健脾理气药诱导人肝癌细胞SMMC7721凋亡的研究

Study on the apoptosis and cell cycle arrest in human liver cancer SMMC7721 cells induced by Jianpiliqi herbs

目的观察健脾理气药的诱导凋亡效应,为其临床应用进一步提供依据。方法采用血清药理学方法研究中药的体外效应,应用四甲基偶氮唑蓝(MTT)比色法检测含中药兔血清对肝癌细胞的抑制效应,以Annexin V标记法、DNA含量测定、电子显微镜方法检测含中药血清诱导凋亡及细胞周期阻滞效应。免疫组化法观察含中药血清对P53,P21^(WAF1)/CIP1)蛋白的影响,RT-PCR法观察含中药血清对P21^(WAF1/CIP1) mRNA表达水平的影响。结果含中药血清有一定的抑制肝癌细胞作用,其作用3d的抑制率为6.6％,作用6d的抑制率为36.2％。含中药血清作用2d诱导9.8％±4.0％的肝癌细胞凋亡,使细胞周期阻滞于S期,并上调P53蛋白、P21^(WAF1/CIP1) mRNA及蛋白的表达。结论健脾理气药具一定的诱导凋亡及抑制肝癌细胞效应,上调p53,p21^(WAF1/CIP1)基因的表达为分子机制。

AIM To evaluate the inducing effects of Jianpiliqi herbs on apoptosis and to offer experimental basis for its clinical application.METHODS The effects of Jianpiliqi herbs were investigated in vitro using serum pharmacological approach. The inhibiting effect of Jianpiliqi herbs containing rabbit serum on SMMC7721 cells was observed using tetrazolium dye (MTT) assay. Apoptosis and cell cycle arrest induced by Jianpiliqi herbs were determined with Annexin V-affinity assay, DNA content analysis and electron microscopy. Effects of Jianpiliqi herbs on p53 and P21WAF1/CIP1 protein expression were assessed by cell culture and immunohistochemistry. With RT-PCR assay, changes in expression of P21WAF1/CIP1 mRNA after Jianpiliqi herbs treatment were observed.RESULTS The serum containing Jianpiliqi herbs could inhibit the proliferation of cancer cells. The inhibiting rate was 6.6% for 3-day treatment and 36.2% for 6-day treatment. The apoptosis rate in SMMC7721 cells induced by Jianpiliqi herbs was 9.8%±4.0%. Jianpiliqi herbs arrested cell cycle at S phase, and increased the expression of p53 protein, P21WAF1/CIP1 mRNA and protein. CONCLUSION Jianpiliqi herbs have the effects of inducing apoptosis and inhibiting the proliferation of livercancer cells. It can enhance the expression of p53 and p21wAF1/CIP1 gene, which might be the molecular mechanism.