hBMP-2基因改良修饰骨髓间充质干细胞和人脐血间充质干细胞研究

目的研究hBMP-2基因改良修饰骨髓和人脐血间充质干细胞的方法，并比较修饰结果。 方法联合应用密度梯度离心法和贴壁培养法分离、培养骨髓和人脐血间充质干细胞，通过高效非脂质体试剂X-treme GENE介导重组hBMP-2质粒转染骨髓和人脐血间充质干细胞，倒置荧光显微镜检测荧光强度并计算转染效率，qPCR检测hBMP-2及软骨连接蛋白在两种细胞中的表达，免疫组化检测细胞中Ⅱ型胶原的变化。 结果成功分离、培养出骨髓和人脐血间充质干细胞，两种细胞具有不同的生长特性。高效非脂质体试剂介导的重组hBMP-2质粒成功转染骨髓和人脐血间充质干细胞，转染骨髓间充质干细胞效率[（18.44±5.94）%]低于人脐血间充质干细胞[（27.74±7.59）%]，且差异有统计学意义（t=3.027，P<0.05）。转染后的两种细胞均检测到hBMP-2、软骨连接蛋白及Ⅱ型胶原的表达。 结论hBMP-2基因可以有效改良修饰骨髓和人脐血间充质干细胞，且能促进其向软骨细胞方向分化。

Research on bone marrow derived-mesenchymal stem cells and human umbilical cord blood derived-mesenchymal stem cells modified with the gene of human bone morphogenetic protein-2

ObjectiveTo study the modification of bone marrow and human umbilical cord blood mesenchymal stem cells with hBMP-2 gene and compare the modification results. MethodsBone marrow and human umbilical cord blood mesenchymal stem cells were separated and cultured by density gradient centrifugation and adherent culture. The recombinant hBMP-2 plasmid was transfected into bone marrow and human umbilical cord blood mesenchymal stem cells by high-performance non-liposome reagent X-treme GENE. The fluorescence intensity was measured by inverted fluorescence microscopy and the transfection efficiency was calculated. The hBMP was detected by qPCR. The expression of-2 and chondronectin in two kinds of cells was detected by immunohistochemistry. ResultsBone marrow and human umbilical cord blood mesenchymal stem cells were successfully isolated and cultured. The two cells had different growth characteristics. The recombinant hBMP-2 plasmid mediated by high-performance non-liposome reagent was successfully transfected into bone marrow and human umbilical cord blood mesenchymal stem cells. The efficiency of transfection of bone marrow mesenchymal stem cells (18.44±5.94)% was lower than that of human umbilical cord blood mesenchymal stem cells (27.74±7.59)%, and the difference was statistically significant (t=3.027, P<0.05). The expression of hBMP-2, cartilage connectin and collagen type II were detected in both transfected cells. ConclusionhBMP-2 gene can effectively modify bone marrow and human umbilical cord blood mesenchymal stem cells and promote chondrocyte differentiation.