《Journal of Oral and Maxillofacial Surgery》 ›› 2015, Vol. 25 ›› Issue (3): 179-. doi: 10.3969/j.issn.1005-4979.2015.03.004

• Basic Scientific Study • Previous Articles     Next Articles

Osteogenic Differentiation of MC3T3-E1 Cells Induced by pBMP-2 Transfection Mediated by Nonviral Vector

LU Kai-ge, XU Ling, ZHANG Fu-qiang   

  1. Department of Prosthodontics, Ninth People’s Hospital , Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
  • Online:2015-06-28 Published:2015-12-01

非病毒载体介导pBMP-2转染MC3T3-E1细胞的体外成骨分化研究

吕凯歌,徐玲,张富强   

  1. 上海交通大学医学院附属第九人民医院口腔修复科,上海市口腔医学重点实验室,上海   200011
  • 通讯作者: 张富强,主任医师 E-mail:fredzc@online.sh.cn
  • 作者简介:吕凯歌(1980—),女,河南许昌人,住院医师,博士. E-mail:lvkaige@126.com
  • 基金资助:

    国家自然科学基金(30772431)

Abstract: 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 protein gene (pEGFP). Gene transfection conditions initially optimized by varying GenEscortTM 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 expression 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.

Key words: nonviral vector, polyethylenimine, MC3T3-E1 cells, bone morphogenetic protein-2, 0steogenic differentiation

摘要: 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。

关键词: 非病毒载体;  , 聚乙烯亚胺;  , MC3T3-E1细胞;  , 骨形态发生蛋白2;  , 成骨分化

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