非病毒载体介导pBMP-2转染MC3T3-E1细胞的体外成骨分化研究

摘要

Objective:To study the effect of plasmids containing bone morphogenetic protein-2 (pBMP-2) transfection with GenEscortTM II on the proliferation and osteogenic differentiation of MC3T3-E1 cells. Methods:Passage 2 of the recovered cryopreserved osteoblasts MC3T3-E1 cell line were transfected with plasmids containing enhanced green fluorescent pro-tein gene (pEGFP). Gene transfection conditions initially optimized by varying GenEscort TM II/plasmid ratios were analyzed by fluorescent microscopy, and flow cytometry was used for quantitative evaluation. After MC3T3-E1 cells were transfected with pBMP-2 through nonviral vector GenEscortTM II, the proliferation of MC3T3-E1 cells was tested with MTT assay, the mineralization was investigated using alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining, and the ex-pression of mRNA for osteoblast gene markers such as ALP, bone sialoprotein (BSP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) were analyzed with real-time polymerase chain reaction (Real-time PCR) at 3-d, 6-d, and 9-d respectively. Results: Results showed that gene transfection efficiency reached up to 35.02 ± 4.42 as demonstrated by EGFP expression.The proliferation of MC3T3-E1 cells was not impacted by gene transfection (P>0.05). ALP staining and ARS staining were more intensive with pBMP-2 gene transfection. The mRNA expression of ALP, BSP, Runx2, OCN and OPN up-regulated in pBMP-2 transfection group at 6-d (P<0.05). Conclusion:pBMP-2 transfection mediated by GenEscortTM II could promote the osteogenic differentiation of MC3T3-E1 cells.%目的：探讨非病毒载体GenEscortTM II介导质粒骨形态发生蛋白2（pBMP-2）转染对MC3T3-E1细胞增殖和体外成骨分化的影响。方法：以非病毒载体GenEscortTM II介导报告基因质粒增强型绿色荧光蛋白（pEGFP）转染MC3T3-E1细胞，优化转染条件，荧光显微镜和流式细胞仪评价转染效率。 pBMP-2转染MC3T3-E1细胞后，MTT法检测细胞增殖情况，染色法检测碱性磷酸酶（ALP）和茜素红S（ARS）的表达，Real-time PCR 分析成骨细胞标志基因ALP、骨涎蛋白（BSP）、Runt 相关转录因子2（Runx2）、骨钙素（OCN）和骨桥蛋白（OPN）的表达。结果： GenEscortTM II介导pEGFP转染MC3T3-E1细胞后，转染效率达35.02±4.42，MTT结果示基因转染对细胞增殖无影响（P>0.05）。pBMP-2转染组的ALP、ARS表达较pEGFP转染组和未转染组显著，同时Real-time PCR检测结果示，转染后6 d， pEGFP转染组和未转染组相比，pBMP-2转染组的成骨基因ALP、BSP、Runx2、OCN、OPN的表达量差异有显著性意义(P<0.05)。结论：非病毒载体GenEscortTM II介导BMP-2转染MC3T3-E1细胞可诱导其向成骨方向分化。