慢病毒介导骨形态发生蛋白-7基因的干细胞用于组织工程骨构建的实验研究

目的 建立能够稳定表达骨形态发生蛋白-7(BMP-7)的骨髓基质干细胞(BMSCs),观察其成骨分化,并与纳米羟基磷灰石胶原(nHAC)材料复合培养体外构建组织工程骨的可行性.方法 实验分4组:BMP-7和eGFI基因转染组(A组)、eGFP基因转染组(B组)、常规成骨诱导组(C组)、未转染组(D组).用G418筛选获得阳性细胞后接种于nHAC支架体外复合培养.荧光显微镜下观察eGFP表达,判断转染效率;以逆转录-聚合酶链反应(RTPCR)、ELISA检测目的基因表达,四唑盐(MTT)实验检测细胞活力,碱性磷酸酶(ALP)活性和Gomori染色榆测成骨情况;扫描电镜观察细胞在支架材料中的黏附、生长状况.结果 慢病毒24 h对BMSCs的转染率约为90%.RTPCR检测到A组在1300 bp处出现特异性条带,其他组结果阴性;ELISA检测24 h BMP-7蛋白含量为(150.2±18.3)pg/mL,种植支架复合培养8周后BMP-7含量为(76.6±7.4)pg/mL;MTT实验显示细胞活性与未转染组比较差异无统计学意义(P>0.05);ALP活性以16 d最强;扫描电镜见细胞种植nHAC支架后黏附、生长良好.结论 BMP-7可在BMSCs内稳定表达,并诱导其向成骨分化;与nHAC复合培养可构建组织工程骨.

Bone marrow stromal cells transferred with lentiviral-mediated bone morphogenetic protein-7 gene cultured in vitro in nano-hydroxyapatite/collagen scaffold

Objective To observe osteogenous differentiation of modified bone marrow stromal cells (BMSCs)which can express bone morphogenetic protein-7(BMP-7)stably and are cultured in vitro in the scaffold of nanohvdroxyapatite/collagen(nHAC)material. Methods BMP-7 and eGFP genes were transferred through lentiviruses into the BMSCs of SD rats.The modified BMSCs were selected out by G418 and combined with the nHAC scaffold to culture in vitro.The transfection efficiency and proliferation of BMSCs were evaluated bv fluorescence microscopy and MTT method after transfection with LVBMP-7+eGFP gene.The expression of BMP-7 was tested by RTPCR and ELISA.ALP testing and staining of Gomori were used to test osteogenous differentiation.The adhesion and growth of BMSCs on the nHAC scaffold were observed by scanning electronic microscopy(SEM).Results The transfection efficiency was about 90%in the first 24 hours.There was no significant difference in proliferation between the infected and non-infected BMSCs(P>0.05).RTPCR suggested that BMP-7 gene was located at 1 300 bp in the transfeetion group hut not in the other grouDs.ELISA revealed that the BMP-7 expression was(150.2±18.3)pg/mL The strongest AIP activity was observed nearlv on 16th dav.The adhesion and growth were fine on the nHAC scaffold.ConclusionsBMP-7 gene can be stably expressed in BMSCs,and Can enhance the capacity of osteogenous differentiation.The BMSCs modified by BMP-7 can be used in combination with nHAC to construct a tissue engineered bone.