基因转染BMSC与BGC的体外生物相容性的实验研究

目的 研究人骨形态发生蛋白-2(human bone morphogenetic protein-2,hBMP-2)基因转染的兔骨髓基质细胞(bone marrow stromal cells,BMSCs)与生物活性陶瓷(Bioactive glass ceramics,BGC)体外培养条件下的生物相容性。方法抽取成兔胫骨骨髓组织,置于含10％胎牛血清的高糖DMEM培养基中培养,脂质体法介导将hBMP-2基因转入培养的BMSCs,G418筛选,取阳性克隆,继续培养。分为实验组(转染的细胞与BGC复合),对照组(单纯转染的细胞),不同时间用倒置相差显微镜观察。MTT法进行细胞增殖测定,并进行细胞微量蛋白含量和碱性磷酸酶的定量检测。结果原位杂交证实基因转染成功,形态特征及生物学行为证实转染的细胞无致瘤性,转染的BMSCs体外培养时复合或不复合BGC均生长良好,BGC利于细胞的贴附、生长与增殖,并对细胞的功能无不良影响。结论 基因转染的BMSCs可以作为一种新型的种子细胞,BGC是较理想的骨组织工程支架材料,基因转染的BMSCs复合BGC用于骨缺损的修复,具有广阔的临床应用前景。

Biocompatibility of Gene Transfection Bone Marrow Stromal Cells with Bioactive Glass Ceramics in Vitro

Objective To study the biocompatibility of human bone morphogenetic protein-2 gene transfection bone marrow stromal cells with biodegradable polymers in vitro culture.Methods Bone marrow stromal cells were obtained from the tibia of healthy adult rabbits and cultured in Dulbecco's modified medium containing 10% fetal bovine serum.PcDNA3-hBMP2 was constructed and with the help of profectamine,the cultured above passage 2 cells were transfected with PcDNA3-BMP-2.The transfected cells were selected by G418 and the positive clone was collected and expanded.The expanded cells were then divided into BGC group(in which the cells were cultured with BGC) and transfected cells group (without BGC).The proliferation and differentiation of all the transfected cells were observed at different time point under inverted phase contrast microscope .Proliferation of transfected cells were evaluated by MTT assay,and activity of alkaline phosphatase and total micro-protein contents in these cells were quantitatively detected.Results The cultured BMSCs were proved to be successfully transfected by way of in situ hybridization and the transfected cells were proved non-neoplastigenic by the evidence of cellular morphology and behavioral biology.The transfected cells grow well in vitro ,regardless of the presence of BGC,with biological and morphological characteristics similar to those of normal transfected cells.BGC improved the adhesion,growth and proliferation of the transfected cells,showing no adverse effects on cell functions.Conclusions The gene transfected BMSCs may be a new seed cells,and BGC is an optimal scaffold material for bone tissue engineering,which may potentially find clinical application for bone defect repair.