上海口腔医学

上海口腔医学 ›› 2016, Vol. 25 ›› Issue (5): 522-526.

• 论著 • 上一篇    下一篇

冻干对BMP-2基因重组慢病毒载体生物学效应的影响

韦晓玲1, 刘道杨2, 潘杰3, 陆珮珺4, 赵君4   

  1. 1.上海市口腔病防治院·上海市口腔医院 牙体牙髓病科,上海 200001；
    2.南京医科大学口腔医院 口腔颌面外科,江苏 南京 210029；
    3.上海市口腔病防治院·上海市口腔医院 口腔正畸科,上海 200001；
    4.上海交通大学医学院附属第九人民医院·口腔医学院 口腔正畸科,上海 200011
  • 收稿日期:2015-12-07 修回日期:2016-03-06 出版日期:2016-10-25 发布日期:2016-11-10
  • 通讯作者: 赵君, E-mail: yuzj_260@hotmail.com
  • 作者简介:韦晓玲（1981-）,女,硕士,主治医师,E-mail:sammi510@126.com
  • 基金资助:
    国家自然科学基金（81200815）

Effects of lyophilized on the biological activities of lentiviral vector of bone morphogenetic protein 2

WEI Xiao-ling1, LIU Dao-yang2, PAN Jie3, LU Pei-jun4, ZHAO Jun4   

  1. 1.Department of Endodontics, Shanghai Stomatological Disease Center, Shanghai Stomatological Disease Hospital. Shanghai 200001;
    2.Department of Oral and Maxillofacial Surgery, Affiliated Stomatological Hospital of Nanjing Medical University.Nanjing 210029, Jiangsu Province;
    3.Department of Orthodontics,Shanghai Stomatological Disease Center, Shanghai Stomatological Disease Hospital. Shanghai 200001;
    4.Department of Orthodontics, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine. Shanghai 200011, China
  • Received:2015-12-07 Revised:2016-03-06 Online:2016-10-25 Published:2016-11-10

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摘要： 目的研究冻干对BMP-2重组慢病毒载体（Lenti-BMP-2）生物学效应的影响。方法利用基因重组技术构建lenti-BMP-2。以感染复数（multiplicity of infection,MOI）10、25、50、100、200 转染大鼠骨髓基质细胞（BMSCs）,X-gal染色确定最佳MOI值。在适宜条件下,将lenti-BMP-2与10%海藻糖配比的冻干保护剂混合制成冻干剂型。采用MTS试剂盒观察冻干前、后lenti-BMP-2对BMSCs增殖的影响。应用ELISA法检测冻干lenti-BMP-2转染大鼠BMSCs后细胞中BMP-2蛋白的表达变化,实时定量PCR检测冻干对lenti-BMP-2转染BMSCs后成骨标志物Runx-2、Col1、OCN、OPN表达水平的影响。采用SPSS13.0软件包对数据进行统计学分析。结果X-gal染色显示,MOI为100 pfu/细胞时,转染效率趋于稳定,冻干前、后lenti-BMP-2对BMSCs增殖无显著影响（P>0.05）。ELISA法检测显示,利用冻干后的lenti-BMP-2转染BMSCs,可持续稳定地分泌BMP-2蛋白,冻干不会影响BMP -2蛋白的分泌活性。实时定量PCR检测结果显示,冻干前和冻干后组被转染的BMSCs细胞中,Runx-2、Col1、OCN、OPN表达水平无显著差异,说明冻干不会影响BMP-2促进成骨标志物的表达（P>0.05）。结论海藻糖冻干剂型的lenti-BMP-2能够长期保持lenti-BMP-2的高生物活性,是一种有效可靠的储存方法。

关键词: BMP-2, 重组慢病毒, 冷冻干燥, 生物学活性

Abstract: PURPOSE: To investigate the influence of lyophilization on the biological activity of recombinant lentiviral vectors of bone morphogenetic protein 2(BMP-2). METHODS: Recombinant lenti-BMP-2 was constructed. lenti-BMP-2 was transfected with rat bone marrow stromal cells (BMSCs) by multiplicity infection (MOI) of 10, 25, 50, 100, 200. The infection efficiency was observed by X-gal staining. Under suitable conditions, the lenti-BMP-2 and 10% trehalose ratio of lyophilized protective agent was mixed into the lyophilization form. Before and after lyophilization, the effect of lenti-BMP-2 on the proliferation of BMSCs was evaluated by MTS assay. The expression of BMP-2 protein in the cells of lyophilized lenti-BMP-2 was detected by ELISA method. The expression of Runx-2, OCN, Col1 and OPN in BMSCs was detected by real-time PCR after transfection of lyophilized lenti-BMP-2. SPSS13.0 software package was used for statistical analysis. RESULTS: X-gal staining showed an MOI of 100 pfu/cell, and stable transfection efficiency. Before and after lyophilization, no significant change was observed in regard to the effect of lyophilized lenti-BMP-2 on BMSCs proliferation (P>0.05). ELISA method showed that BMSCs transfected by lyophilized lenti-BMP-2 could express BMP-2 protein continuously and stably at a high level. Before and after lyophilization, the result of real-time PCR showed that no significant difference in the expression of OPN,Col1,OCN and Runx-2 in BMSCs (P>0.05). CONCLUSIONS: Lyophilized lenti-BMP-2 with trehalose can maintain high activity for a long time as an effective and reliable storage method.

Key words: Bone morphogenetic protein 2, Lentiviral vector, Lyophilization, Biological activities

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