胰岛素样生长因子1基因转染兔脂肪间质干细胞的增殖  

Effects of insulin-like growth factor 1 gene transfection on proliferation of rabbit adipose mesenchymal stem cells

作　　者：安春厚[1] 程扬[1] 原泉[1] 李建军[1] 

机构地区：[1]中国医科大学附属盛京医院骨科,辽宁省沈阳市110004

出　　处：《中国组织工程研究与临床康复》2009年第23期4433-4436,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30772216)~~

摘　　要：背景：基因工程是目前软骨修复的主要方法，脂肪间质干细胞以其来源丰富、取材容易、免疫相容性良好、体外培养可以较长时间保持分化表型、有较强的向软骨细胞转化的能力而成为理想的种子细胞。目的：探讨胰岛素样生长因子1基因转染对兔脂肪间质干细胞增殖的影响。设计、时间及地点：细胞学基因转染体外观察，于2006-03／2007-03在中国医科大学细胞生物实验室完成。材料：成年新西兰大白兔3只，由中国医科大学实验动物中，心提供。含有全长人胰岛素样生长因子1cDNA片段的质粒pcDNA3．1．hlGF-1由吉林大学徐莘香教授惠赠。方法：取兔颈后皮下脂肪，Ⅰ型胶原酶消化法体外分离培养兔脂肪间质干细胞。取传至第2代细胞，以．0．5×10^6/孔密度接种于6孔板，细胞融合至90％-95％时随机分为未转染组、转染空载体pcONA3．1组、转染质粒pcDNA3．1-hlGF-1组，采用脂质体介导基因转染法进行质粒pcDNA3．1-IGF-1转染，经G418筛选后培养4周。主要观察指标：采用RT-PCR及Westernblot方法检测胰岛素样生长因子1的表达情况，MTT法及流式细胞仪检测细胞增殖情况。结果：转染pcDNA3．1-hlGF-1组的脂肪间质干细胞内有大量胰岛素样生长因子1mRNA及蛋白表达，未转染组及转染空载体pcDNA3．1组无表达。细胞增殖曲线显示，转染质粒pcDNA31-hlGF-1组的脂肪间质干细胞增殖速度较其他2组细胞明显加快。流式细胞仪检测显示，转染质粒pcDNA3．1-hlGF．1组的脂肪间质干细胞较其他2组细胞的S期比例显著增加（户〈0．05），G1期比例显著下降（P〈0．05）。结论：质粒pcDNA3．1-IGF-1转染兔脂肪间质干细胞后，可以获得稳定表达，并能明显促进脂肪间质干细胞的增殖。BACKGROUND： Genetic engineering is a main method to repair cartilage damage. Adipose mesenchymal stem cells （ADSCs） are ideal seed cells and characterized by abundant source, easy material collection, good immune compatibility, long-term differentiation phenotype cultured in vitro, and strong ability to transformation into chondrocytes. OBJECTIVE： To study the effect of human insulin-like growth factor 1 （IGF-1） gene transfection on the proliferation of rabbit ADSCs. DESIGN, TIME AND SETTING： The cytological, gene transfection, in vitro, study was performed at the Laboratory of Cell Biology China Medical University from March 2006 to March 2007. MATERIALS： Three adult New Zealand rabbits were supplied by Experimental Animal Center, China Medical University. The plasmid of pcDNA3.1-hlGF-1 containing total length human IGF-1 cDNA fragment was presented by Professor Xu from Jilin University. METHODS： Subcutaneous fat from rabbit posterior neck was obtained. ADSCs were separated in vitro by coltege-1 enzyme digestion method. Cells at passage 2 （0.5×10^8/well） were incubated in a 6-well plate. When 90%-95% confluency, ADSCs were randomized into non-transfection, pcDNA3.1, and pcDNA3.1-hlGF-1 groups. The plasmid of pcDNA3.1-hlGF-1 was transfected into ADSCs by using Lipofectamine 2000. The positive cell clones were selected by G418 and cultured for 4 weeks. MAIN OUTCOME MEASURES： Expression of IGF-1 was quantified by RT-PCR and Western blot assay. Proliferation of ADSCs was determined by MTT assay and flow cytometer analysis. RESULTS： IGF-1 mRNA and protein only expressed in ADSCs transfected with pcDNA3.1-hlGF-1. M-IT assay showed that the proliferation speed of the group transfected by pcDNA3.1-hlGF-1 was faster than those of transfected by pcDNA3.1 and ADSCs without transfection. Flow cytometry demonstrated that compared with the groups of transfected by pcDNA3.1 and ADSCs without transfection, the cellular proportion in S phase of the group of transfected by pcDNA3.1-hlGF-1 was si

关 键 词：脂肪间质干细胞 胰岛素样生长因子1 基因转染 细胞周期 

分 类 号：R394.2[医药卫生—医学遗传学]