中药胃康宁对胃癌细胞生长与胃癌细胞周期调控因子的影响

Effect of Weikangning on Growth and Cell Cycle Regulators of Gastric Cancer Cell

目的观察中药胃康宁含药血清对胃癌细胞生长与胃癌细胞周期调控因子CyclinD2、CyclinE、Cdk2(Cyclin dependant kinase2,CDK2)Cdk4、Cdk6和p16INK4a、p27KIP1mRNA及其蛋白表达的作用。方法分别将高、中、低不同剂量的中药胃康宁制剂对SD大鼠进行灌胃,制备含药血清,然后分别用不同剂量含药血清培养胃癌细胞,用流式细胞仪检测细胞周期的变化;采用免疫组织化学SABC法及RT-PCR法检测各组胃癌细胞中CyclinD2、CyclinE、Cdk2、Cdk4、Cdk6和p16INK4a、p27KIP1蛋白和mRNA的表达。结果中药胃康宁能将增殖过程中的MGC-803胃癌细胞阻滞于G0-G1期,使之不进入或延迟进入S期(即DNA合成期),抑制胃癌细胞的生长;免疫组化法结果显示,与空白对照组比较,中药胃康宁高、中剂量组CyclinE和Cdk2,CyclinD2和Cdk4、Cdk6蛋白均有明显升高,而p27KIP1、p16INK4a蛋白则显著降低(P<0·01);RT-PCR结果显示,中药胃康宁高、中剂量组均能明显降低CyclinD2、CyclinE、Cdk2和Cdk4、Cdk6OPTD值,同时升高p27KIP1、p16INK4aOPTDI值(P<0·01)。结论中药胃康宁可抑制胃癌细胞促细胞周期相关因子CyclinD2、CyclinE、Cdk2、Cdk4和Cdk6的表达,同时增强胃癌细胞周期抑制因子p27KIP1、p16INK4a的表达,这可能是中药胃康宁抑制胃癌细胞生长的作用机制。

Objective To study the effect of Weikangning （WKN） containing serum on growth and cell cycle regulators of gastric cancer cell. Methods WKN containing drug serum was prepared by gastric perfusion of WKN in different dosages to SD rats. Gastric cancer cells were cultured in medium contained the drug serum with different concentrations of WKN. The change of cell cycle was detected by flow cytometry, and the mRNA and protein expressions of CyclinD2, CyclinE, Cyclin-dependant kinase2 （CdK2.）, Cdk4, Cdk6 and p16INK4a and p27^KIP1 were detected with immunohistochemistric （IHC） SABC method and RT-PCR. Results After WKN inter- vention, the cancer cells were constrained in stage Go/G1, unable or retardatory to enter stage G （ namely, the DNA synthesis stage）. IHC examination showed the grey scale values of CyctinD2, CyctinE, Cdk2, Cdk4 and Cdk6 were higher, and that of p16INK4a and p27^KIP1 were lower in cells after moderate/high dosage WKN intervention than those in the blank control （P〈0.01） ; RT-PCR showed the OPTD values of CyclinD2, CyclinE, Cdk2, Cdk4 and Cdk6 were lower, and that of p16INK4a and p27^KIP1 were higher in cells after moderate/high dosage WKN intervention than those in the blank control （ P 〈0.01 ）. Cenelusien WKN can inhibit the expressions of cell cycle promoting related factors of gastric cancer cell, including CyclinD2, CyclinE, Cdk2,Cdk4 and Cdk6, also enhance the expressions of cell cycle inhibiting factors of gastric cancer cell, as p16INK4a and p27^KIP1, which may be the mechanism of action of the remedy in preventing the growth of gastric cancer cells.