慢病毒介导的BMP-2基因转染大鼠BMSCs及对BMSCs增殖的影响  

A Study of Transfecting Lentivirus Mediated BMP-2 into Murine BMSCs and Its Effect on the Proliferation of BMSCs

作　　者：严丹红[1] 姚骅珊[1] 方月琴[1] 王远鹏 马文慧 李保根 WANG Yuanpeng;MA Wenhui;LI Baogen(College of Medical Science and Technology,Suzhou Chien-Shiung Institute of Technology,Taicang 215411,China)

机构地区：[1]苏州健雄职业技术学院医药科技学院,太仓215411

出　　处：《江苏工程职业技术学院学报》2022年第2期21-25,共5页Journal of Jiangsu College of Engineering and Technology

基　　金：江苏省高校自然科学研究面上项目(编号:19KJB310017);苏州健雄职业技术学院基金项目(编号:2019YBZK001);2021年江苏高校“青蓝工程”优秀青年骨干教师培养对象资助项目。

摘　　要：为考察BMP-2(骨形态发生蛋白2,Bone Morphogenetic Protein 2,简称BMP-2)基因转染大鼠BMSCs(骨髓间充质干细胞,Bone Mesenchymal Stem Cells,简称BMSCs)目的基因表达及对BMSCs增殖的影响,通过实验将BMSCs分离培养并进行扩增,构建携带含有BMP-2基因的重组慢病毒表达载体,转染至BMSCs,利用倒置显微镜观察转染后绿色荧光蛋白基因的表达,通过RT-PCR(反转录聚合酶链反应,Reverse Transcription-Polymerase Chain Reaction,简称RT-PCR)检测转染后细胞BMP-2的表达水平,采用蛋白质印迹法(Western Blotting)分析BMP-2在细胞内的表达情况,使用ELISA(酶联免疫吸附测定,Enzyme Linked Immunosorbent Assay,简称ELISA)测定细胞培养上清液中目的蛋白质量浓度,细胞毒性实验MTT(噻唑蓝3-(4,5)-dimethylthi-ahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,简称MTT)法测定BMSCs增殖能力。结果表明,实验成功构建了含有BMP-2基因的重组慢病毒表达载体,倒置荧光显微镜下观察到转染BMSCs发出稳定的绿色荧光信号,转染细胞的BMP-2 mRNA(Messenger RNA,信使核糖核酸)表达水平明显高于未转染细胞,转染细胞裂解液中检测出BMP-2条带,转染细胞的培养上清液中BMP-2值明显高于未转染细胞的。转染BMSCs的增殖速度显著高于空载体BMSCs和未转染BMSCs。To study both the target gene expression of murine bone marrow mesenchymal stem cells(BMSCs)into which bone morphogenetic protein 2(BMP-2) was transfected and the effect of the transfection on the proliferation of BMSCs,a recombinant lentiviral expression vector carrying BMP-2 gene was constructed and then transfected into BMSCs which were cultured separately in the experiment and expanded. The expression of green fluorescent protein after the transfection was observed with an inverted microscopy. The expression levels of BMP-2in the transfected cells were measured by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and its expression conditions were analyzed by Western Blotting. Enzyme Linked Immunosorbent Assay(ELISA) was used to determine the concentration of the target protein in the cell culture supernatant, and Cytotoxicity test MTT(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide)to determine the proliferative capacity of BMSCs. The experimental results showed that the recombinant lentiviral expression vector carrying BMP-2 gene was successfully constructed;a stable green fluorescent signal from transfected BMSCs was observed under the inverted microscopy;the expression levels of BMP-2 mRNA(Messenger RNA) were obviously higher in transfected cells than in non-transfected ones;So were the values of BMP-2 in the culture supernatant of transfected cells;BMP-2 antibody protein expression was high in transfected BMSCs whose proliferation rate was significantly higher than that of non-transfected BMSCs and BMSCs with empty vector.

关 键 词：骨髓间充质干细胞 细胞转染 细胞增殖 慢病毒 

分 类 号：R684[医药卫生—骨科学]