Cyclin G1介导孕激素对子宫内膜上皮细胞增殖的负调控作用实验研究  

Cyclin G1 mediate inhibitory effect of progesterone on proliferation of EECs

作　　者：丁小玲[1] 方明[1] 岳利民[1] 于海礼[1] 袁东智[1] 刘芳[1] 张金虎[1] 何亚平[1] 

机构地区：[1]四川大学华西基础医学与法医学院生理教研室,四川成都610041

出　　处：《西部医学》2010年第12期2191-2194,共4页Medical Journal of West China

基　　金：国家自然科学基金资助项目(30770813);四川省科学委员会基金资助项目(2006J13-090)

摘　　要：目的探讨孕激素依赖的cyclin G1是否介导了孕激素对子宫内膜上皮细胞增殖的负调控作用。方法运用RNA干扰技术(RNAi)使cyclin G1基因表达沉默,观察孕激素所致小鼠子宫内膜上皮细胞增殖抑制作用的变化。针对cyclin G1基因设计并化学合成cyclin G1的小干扰RNA(G1-siRNA)和与cyclin G1序列无同源性的siRNA(con-trol-siRNA)作为阴性对照,两种siRNA均在其正义链的5’端加上荧光标记物Cy3。随机选取9只去势12天的成年雌性小鼠,连续2天皮下注射雌激素(100ng/次.只)。末次皮下注射雌激素19小时后,将Lipofectamin 2000与G1-siRNA(200nmol/ml)和control-si RNA(200nmol/ml)的混合液分别注射入同一小鼠的两侧子宫。5小时后,实验小鼠皮下注射孕激素(2 mg/只)。于孕激素注射24小时后,切取两侧子宫。冰冻切片后,荧光显微镜观察,以在子宫内膜上皮细胞中见到Cy3发出红色荧光作为判断RNA被子宫内膜层有效吸收的依据。确定siRNA被有效吸收后,应用免疫组化检测对照组和实验组子宫内膜中cyclin G1的表达和反映细胞增殖活性的增殖细胞核抗原(PCNA)的表达。结果免疫组织化学染色结果显示,实验组子宫内膜上皮细胞内cyclin G1表达明显减弱,而PCNA的蛋白表达明显增强,差异具有统计学意义(P<0.05)。结论cyclin G1至少部分介导了孕激素对子宫内膜上皮细胞增殖的负调控作用,为进一步探索和治疗子宫内膜癌提供了新的依据。Objective To study the negative regulation of progesterone-dependent cyclin G1 on proliferation of endometrial epithelial cells（EECs）.Methods We knocked down the expression of cyclin G1 gene in EECs with technology of RNA interference（RNAi） and observed the change of their proliferative activities inhibited by progesterone in vivo.siRNA targeting murine cyclin G1 and control siRNA were designed and artificially synthesized.siRNAs were labeled with Cy3 at the 5＇-end of the sense strand to investigate cellular uptake in EECs.9 Kunming female mice were ovariectomized and rested for 12 days before they were primed with 100 ng of E2 in peanut oil by sc injection for 2 days.19 hr after the last E2 injection,two types of siRNA dilutions（200nmol/ml） were mixed with Lipofectamin2000 and cyclinG1 siRNA-Lipofectamine 2000 complexes was directly injected into one side of uterine horns,and control siRNA-Lipofectamine 2000 complexes for another side.5 hours later,mice were treated with a single injection of progesterone（2mg/mouse）.24hr after the last injection,the uteri were collected.Frozen sections of the uteri were used for observing the transfection efficiency of EECs under fluorescence microscope.After make sure that complexes was efficiently taken up by EECs,the inhibitory effect of siRNA on the expression of cyclin G1 and proliferating cell nuclear antigen（PCNA） reflecting the proliferative activity of EECs were detected by immunohistochemical method.Results Compared with those in control sides,specific siRNA targeting cyclin G1 significantly reduced cyclin G1 expression,but intensity of PCNA protein expression in EECs were significantly high.Conclusion Progesterone-dependent cyclin G1 as a negative factor in regulating cell cycle at least in part mediates the inhibitory effect of progesterone on proliferation of EECs.

关 键 词：细胞周期蛋白G1(cyclin G1) 子宫内膜 孕激素 siRNA 

分 类 号：R339.22[医药卫生—人体生理学]