CAJ | 학술논문

目的研究粘着斑激酶磷酸化在细胞外基质成份诱导平滑肌细胞粘附和迁移中的作用.方法通过纤粘连蛋白(fibronectin, FN)诱导培养的平滑肌细胞粘附迁移,以免疫沉淀和Western blot方法检测粘着斑激酶(focal adhesion kinase, FAK)及其磷酸化的表达量.将FAK反义寡核苷酸(antisense oligodeoxynucleotides, ODNs)经脂质体转染细胞,观察对FAK磷酸化、细胞粘附铺展和迁移的影响.结果 FN在显著诱导平滑肌细胞粘附和迁移时,FAK也呈明显表达,20?μg/ml FN可使其磷酸化处于较高表达量.脂质体可有效地介导ODNs转染,转染效率为86.7%±4.5%,FAK磷酸化表达量明显减少,5-60?μg/ml不同浓度FN组,细胞铺展率减少17.89%-27.67%,10、 20、 4 0和60?μg/ml FN组迁移细胞数也分别显著减少23.26%、21.63%、19.31%、17.88%(P <0.05).结论活化的FAK是细胞外基质诱导SMCs粘附和迁移的重要信号分子,由其介导的信号转导促进了这一过程,反义FAK ODNs可有效地对此进行抑制.
목적연구점착반격매린산화재세포외기질성빈유도평활기세포점부화천이중적작용.방법통과섬점련단백(fibronectin, FN)유도배양적평활기세포점부천이,이면역침정화Western blot방법검측점착반격매(focal adhesion kinase, FAK)급기린산화적표체량.장FAK반의과핵감산(antisense oligodeoxynucleotides, ODNs)경지질체전염세포,관찰대FAK린산화、세포점부포전화천이적영향.결과 FN재현저유도평활기세포점부화천이시,FAK야정명현표체,20?μg/ml FN가사기린산화처우교고표체량.지질체가유효지개도ODNs전염,전염효솔위86.7%±4.5%,FAK린산화표체량명현감소,5-60?μg/ml불동농도FN조,세포포전솔감소17.89%-27.67%,10、 20、 4 0화60?μg/ml FN조천이세포수야분별현저감소23.26%、21.63%、19.31%、17.88%(P <0.05).결론활화적FAK시세포외기질유도SMCs점부화천이적중요신호분자,유기개도적신호전도촉진료저일과정,반의FAK ODNs가유효지대차진행억제.
Objective To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin.Methods Adhesion and migration of cultured SMCs were stimulated by different concentrations of fibronectin (FN), FAK and its phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. SMCs adhesion and migration were also measured by morphological enumeration and modified Boyden Chambers, respectively.Results FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20μg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5-60μg/ml FN groups was reduced by 17.89%-27.67%. Cell migration stimulated by FN at 10, 20, 40 and 60μg/ml were reduced by 23.26%, 21.63%, 19.31% and 17.88%, respectively (P<0.05).Conclusions FAK phosphorylation and FAK-mediated signal transduction play important roles in SMCs adhesion and migration stimulated by ECM. The process can be inhibited effectively by FAK antisense ODNs.