人胰岛素样生长因子1基因转染脂肪源性干细胞的效应.docx

中国组织工程研究??2013,?Vol. 17??Issue (6): 1018-1023????DOI: 10.3969/j.issn.2095-4344.2013.06.012脂肪干细胞人胰岛素样生长因子1基因转染脂肪源性干细胞的效应Effects of human insulin-like growth factor 1 gene trasfection on adipose- derived stem cells全文:?PDF?(461 KB)摘要?背景：人胰岛素样生长因子1基因对脂肪源性干细胞的增殖和分化也可能会产生有效作用。目的：验证人胰岛素样生长因子1基因转染对体外培养的脂肪源性干细胞的效应。方法：构建含人胰岛素样生长因子1基因的双顺反子真核表达载体pIRES2-EGFP-hIGF-1，利用阳离子脂质体Lipofectamine 2000介导转染体外培养的人脂肪源性干细胞。观察基因转染后细胞增殖及形态的变化，倒置荧光显微镜观测标记基因增强绿色荧光蛋白的表达并计算转染效率，酶联免疫吸附试验检测培养上清中人胰岛素样生长因子1的浓度，免疫组织化学染色及RT-PCR检测人胰岛素样生长因子1的表达，流式细胞仪检测转染前后细胞周期的变化。结果与结论：测序及酶切证实真核表达载体pIRES2-EGFP-hIGF-1构建正确。体外培养的脂肪源性干细胞为多种形态并存，转染后6 h检测到有EGFP的表达，至60 h达到高峰，转染效率为(16±3)%。细胞上清中人胰岛素样生长因子1的浓度在60 h达到22.65 μg/L。免疫组织化学染色及RT-PCR均检测到人胰岛素样生长因子1的表达。转染后的细胞分裂增殖加快，细胞群体倍增时间缩短，S期细胞比例增多。证实人胰岛素样生长因子1基因可有效转染脂肪源性干细胞并表达人胰岛素样生长因子1蛋白质，同时可促进细胞增殖。关键词?：?干细胞；?脂肪干细胞；?胰岛素样生长因子；?脂肪源性干细胞；?基因；?转染；?细胞增殖；?国家自然科学基金；?干细胞图片文章； ??Abstract：BACKGROUND:?Human insulin-like growth factor 1 gene produces effects on the proliferation and differentiation of adipose-derived stem cells.OBJECTIVE:?To investigate the possible effects of human insulin-like growth factor 1 gene transfection on the adipose-derived stem cells cultured in vitro.METHODS:?The cDNA for human insulin-like growth factor 1 was cloned into the recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF-1. The adipose-derived stem cells were derived from adipose tissue and cultured in vitro, and then the plasmid was transfected into cells by Lipofectamine 2000. After gene transfection, changes in cell proliferation and morphology were observed. Through the use of inverted fluorescent microscopy, the marker gene coding enhanced green fluorescent protein was observed and the transfection efficiency was determined. The human insulin growth factor 1 concentration in the supernatant fluids was measured by ELISA. The expression of human insulin-like growth factor 1 was detected by immunohistochemical staining and RT-PCR. The changes in cell cycle before and after transfection were assessed by flow