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中药WCA对人胃癌细胞SGC-7901体内作用基因的筛选与验证

Keywords: 胃癌,健脾中药,基因表达,cDNA微阵列,实时定量聚合酶链反应

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Abstract:

目的:探讨以四君子汤等健脾中药为基础的复方胃肠安(WCA)对胃癌细胞的体内作用分子机制。方法:采用人胃癌细胞SGC-7901裸小鼠皮下移植瘤模型系统评价WCA对胃癌细胞的体内抑瘤作用;链霉素抗生物素-过氧化酶法(SP法)检测增殖细胞核抗原PCNA表达,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL法)检测凋亡指数以及SP法检测caspase-3的活化表达;cDNAarray筛选出WCA组N.S.对照组差异表达2倍以上基因,Real-timeQuantitativePCR验证与增殖、凋亡、分化及信号转导密切相关的19条基因的表达;SP法检测部分有差异表达的基因在细胞内的蛋白表达。结果:与对照组比较,WCA组对皮下移植瘤生长的平均抑制率为(44.32+5.67)%,3次实验WCA组的平均瘤重分别为(0.99±0.76),(1.02±0.16),(0.88±0.47)g,分别低于对照组的(1.93±0.58),(1.65±0.46),(1.63±0.52)g(P<0.05);WCA组移植瘤PCNA阳性表达率(35.73±6.01)%、强阳性表达率(3.39±1.48)%和总阳性率(39.03±7.37)%均分别低于对照组的(53.48±9.34)%,(8.78±5.43)%,(62.26±13.80)%(P<0.05);凋亡指数为(9.72±4.51)%高于对照组的(2.45%±1.37)%(TUNEL法);WCA组活化caspase-3表达阳性率为(5.20±2.26)%,较对照组的(2.82±1.84)%高(P=0.0367);与对照组比较,全基因表达谱芯片筛选出WCA组共45个2倍以上差异表达基因,其中上调表达24个,下调表达21个。35个已克隆基因,10个表达序列标签(expressionsequencetag,EST);2-ΔΔCT法对19条目标基因的表达量进行评估,与对照组比较,表达量显著差异4倍以上的有Stat3(2-ΔΔCT=0.16),RIPX(2-ΔΔCT=0.18),ROD1(2-ΔΔCT=0.23),bcl-2(2-ΔΔCT=0.10),均为下调表达作用;WCA组P-Stat3阳性表达率(35.93±12.67)%、强阳性表达率(3.64%±1.72)%和总阳性率(39.57±13.31)%均分别低于对照组的(56.49±9.34)%,(13.16±7.06)%,(69.65±10.80)%,差异有显著性;bcl-2蛋白阳性表达率(1.62±0.82)%低于对照组的(7.53±3.73)%(P<0.05)。结论:健脾中药复方WCA在体内可抑制胃癌细胞增殖,诱导凋亡;WCA对胃癌细胞SGC-7901在核酸层次上有影响作用的已知基因主要为Stat3,RIPX,ROD1,bcl-2,均为下调表达作用;在蛋白表达水平的作用机制与抑制磷酸化的Stat3,bcl-2蛋白在细胞内的表达,上调活化caspase-3的表达有关。

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