摘要
目的探讨接合物蛋白Crk和CrkL在卵巢癌细胞株MCAS细胞增殖中的不同作用。方法采用免疫共沉淀法检测上皮性卵巢癌细胞株MACS中Crk和CrkL蛋白与已知的与肿瘤增殖相关的上下游结合蛋白的内源性结合;用可调节性siRNA敲低MACS细胞中内源性的Crk及CrkL,观察二者对细胞生长及存活能力的影响。结果免疫共沉淀显示卵巢癌细胞株MACS中,Crk与影响细胞生长增殖的上游蛋白p130Cas及下游蛋白Sos的结合明显强于CrkL,尽管Crk与CrkL表现出部分相同的免疫原性。免疫印迹显示在加入Dox后即Crk或CrkL表达沉默被启动。Crki/CrkLi细胞中加入Dox后Crk与CrkL表达明显降低[Dox+:Crk(0.021±0.001),CrkL(0.676±0.005);Dox-:Crk(0.544±0.003),CrkL(1.282±0.007),P<0.05]。对可调节性siRNA构建之可控性Crki/CrkLi细胞的连续动态观察发现,Crki细胞表现出细胞增殖力受限仍保存生存活力,而CrkLi细胞则表现出逐渐脱壁至死亡。结论 Crk及CrkL在卵巢癌细胞MCAS的恶性增殖中扮演着不同的作用,Crk蛋白的过度表达可能直接影响着卵巢癌细胞恶性增殖,而CrkL则可能更多地参与了细胞基本生存相关的生物行为。
Objective To verify the different roles of adaptor proteins Crk and Crk-like（CrkL） in the proliferation in ovarian cancer cell line MCAS.Methods Co-immunoprecipitation was performed to detect Crk and CrkL endogenous binding proteins that were known to be associated with tumor malignant proliferation in ovarian cancer cell line MCAS.Inducible siRNA was employed to knock down endogenous Crk and CrkL expression in MCAS cells,respectively,and the growth and viability of the inducible Crk/CrkL-knockdown MCAS cells（MACS-Crki/CrkLi） were observed.Results Crk rather than CrkL strongly bound to p130Cas and Sos,which were reported to be related with cell proliferation,despite CrkL and Crk had partially the same immunogenicity.The result of Western blotting showed that Crk/CrkL started to be silenced and their expression significantly decreased when exposed to doxycycline（Dox） in MACS-Crki/CrkLi cells（Dox＋： Crk 0.021±0.001,CrkL 0.676±0.005;Dox-： Crk 0.544±0.003,CrkL 1.282±0.007;P〈0.05）.Serial observation of the MACS-Crki/CrkLi cells demonstrated that the Dox-induced CrkL knockdown cells presented with gradual detachment from plastic dish and even death,as compared with the Dox-induced Crk knockdown cells with reduced cell growth but remaining viability.Conclusion As compared with the role of Crk in malignant proliferation of MCAS cells,CrkL may be involved in some basic biological events associated with MCAS cell survival.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2012年第12期1185-1188,共4页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(30672432
30772330)
重庆医科大学第一医院医学科学基金(YXJJ2009-06)~~
