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

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(ultraviolet 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)、HaCaT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cFos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。

Objective：To research the effect of gypenosides-containing serum on the expression of proto-oncogene c-Fos and c-Jun in ultraviolet irradiated ceils. 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 andⅡ,Ⅲ,Ⅳ,Ⅴ,Ⅵ groups of HSF underwent UVB and UVA radiation. Supematant of 4 groups HaCaT was put into HSF Ⅲ- Ⅵ groups in turn. Six groups for measurement were made：blank control group（ Ⅰ group）,UVA model group（ Ⅱ group）, HaCaT supematant A group（ Ⅲ group）,HaCaT supernatant B group（ Ⅳ group）,HaCaT supernatant C group（ Ⅴ group） ,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 group H than in group Ⅰ （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.