文章摘要
林俊安,李〓翔,张〓宁,殷国勇.GIT1-WT及GIT1-Y293F慢病毒载体的构建及其对成骨细胞迁移能力的影响[J].南京医科大学学报,2010,(6):796~800
GIT1-WT及GIT1-Y293F慢病毒载体的构建及其对成骨细胞迁移能力的影响
Construction of GIT1-WT and GIT1-Y293F lentivirus vectors and investigation the role of GIT1-Y293F in osteoblasts migration
投稿时间:2010-04-20  
DOI:10.7655
中文关键词: G蛋白偶联受体激酶结合蛋白1  成骨细胞  慢病毒载体
英文关键词: G protein coupled receptor kinase interacting protein(GIT1)  lentivirus vector  ostroblasts migration
基金项目:江苏省兴卫工程基金(RC2007059)
作者单位
林俊安 南京医科大学第一附属医院骨科,江苏 南京〓210029 
李〓翔  
张〓宁  
殷国勇  
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中文摘要:
      目的:设计构建G蛋白偶联受体激酶结合蛋白1(G protein coupled receptor kinase interacting protein 1,GIT1)野生型(WT)及点突变型(Y293F)慢病毒载体,探讨293位点对GIT1功能的影响?方法:用PCR从小鼠cDNA文库中扩增GIT1-WT,将其连接到慢病毒载体PLJM-GFP中,构建质粒PLJM-GFP-GIT1-WT,测序鉴定?利用TaKaRa MutanBEST Kit试剂盒,对PLJM-GFP-GIT1-WT进行定点突变,构建质粒PLJM-GFP-GIT1-Y293F,测序鉴定?重组慢病毒载体转染包装细胞293T,获取病毒上清感染培养至第4代的小鼠成骨细胞?划痕愈合试验检测成骨细胞迁移能力的变化?结果:通过PCR鉴定?双酶切鉴定及测序鉴定,成功构建了PLJM-GFP-GIT1-WT与PLJM-GFP-GIT1-Y293F?划痕愈合试验观察,与PLJM-GFP-GIT1-WT相比,PLJM-GFP-GIT1-Y293F明显抑制成骨细胞迁移?结论:GIT1功能的正常发挥有赖于293位点适时的磷酸化?
英文摘要:
      Objective:To construct GIT1-WT(full-length avian GIT1)and GIT1-Y293F lentivirus vectors and to investigate the role of 293 site in GIT1. Methods:The GIT1-WT gene was obtained by polymerase chain traction(PCR)from mouse gene bank. The PCR products were inserted into PLJM-GFP plasmid. The PLJM-GFP-GIT1-Y293F was obtained by TaKaRa MutanBEST Kit. Both PLJM-GFP-GIT1-WT and PLJM-GFP-GIT1-Y293F were evaluated by PCR and sequencing. The virus obtained from transfected 293T cells was infected into osteoblasts. The role of GIT1-Y293F in ostroblasts migration was determined by wound healing assay. Results:The PLJM-GFP-GIT1-WT and PLJM-GFP-GIT1-Y293F were constructed successfully. The results of wound healing assay showed that GIT1-Y293F lentivirus vectors significantly inhibited osteoblast migration compared with GIT1-WT. Conclusion:The functions of GIT1 dependent on the phosphorylation of 293 sites.
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