CHO细胞模型中整合素β3胞浆段尾部NITY基序对αⅡbβ3介导的细胞功能的影响  

Effect of the Integrin β3 Cytoplasmic NITY Motif on αⅡbβ3-Mediated Cell Functions in CHO Cell Model

作　　者：杨纪春[1] 施小凤[1] 黄建松[1] 龙章彪[1] 肖兵[1] 阮铮[1] 奚晓东[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院上海血液学研究所医学基因组学国家重点实验室,上海200025

出　　处：《中国实验血液学杂志》2015年第3期768-773,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金项目(81270594);中国博士后科学基金项(2013M541528)

摘　　要：目的:探讨整合素β3胞浆段尾部NITY基序对αⅡbβ3介导的细胞功能的影响。方法:利用中国仓鼠卵巢(CHO)细胞模型建立共表达人类野生型整合素αⅡb和野生型β3或突变型β3ΔNITY(β3胞浆段缺失NITY基序)稳转的细胞株,通过细胞在固相化纤维蛋白原上的黏附及伸展试验检测各稳转细胞株的黏附及伸展功能,通过免疫共沉淀方法检测蛋白相互作用。结果:成功建立了CHO-αⅡbβ3和CHO-αⅡbβ3ΔNITY细胞株,稳定表达野生型整合素αⅡbβ3的CHO细胞具有在固相化纤维蛋白原上的黏附和伸展能力,与CHO-αⅡbβ3细胞株相比,CHO-αⅡbβ3ΔNITY细胞株的黏附能力减弱,但是黏附后的伸展能力无明显改变。免疫共沉淀结果显示,kindlin-2能够结合野生型整合素β3,但与突变型整合素β3结合的量显著减少。结论:整合素β3胞浆段缺失NITY基序引起细胞黏附功能受损,这种缺失突变导致整合素β3与kindlin-2蛋白的结合受到抑制,从而部分抑制了整合素β3的信号转导。Objlective： To investigate the effect of integrin β3 cytoplasmic NITY motif on α Ⅱ bβ3-mediated cell functions. Methods ： Stable Chinese hamster ovary （CHO） cell lines that co-express human wild type integrin a Ⅱ b and wild type β3 or mutant β3ANITY （ β3 deleting cytoplasmic NITY motif） were established. Expression of αα Ⅱ b and β3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions. Results： CHO-α Ⅱ bβ3 ,CHO-α Ⅱ bβ3ANITY cells were successfully established. The CHO cells transfected with wild type α Ⅱ bβ3 had the ability of adhesion and spreading. Compared with CHO-α 11 bβ3 cells, CHO-α lI bβ3ANITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered ceils. The coimmunoprecipitation showed that kindlin-2 associated with wild type integdn α Ⅱ bβ3. The 133ANITY mutation substantially reduced kindlin-2 association. Conclusion： Deletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin β3, thereby partially inhibit the integrin β3 signaling.

关 键 词：整合素Β3 CHO细胞模型 β3胞浆NITY基序 αⅡbβ3介导的细胞功能 

分 类 号：R331.143[医药卫生—人体生理学]