞股蓝总皂苷对紫外线照射的人皮肤细胞原癌基因表达的影响

Effect of gypenosides on expression of proto-oncogene in skin cells following ultraviolet irradiation

摘要:

目的：探讨绞股蓝总皂苷对紫外线照射的人皮肤相关原癌基因c-Fos和c-Jun表达的影响。方法：制备空白大鼠血清和绞股蓝含药血清。将人永生化角质形成细胞（human immortal skin keratinocytes，HaCaT）40瓶随机分为 4 组（A～D组），每组 10 瓶，将人皮肤成纤维细胞（human skin fibroblast，HSF）60 瓶随机分为 6 组（Ⅰ～Ⅵ组），每组10瓶。分别用中波紫外线（ultra－violet B，UVB）对 HaCaT 细胞 B～D 组、用长波紫外线（ultraviolet A，UVA）对 HSF 细胞Ⅱ～Ⅵ 组进行照射。将 HaCaT 4组细胞培养上清液分别加入HSF细胞的 Ⅲ～Ⅵ组。最终制成用于检测的6组细胞，分别为空白对照组（Ⅰ组）、UVA模型组（Ⅱ组）、Ha－CaT 培养上清液A组（Ⅲ组）、HaCaT 培养上清液B组（Ⅳ组）、 HaCaT 培养上清液C组（Ⅴ组）、HaCaT 培养上清液D组（Ⅵ组）。采用反转录聚合酶链反应（reverse transcription-PRC，RT-PCR）和蛋白免疫印迹（Western blot）方法检测Ⅰ～Ⅵ 组细胞c-Fos和c-Jun基因和蛋白的表达水平。结果：与Ⅰ组比较，Ⅱ组细胞c-Fos和c-Jun基因和蛋白的表达水平明显上调（各组P值均为0.000）；与Ⅱ组比较，Ⅳ组c-Fos和c-Jun 基因和蛋白表达均上调（Pc-Fos基因＝0.016、Pc-Fos蛋白＝0.001、Pc-Jun基因＝0.005、Pc-Jun蛋白＝0.000），而 Ⅲ 组变化不明显；与Ⅳ组比较，Ⅵ组c-Fos和c-Jun基因和蛋白表达均下调（各组P值均为0.000），而Ⅴ组变化不明显（Pc-Fos基因＝0.081、Pc-Fos蛋白＝0.088、Pc-Jun基因＝0.109、Pc-Jun蛋白＝0.026）。结论：经UVB照射的HaCaT细胞分泌细胞因子促进经UVA照射的HSF细胞原癌基因c-Fos和c-Jun 基因和蛋白的表达。绞股蓝总皂苷对UVB照射HaCaT细胞培养上清液作用的UVA照射HSF细胞中原癌基因c-Fos和c-Jun的表达具有抑制作用。

Abstract:

Objective：To research the effect of gypenosides－containing serum on the expression of proto-oncogene c-Fos and c-Jun in ultraviolet irradiated cells. Methods：Blank serum and gypenosides-containing serum were made. Human immortal skin keratinocytes （HaCaT） were divided into 4 groups（A-D），10 bottles in each group. Human skin fibroblast（HSF） were divided into 6 groups（Ⅰ-Ⅵ），10 bottles in each group. B，C，D groups of irradiated HaCaT and Ⅱ，Ⅲ，Ⅳ，Ⅴ，Ⅵ groups of HSF underwent UVB and UVA radiation. Supernatant of 4 groups HaCaT was put into HSF Ⅲ-Ⅵ groups in turn. Six groups for measurement were made：blank control group（Ⅰgroup），UVA model group（Ⅱgroup），HaCaT supernatant A group（Ⅲ group），HaCaT supernatant B group（Ⅳ group），HaCaT supernatant C group（Ⅴgroup），HaCaT supernatant D group（Ⅵ group）. Expression levels of c-Fos and c-Jun mRNA and protein in each group were measured by RT-PCR and Western blot. Results：c-Fos and c-Jun mRNA and protein expression was significantly higher in groupⅡ than in groupⅠ（all P =0.000）. c-Fos and c-Jun mRNA and protein expression was significantly higher in group Ⅳ than in group Ⅱ（Pc-Fos mRNA＝0.016，Pc-Fos protein＝0.001，Pc-Jun mRNA＝0.005，Pc-Jun protein＝0.000）. Changes in group Ⅲ were not significant. c-Fos and c-Jun mRNA and protein expression was significantly lower in group Ⅵ than in group Ⅳ（all P =0.000）. Changes in group Ⅴ were not significant（Pc-Fos mRNA＝0.081，Pc-Fos protein＝0.088，Pc-Jun mRNA＝0.109，Pc-Jun protein＝0.026）. Conclusions：The UVB irradiated HaCaT cell secrete cytokine to increase c-Fos and c-Jun mRNA and protein expression in UVA irradiated cell HSF. Gypenosides can inhibit c-Fos and c-Jun mRNA and protein expression of UVA irradiated HSF cells which are affected by UVB irradiated HaCaT supernatant.