聚乙烯亚胺介导骨形态发生蛋白2基因转染兔脂肪干细胞的研究

Osteoblastic differentiation of rabbit adipose-derived stem cells transfected by polyethylenimine-me diated bone morphogenetic protein-2 gene in vitro

目的 观察聚乙烯亚胺作为载体介导骨形态发生蛋白-2（BMP-2）基因转染脂肪干细胞（ADSCs）的基因表达,探讨转染后细胞的成骨分化效果。方法 取成年日本大耳白兔的颈部脂肪10 g,经体外分离培养的方法获得ADSCs,并对其进行表型鉴定,用相对分子质量为13×10-3的聚乙烯亚胺介导BMP-2基因转染至ADSCs,G418筛选获得阳性细胞克隆。逆转录-聚合酶链反应（RT-PCR）检测BMP-2 mRNA表达,酶联免疫吸附试验（ELISA）法检测BMP-2蛋白表达分泌,Western blot法检测骨钙素和I型胶原蛋白的含量,碱性磷酸酶定量检测,并行茜素红染色,未转染组作对照。结果 从兔脂肪组织中分离出来的第3、6代ADSCs均高表达CD29、CD44,阳性率超过90%,低表达CD34、CD45,阳性率不超过5%,转染细胞有BMP-2 mRNA的转录及其蛋白的表达,碱性磷酸酶、骨钙素和I型胶原蛋白的含量增加,茜素红染色呈阳性反应,对照组呈阴性。结论 聚乙烯亚胺可介导BMP-2基因转染兔ADSCs,BMP-2基因在ADSCs中有效表达并诱导其定向成骨分化。

Objective To investigate the expression of human bone morphogenetic protein-2 （BMP-2） gene transfected to rabbit adipose-derived stem cells （ADSCs） mediated by polyethylenimine and assess the effect of osteoblastic differentiation of the cells. Methods A total of 10 g adipose tissue was collected from the neck of adult Japanese white rabbits, and after isolation and culture in vitro ADSCs were obtained, and identified phenotypically. BMP-2 was transfected into rabbit ADSCs mediated by polyethyl- enimine （ 13 x 10^3 ）. Positive cell clones were selected with G-418. The expression of BMP-2 mRNA and protein was detected by reverse transcription-polymerase chain reaction （RT-PCR） and enzyme-linked im- munosorbent assay （ELISA） respectively. The expression of osteocalcin and collagen I was detected by Western blotting. Alkaline phosphatase was detected with ALP kit, and stained with alizarin red staining. The untransfeeted groups served as the control groups. Results ADSCs were successfully isolated from rab- bit adipose tissue. ADSCs at passage 3 and 6 highly expressed CD29 and CD44 （the positive rate being more than 90% ） and low expressed CD34 and CD45 （the positive rate being less than 5% ）. The expression of BMP-2 mRNA and protein was detectable and that of alkaline phosphatase, osteocalcin and collagen I increased in transfected ADSCs, and Alizarin red staining was postive. Conclusion It is feasible that polyethylenimine-mediated BMP-2 gene can be transfected into rabbit ADSCs. BMP-2 gene was effectively expressed in ADSCs and induce directional osteogenic differentiation of ADSCs.