G蛋白偶联受体激酶结合蛋白1发夹结构通过影响Paxillin的功能抑制成骨细胞迁移

目的探索G蛋白偶联受体激酶结合蛋白1（Gprotein coupled receptor kinase interacting protein 1，GIT1）的RNA发夹结构（hairpin）（GIT1-RNAh）对成骨细胞迁移的影响，并分析其机制。方法取培养至第6代的鼠成骨细胞，随机分成两组，分别用包含GIT1-RNAh（实验组）和绿色荧光蛋白（green fluoresence protein，GFP）的RNA发夹结构（GFP—RNAh）的腺病毒（对照组）感染12h后，再将每组分成有、无血小板源性生长因子（platelet drived growth factor，PDGF）刺激的两组。免疫荧光染色方法检测成骨细胞内源性GIT1蛋白表达和Paxillin的位置。Western Blot检测Paxillin的磷酸化。构建包含蓝色荧光蛋白的GIT1-RNAh（CFP—GIT1-RNAh）实验组和GFP—RNAh（CFP—GFP—RNAh）（对照组），免疫荧光双染的方法检测CFP—GIT1-RNAh和CFP—GFP—RNAh对Paxillin位置的特异性作用。用划痕愈合法检测GIT1-RNAh（实验组）和GFP—RNAh（对照组）腺病毒对成骨细胞在PDGF刺激下的迁移能力的影响。结果免疫组织化学观察，实验组和对照组比较，明显抑制成骨细胞内源性的GIT1蛋白表达和扰乱Paxillin的分布。Western Blot观察，和对照组相比，实验组明显抑制了Paxiliin的磷酸化（P〈0．05）。划痕愈合法检测观察，和对照组相比，实验组明显抑制成骨细胞的迁移。结论GIT1-RNAh通过扰乱Paxillin的分布和其磷酸化从而抑制成骨细胞的迁移。

MECHANISM OF G PROTEIN COUPLED RECEPTOR KINASE INTERACTING PROTEIN 1 RNA HAIRPIN INHIBITING OSTEOBLASTS MIGRATION

Objective To study the function and mechanism of G protein coupled receptor kinase interacting protein 1(GIT1) RNA hairpin (GIT1-RNAh) in osteoblast migration. Methods The sixth passage osteoblasts were divided into 2 groups and were infected by GIT1-RNAh (experimental group) and green fluoresence protein RNA hairpin (GFP-RNAh) (control group) adenovirus for 12 hours respectively. Each group was further classfied into two groups according to with or without platelet-drived growth factor (PDGF) stimulation. The GIT1 expression and Paxillin distribution was analyzed by immunofluorescence staining. Paxillin phosphorylation was detected by Western Blot. The localization of Paxillin was determined by co-immunofluorescence staining after transfection with cyanine fluorescence protein tagged GIT1-RNAh (CFP-GIT1-RNAh)(experimental group) and GFP-RNAh (CFP-GFP-RNAh)(control group). The role of GIT1-RNAh (experimental group) and GFP-RNAh (control group) adenovirus in osteoblasts migration was determined by wound healing assay. Results Immunofluorescence staining results showed that the GIT1-RNAh significantly inhibited endogenous GIT1 expression, interfered Paxillin distribution. Western Blot results showed that Paxillin phosporylation was obviously inhibited in osteoblasts infected with GIT1-RNAh adenovirus (P<0.05). The wound healing assay results showed that GIT1-RNAh adenovirus significantly inhibited osteoblast migration induced by PDGF. Conclusion GIT1-RNAh inhibits osteoblasts migration by interfering paxillin distribution and decrease Paxillin phosphorylation.