非病毒载体介导pBMP-2转染MC3T3-E1细胞的体外成骨分化研究

doi:10.3969/j.issn.1005-4979.2015.03.004

《口腔颌面外科杂志》 ›› 2015, Vol. 25 ›› Issue (3): 179-. doi: 10.3969/j.issn.1005-4979.2015.03.004

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

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

出版日期:2015-06-28 发布日期:2015-12-01
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)

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

LU Kai-ge, XU Ling, ZHANG Fu-qiang

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

摘要/Abstract

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

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

中图分类号:

R782

吕凯歌，徐玲，张富强. 非病毒载体介导pBMP-2转染MC3T3-E1细胞的体外成骨分化研究[J]. 《口腔颌面外科杂志》, 2015, 25(3): 179-.

LU Kai-ge, XU Ling, ZHANG Fu-qiang. Osteogenic Differentiation of MC3T3-E1 Cells Induced by pBMP-2 Transfection Mediated by Nonviral Vector[J]. 《Journal of Oral and Maxillofacial Surgery》, 2015, 25(3): 179-.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2015/V25/I3/179

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非病毒载体介导pBMP-2转染MC3T3-E1细胞的体外成骨分化研究

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非病毒载体介导pBMP-2转染MC3T3-E1细胞的体外成骨分化研究

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

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