摘要
目的 研究胰岛素受体（IR）亚型（包括IR-A和IR-B）在子宫内膜癌细胞及组织中的表达情况,并初步探讨IR-A在子宫内膜癌细胞增殖中的作用.方法 选取4种不同子宫内膜癌细胞系HEC-1-A、Ishikawa、RL95-2、KLE细胞,以乳腺癌细胞系MCF-7细胞和肝癌细胞系Hep-G2细胞为阳性对照;收集2007年11月-2009年7月间北京大学人民医院收治的42例子宫内膜癌患者的癌组织标本,以同期收治的15例因卵巢肿瘤行子宫切除术患者的正常子宫内膜组织标本作为对照.（1）采用逆转录（RT）-PCR技术检测4种不同子宫内膜癌细胞及组织中IR亚型的表达形式,应用实时定量 PCR技术检测其IR及IR-A mRNA的表达水平,采用Spearman等级相关分析方法对影响子宫内膜癌组织中IR及IR-A mRNA表达的临床病理因素进行分析.（2）构建稳定过度表达IR-A基因的RL95-2细胞系RL95-2（IR-A）细胞,采用非放射性细胞增殖检测法—MTS法检测4种不同子宫内膜癌细胞和RL95-2（IR-A）细胞培养1～7d的增殖情况.结果（1）RT-PCR技术检测显示,在子宫内膜癌 HEC-1-A、Ishikawa、RL95-2、KLE细胞及组织中,IR均以IR-A和IR-B两种亚型共同表达.实时定量PCR技术检测显示,在子宫内膜癌细胞中,IR mRNA的表达水平以RL95-2细胞最高,其次为Ishikawa、KLE和HEC-1-A细胞,其IR mRNA的表达水平分别为（26.54±1.82）×10-4、（15.44±3.29）×10-4、（10.14 ±0.10）×10-4和（2.63 ±0.23）×10-4,4者比较,差异有统计学意义（P＜0.01）;IR-A mRNA的表达水平以Ishikawa细胞最高,其次为KLE、RL95-2和HEC-1-A细胞,其IR-A mRNA的表达水平分别为（12.07 ±3.31）×10-4、（4.68 ±0.63）×10-4、（3.03±0.22）×10-4和（1.46±0.03）×10-4,4者比较,差异有统计学意义（P＜0.01）.在子宫内膜癌组织中,IR及IR-A mRNA的表达水平分别为0.017±0.013、0.011±0.010,分别与对照组（分别为0.015±0.014、0.010±0.012）比较,差异均无统计学意义（P=0.662,P=0.780）.子宫内膜癌组织中IR及IR-A mRNA的表达水平与手术病理分期、病理分级、肌层浸润、淋巴管癌栓、淋巴结转移及ER、PR、PTEN 基因的表达均无明显相关性（P均＞0.05）;IR-A mRNA的表达水平与有无2型糖尿病明显相关（P=0.031）,但IR mRNA的表达水平与有无2型糖尿病无明显相关性（P =0.438）.（2）本研究成功构建过度表达IR-A基因的RL95-2细胞系RL95-2（IR-A）细胞.MTS法检测显示,分别培养1～7d 后,4种不同子宫内膜癌细胞中以Ishikawa细胞的增殖速度最快,其次依次为HFC-1-A、KLE和RL95-2细胞,4种细胞各时间点分别比较, 差异均有统计学意义（P＜0.01）;稳定转染后的RL95-2（IR-A）细胞的增殖速度明显快于未转染的RL95-2细胞,差异有统计学意义 （P＜0.01）.结论 子宫内膜癌细胞及组织中IR均以IR-A和IR-B两种亚型共同表达,过度表达IR-A基因可促进子宫内膜癌细胞的增殖.
Objective To investigate the expression of insulin receptor isoforms（IR,including IR-A and IR-B）in endometrial carcinoma（EC）,and explore the role of IR-A in the growth of endometrial carcinoma cells.Methods The expression of IR isoforms were detected by reverse transcription（RT）-PCR and real-time PCR in 4 different endometrial cancer cell lines（HEC-1-A,Ishikawa,RL95-2 and KLE）,with human breast cancer cell line MCF-7 cells and hepatocellular carcinoma cell line Hep-G2 as positive control and in the endometrial cancer tissue specimens of 42 cases,admitted in Peking University People＇s Hospital from November 2007 to July 2009,with normal endometrial tissues from 15 cases of ovarian neoplasms at the same period as controls.The relationships among the expression of IR,IR-A isoforms and the clinicopathological parameters of EC tissues were analyzed by Spearman rank correlation analysis.An eukaryotic IR-A expression plasmid was constructed and transfected into RL95-2 cells[RL95-2（IR-A）]for overexpression of IR-A in RL95-2 cells,in which the expression of IR-A originally was low.Non radioactive cell proliferation assay—MTS was used to determine the proliferation curves in the four EC cells listed above and in RL95-2 or RL95-2（IR-A）.Results（1）There were two isoforms of IR-A and IR-B coexpressed were detected in EC cells and EC tissues.Among four kinds of EC cell lines,the expression level of IR mRNA in RL95-2 cells was the highest,followed by Ishikawa,KLE and HEC-1-A cells,in which the relative IR mRNA expression levels were（26.54 ± 1.82）× 10-4,（15.44 ± 3.29）× 10-4,（10.14 ±0.10）× 10-4 and（2.63 ±0.23）× 10-4,respectively（P 〈0.01）.The expression level of IR-A mRNA was the highest in Ishikawa cells,followed by KLE,RL95-2 and HEC-1-A cells,with the relative expression levels were（12.07 ±3.31）× 10-4,（4.68 ±0.63）× 10-4,（3.03 ±0.22）× 10 4 and（1.46 ±0.03）×10-4,respectively（P 〈0.01）.The relative expression level of IR and IR-A mRNA were 0.017 ±0.013 and 0.011 ±0.010 in the EC tissues,respectively,compared with 0.015 ± 0.014 and 0.010 ± 0.012 in the controls,in which there were no significant differences in the expression level of IR or IR-A mRNA between EC tissues and the control（P =0.662,P =0.780）.The expression of IR and IR-A in EC tissues had no significant relevance with International Federation of Gynecology and Obstetrics（FIGO）stage,cell differentiation,depth of myometrial invasion,invasion of lymph-vascular space,lymph nodes metastasis,the expression status of estrogen receptor,human progesterone receptor,and PTEN gene（all P 〉 0.05）.The expression of IR-A mRNA in EC patients with type 2 diabetes mellitus（DM）was significantly higher than that in patients without type 2 DM（P =0.031）,while there were no statistical correlation between the expression of IR mRNA and type 2 DM（P =0.438）.（2）The proliferation rates of the four kinds of EC cells was positively related with the IR-A expression ratio,with the most growth potential in Ishikawa,followed by HEC-1-A,KLE and RL95-2 cells.The overexpression of IR-A in RL95-2（IR-A）cells showed a significant proliferation-promoting effect than that in control RL95-2 cells（P 〈 0.01）.Conclusion There are two isoforms of IR-A and IR-B co-expressed in EC.The overexpression of IR-A may promote the proliferation of EC cells.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2012年第11期839-845,共7页
Chinese Journal of Obstetrics and Gynecology
基金
国家自然科学基金（30973181）
教育部高等学校博士点专项基金（200800010095）
