Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究

doi:10.13418/j.issn.1001-165x.2015.03.017

中国临床解剖学杂志 ›› 2015, Vol. 33 ›› Issue (3): 311-315.doi: 10.13418/j.issn.1001-165x.2015.03.017

Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究

李庆庆¹，　王大平²，　熊建义²，　黄江鸿²，　付志杰²

1. 浙江医院骨一科，杭州 310013; 2.深圳市第二人民医院骨科，深圳 518035

收稿日期:2014-07-09 出版日期:2015-05-25 发布日期:2015-07-24
通讯作者: 王大平，教授，主任医师，博士生导师， E-mail：dapingwang1963@qq.com
作者简介:李庆庆（1987-），男，浙江杭州人，住院医师，硕士，主要从事骨科材料方面研究，Tel：（0571）81595169，E-mail：wwwbchain@126.com
基金资助:
广东省自然科学基金(s2012010008129); 深圳市战略新兴产业发展专项资金项目（ZDSY20120614154551201); 深圳市科技研发资金项目（CXZZ20120614160234842）; 深圳市科技研发资金项目（JCYJ20130401113330839）; 深圳市科技研发资金项目（CXZZ2013032115 2713220）; 深圳市重点实验室提升项目（CXB201104220049A）; 广东省大学生创新训练项目（1056013089）

Research of lentiviral-mediated Runx2 transferred into bone marrow mesenchymal stem cells promoting osteogenic differentiation

LI Qing-qing¹, WANG Da-ping², XIONG Jian-yi², HUANG Jiang-hong², FU Zhi-jie²

1.Department of Orthopaedics, Zhejiang Hospital, Hangzhou 310013, China; 2. Department of Orthopaedics, Shenzhen Second People`s Hospital , Shenzhen 518035, China

Received:2014-07-09 Online:2015-05-25 Published:2015-07-24

摘要/Abstract

摘要：

目的　利用重组Runx2基因的慢病毒感染大鼠骨髓间充质干细胞（BMSCs），使其Runx2基因的表达水平提高，观察BMSCs成骨相关基因的表达情况，研究Runx2基因促进BMSCs的成骨分化情况。方法　PCR扩增Runx2基因，并连接到慢病毒表达载体质粒Pez-lv201，与包装质粒共转染293T细胞进行包装，测得慢病毒液滴度。取4周龄SD大鼠胫骨，分离、培养BMSCs，流式细胞仪鉴定。将Runx2重组慢病毒感染BMSCs。显微镜下观察细胞形态变化；RT-PCR分析BMSCs成骨基因的表达情况。结果　Runx2基因重组慢病毒表达载体质粒酶切和测序鉴定正确。测得慢病毒液滴度为1.6×109 TU/ml。流式细胞仪检测表面抗原CD90和CD105，表达率为99.8%、99.3%。重组慢病毒感染后实验组细胞形态呈成骨样细胞改变；对照组未见明显变化。实验组Runx2、OCN、osteonectin、ALP、BMP-2、OPN基因的表达水平随时间推移而增高；对照组上述基因均无明显表达。结论　利用Runx2重组慢病毒感染BMSCs，使其高表达Runx2基因，可以使OCN、osteonectin、ALP、BMP-2、OPN基因表达增强，说明Runx2基因可以促进BMSCs向成骨方向分化。

关键词: , 组织工程, Runx2, 骨髓间充质干细胞, 慢病毒载体

Abstract:

Objective To obtain the recombinant BMSCs with the reprogramming Runx2 gene mediated by lentiviral vectors, and detect the levels of the osteogenesis-related genes expression, in order to elucidate that Runx2 is able to promote BMSCs' differentiation. Methods Runx2 genes were amplified by RT-PCR and linked to the lentivirus vector, then transfected 293T cells together with packing plasmids. Lentivirus titer was identified. BMSCs were obtained from 4 weeks old SD rats' tibia bone marrow and isolated and were cultured. Analysis of cell surface markers CD90 and CD105 was performed by flow cytometry to confirm the cultured cells were BMSCs, which were finally transfected by Runx2 recombinant lentivirus. The expression of the osteogenic-related gene was studied through RT-PCR. Results Runx2 gene linked with expression vector, was confirmed through enzyme digestion and sequencing. The virus tilter was 1.6×109 TU/ml. The expression percentage of CD90 and CD105 was 99.8%and 99.3%, indicating the successful culture of BMSCs. After being infected with lentivirus, cells of BMSCs-Runx2 became osteogenous. The control BMSCs stayed the same as pre-infected. The expression of Runx2, OCN, osteoectin, ALP, BMP-2, OPN increased over time and achieved the highest on day 14 in experimental groups, while the control group didn't show the expression, verifying that Runx2 is able to promote BMSCs' differentiation.Conclusion Lentiviral-mediated Runx2, which was transfered into bone marrow mesenchymal stem cells, could promote the osteogenic differentiation.

Key words: Tissue engineering, Runx2, Bone mesenchymal stem cells, Lentivirus

李庆庆,王大平,熊建义,黄江鸿,付志杰. Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究[J]. 中国临床解剖学杂志, 2015, 33(3): 311-315.

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Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究

Research of lentiviral-mediated Runx2 transferred into bone marrow mesenchymal stem cells promoting osteogenic differentiation

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