粘着斑激酶和丝裂原活化蛋白激酶对纤粘连蛋白诱导平滑肌细胞迁移和增殖的影响

目的　研究粘着斑激酶和丝裂原活化蛋白激酶在细胞外基质成分诱导血管平滑肌细胞迁移和增殖中的作用。方法　通过纤粘连蛋白 (FN)诱导培养的平滑肌细胞迁移和增殖 ,以免疫沉淀和Western印迹法检测粘着斑激酶 (FAK)和丝裂原活化蛋白激酶 (p4 2 / 4 4MAPK)及其磷酸化的表达量。将FAK反义寡核苷酸 (ODN)经脂质体转染细胞 ,观察其对FAK和p4 2 / 4 4MAPK磷酸化、细胞迁移以及增殖的影响。结果　不同浓度FN(5、10、2 0、4 0、6 0 μg/ml)在有效诱导平滑肌细胞迁移和增殖时FAK和p4 2 / 4 4MAPK也呈明显表达 ,2 0 μg/mlFN可使其磷酸化处于较高的表达量。脂质体可有效地介导ODN转染 ,转染效率为 86 7%± 4 5 %。转染后FAK以及p4 2 / 4 4MAPK磷酸化表达量明显减少 ,10、2 0、4 0和 6 0 μg/mlFN组迁移细胞数也分别显著减少 (2 3 2 6 %、2 1 6 3%、19 31%、17 88% ,P <0 0 5 ) ,5～ 6 0 μg/ml不同浓度的FN组 ,细胞增殖减少 2 7 6 7%～ 4 6 6 7% (P <0 0 5 )。 结论　活化的FAK和p4 2 / 4 4MAPK是细胞外基质诱导平滑肌细胞迁移和增殖的重要信号分子 ,二者之间存在着密切的联系 ,由其介导的信号转导促进了这一过程 ,反义FAKODN可有效地对此进行抑制

Focal adhesion kinase and mitogen activated protein kinase are involved in vascular smooth muscle cells migration and proliferation stimulated by fibronectin

OBJECTIVE: To study the roles of focal adhesion kinase (FAK) and mitogen activated protein kinase (p42/44MAPK) in the process of migration and proliferation of vascular smooth muscle cells (VSMC) stimulated by fibronectin (FN). METHODS: VSMCs were taken from the tunica media of SD rats and cultured. Migration and proliferation of cultured VSMCs were stimulated by different concentrations of FN; FAK, p42/44MAPK and their phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotide (ODN) was transfected into VSMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. VSMC migration and proliferation were also measured by modified Boyden Chamber and (3)H-thymidine respectively. RESULTS: FAK and p42/44MAPK were expressed when VSMC adhesion and migration were successfully simulated by FN (5, 10, 20, 40, and 60 microg/ml), high contents of FAK and p42/44MAPK phosphorylation were detected in groups with 20 microgram/ml FN or more. FAK antisense ODN was transfected efficiently by cationic lipid. Phosphorylation of FAK and p42/44MAPK was inhibited significantly after FAK antisense ODN transfection. Cell migration stimulated by FN of the concentrations of 10, 20, 40, and 60 microg/ml was reduced by 23.26%, 21.63%, 19.31% and 17.88% respectively (P < 0.05). VSMC proliferation in 5 approximately 60 microgram/ml FN groups was reduced by 27.67% approximately 46.67% (P < 0.05). CONCLUSION: FAK and p42/44MAPK play an important role in VSMC migration and proliferation stimulated by extracellular matrix. The process can be inhibited by FAK antisense ODN effectively.