肝细胞生长因子对子宫内膜癌细胞增殖影响实验探究.docVIP

肝细胞生长因子对子宫内膜癌细胞增殖影响实验探究 　 作者：都红蕾，孙奋勇，陈勍，潘秋辉 【摘要】; 目的 观察肝细胞生长因子（HGF）对子宫内膜癌细胞HEC2B增殖的影响及子宫内膜癌细胞HEC2B中HGF受体Cmet的表达，了解HGF对子宫内膜癌细胞的作用及其机制。方法 体外培养人子宫内膜癌细胞HEC2B，用RTPCR的方法检测HEC2B中HGF受体Cmet有表达。对HEC2B进行24 h血清饥饿后用不同剂量的HGF作用于HEC2B。用四甲基偶氮唑盐微量酶反应比色法(MTT法)分析细胞的增殖力,用流式细胞仪检测细胞的增殖周期。结果 Cmet在HEC2B细胞中稳定表达，HGF促进细胞的增殖,并在一定的范围内有剂量依赖关系，各处理组与对照组相比，Plt;0.05。当HGF浓度达到60 ng· mL-1时，增殖水平较对照上调约1.9倍。结论 HGF/Cmet能够在体外促进子宫内膜癌细胞的增殖。 【关键词】; 子宫内膜癌细胞；肝细胞生长因子；肝细胞生长因子受体 　　Abstract:Objective To study the effects of hepatocyte growth factor on endometrial cancer cells, detect the alterations of cell proliferation and expression level of HGF receptor Cmet and understand the molecular mechanism. Methods; Endometrial cancer cell line, HEC2B was cultured in vitro. The expression level of Cmet in HEC2B was determined with RTPCR. HEC2B was treated with HGF at different doses. Cell proliferation was analyzed with MTT assay. Cell cycle distribution was examined with FACS. Statistical evaluations were conducted using ttest. Results; Cmet was expressed in HEC2B cells. Compared with controls, HGF could promoted the proliferation of HEC2B in a dosedependent manner (Plt;0.05). Treatment with 60 ng· mL-1 HGF increased the proliferation level by 1.9 fold. Conclusion; Cmet was expressed stably in HEC2B cells. HGF/Cmet system promoted the formation, development and proliferation of endometrial cancer cells in vitro. 　　Key words:endometrial cancer cell; hepatocyte growth factor; hepatocyte growth factor receptor 　　子宫内膜癌是女性生殖道常见三大恶性肿瘤之一，近20年来在世界范围内子宫内膜癌发病率有上升趋势。肝细胞生长因子(HGF)是一个多功能生长因子。HGF的单一受体Cmet是一跨膜蛋白，由原癌基因Cmet编码。在许多恶性肿瘤中Cmet被过表达［1-6］。HGF/Cmet与子宫内膜癌的关系却鲜有报道。本研究通过观察子宫内膜癌细胞中Cmet的表达及HGF对子宫内膜癌细胞的作用，旨在为临床以HGF／Cmet为靶点治疗子宫内膜癌提供理论基础。 　　1; 材料与方法 　　1.1; 试剂与细胞株 ; 子宫内膜癌细胞株HEC2,购自中科院细胞库;RNase、胰蛋白酶、DMEM 培养基、Trizol、逆转录酶Superscript Ⅲ为Invitrogen公司产品;优质胎牛血清,Gibco公司;HGF购自Ramp;D， Agarose 、G418 由Sigma公司提供。PCR用酶SYBR Green PCR Master Mix购自ABI公司。EPICS AL TRA流式细胞仪,美国Beckman Coulter公司提供。 　　1.2; HEC2B细胞的体外培养与诱导 ; 将细