pIRES2-EGFP-IGF-1真核表达载体转染骨髓基质细胞的动物实验

Experimental study on dog′s bone marrow stem cells transfected by pIRES2-EGFP-IGF-1 gene

目的构建表达胰岛素样生长因子1(IGF-1)的骨髓基质细胞(MSC)的动物模型,为进一步将其用于修复牙周组织缺损奠定基础。方法将真核表达载体pIRES2-EGFP-IGF-1转染MSC,获得阳性细胞克隆,免疫组化、Westernblot鉴定IGF-1蛋白表达水平,ELISA方法检测转染细胞上清液中IGF-1蛋白含量;用转染细胞上清液培养牙周膜细胞(PDLC),甲基噻唑基四唑(MTT)法检测对细胞增殖的影响。结果免疫组化和Westernblot结果证实,转染细胞内有IGF-1蛋白的高表达。ELISA方法检测显示,IGF-1能分泌至细胞外,且能明显促进PDLC的增殖。结论IGF-1基因可成功地转染MSC细胞,获得高表达有活性的IGF-1蛋白的MSC细胞模型,为将其应用于牙周组织工程提供了实验依据。

Objective To establish the bone marrow stem cells （MSC） model which could highly express the insulin-like growth factor 1 （IGF-1） transfected by dog＇s IGF-1 gene. Methods plRES2-EGFP- IGF-1 was transfected into MSC by lipofectamine, Positive clones were selected with G418. The expression of IGF-1 protein in the MSC was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supematant of the transfected MSC was detected by sandwich-in ELISA The periodontal ligament cells （PDLC） were cultured in the supernatant of the transfected MSC, The changes of PDLC＇ proliferation were observed by MTT. Results IGF-1-transfected MSC could apparently express IGF-1. The IGF-1 protein in the supernatant of the transfected MSC was confirmed by sandwich-in ELISA. IGF-1 could promote the PDLC＇ proliferation. Conclusions The MSC transfected by dog＇s IGF-1 gene can highly express IGF-1, which may lay the foundation for further study on periodontal regeneration.