pELNS—BMPs与pELNS—Wnt3a联合构建表达对成骨细胞系成骨效能的作用研究

Osteogenesis capacity of constructing pELNS-BMPs and pELNS-Wnt3a co-expression in osteoblasts

目的构建第三代自体失活的骨形成蛋白（bonemorphogeneticprotein，BMP）-2、4、6、7、9和Wnt3a慢病毒表达载体，并整合入小鼠间质干细胞MC3T3一E1的基因组中，构建成骨诱导因子基因修饰的小鼠间质干细胞，探讨各成骨诱导因子的成骨分化能力及通过双基因联合转染提高成骨分化能力的有效性。方法以cDNA文库为模板，构建BMP．2、4、6、7、9及Wnt3a基因表达质粒载体，酶切鉴定重组体且经测序进一步确定后，与pLP／VSVG、pLPl、pLP2质粒共转染293FT细胞。包装pELNS．BMP-2、4、6、7、9及Wnt3a后转染小鼠间质干细胞MC3T3．E1细胞，GFP荧光证实转染效果。RealtimePCR检测Runx2mRNA的表达水平，鉴定各成骨诱导因子的单独转染效能。双基因联合转染（共8组）MC3T3．E1细胞，GFP荧光证实转染效果；应用ELISA检测MC3T3-E1细胞培养上清中骨钙素（bone出protein，BGP）和碱性磷酸酶（alkalinephosphatase，ALP）的表达水平；应用RealtimePCR检测Runx2mRNA的表达水平；应用Westernblot检测BMP-2、4、6、7、9及Wnt3a蛋白的表达水平，鉴定双基因联合转染的成骨分化效能。结果重组慢病毒pELNS-BMP-2、4、6、7、9及pELNS-Wnt3a构建成功，成功转染MC3T3-E1细胞；Runx2mRNA表达水平：BMP-2〉BMP4-〉BMP-9〉BMP-7〉Wnt3a〉BMP-6。双基因（共8组）联合转染MC3T3-E1细胞，GFP荧光证实转染成功；Runx2mRNA表达水平、BGP和ALP表达水平结果显示，BMP-2与BMP-7共转染成骨效率最高，Westernblot证实BMP-2和BMP-7联合转染MC3T3-E1细胞后BMP-2、4、6、7、9及Wnt3a蛋白表达升高。结论成功构建第三代慢病毒载体pELNS可将BMP．2、4、6、7、9和Wnt3a导人小鼠间质干细胞MC3T3-E1，使其有效表达；各成骨诱导因子能促进MC3T3-E1细胞向成骨细胞分化，BMP-2和BMP-7双基因联合转染能有效提高成骨转化。

Objective To construct the third-generation self-inactivating lentiviral vectors including BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a, then integrate them into the genome of mouse embryonic osteoblast cell, MC3T3-E1, and to explore the capability of osteogenic differentiation of individual bone morphogenetic control factor and the effective ap- proaches to further improve the capability of osteogenic differentiation. Methods The plasmid vectors of gene expression in- cluding BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a were constructed, which were identified through enzyme cutting and further confirmed through sequencing. After packing pELNS-BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a, mouse embryon- ic osteoblast cell, MC3T3-E1 was transfected, and the transfection efficiency was confirmed by GFP fluorescence imaging. The ex- pression level of Runx2 mRNA and the transfection efficiency of individual bone morphogenetic control factor was detected by Re- al time PCR. Eight groups of MC3T3-E1 were dual-gene co-transfected, and the transfection efficiency was verified by GFP fluores- cence imaging. ELISA was adopted to detect the expression level of BGP and ALP in MC3T3-Elcuhure supernatants; Real time PCR was adopted to detect the expression level of Runx2 mRNA; Western blot was adopted to detect the expression level of pro- tein of BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a. Thus, the effectiveness of osteogenic differentiation of dual-gene co- transfection were evaluated. Results The recombination of lentiviruses, pELNS-BMP-2, pELNS-BMP-4, pELNS-BMP-6, pELNS- BMP-7, pELNS-BMP-9 and pELNS-Wnt3a were successfully constructed. MC3T3-E1 was successfully transfected. The expres- sion levels of Runx2 mRNA were： BMP-2 〉 BMP-4 〉 BMP-9 〉 BMP-7 〉 Wnt3a 〉 BMP-6. Successful transfection of the dual-gene co-transfection of eight groups of MC3T3-E1 were verified by GFP fluorescence imaging. The expression level of Runx2 mRNA, the expression of BGP and ALP showed BMP-2 and BMP-7 co-transfection group was the most efficient in osteogenesis transfection. Western blot revealed thatthe protein expression of BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a increased after cotransfeetion of MC3T3-E1 by BMP-2 and BMP-7. Conclusion The third-generation lentiviral vector, pELNS can lead BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a into MC3T3-E1, mouse embryonic osteoblast cell, and stabilize its expression. Individual bone morphogenetie control factors can promote MC3T3-EI＇s differentiation to osteoblasts. The dual-gene co-transfeetion of BMP-2 and BMP-7 can effectively promote osteoblast conversion, which provides significant theoretical basis and technical support for remodeling of tissue engineering bone.