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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

22卷

期数:

2016年03期

页码:

208-212

栏目:

基础医学

出版日期:

2016-05-20

文章信息/Info

Title:

Construction of a single vector in BMP-2 expression in a controlled manner and its expression in ADSCs

文章编号:

1006-8147（2016）03-0208-05

作者:

王鹏¹; 苗军²; 方成¹; 胡永成²; 陈晓鹏¹

?（1. 天津医科大学研究生院 ，天津 300070；2. 天津市天津医院骨科，天津 300211）

Author(s):

?WANG Peng¹ ;  MIAO Jun²;  FANG Cheng¹;  HU Yong-cheng²;  ZHANG Rong-xin¹

?（1.Graduate School， Tianjin Medical University, Tiamjin 300070,China; 2.Department of Orthopedics, Tianjin Hospital, Tianjin 300211,China）

关键词:

骨形态发生蛋白-2; 基因调控; 脂肪基质干细胞; 慢病毒表达载体

Keywords:

bone morphogenetic protein-2;  gene controlling;  doxycyline;  adipose derived stromal cells;  lentiviral vectors

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分类号:

R68

DOI:

-

文献标志码:

A

摘要:

目的：拟构建可调控骨形态发生蛋白（BMP-2）表达的单质粒慢病毒表达载体，为研究骨修复过程中的成骨性能打下基础。方法：通过基因重组技术获取含BMP-2的慢病毒表达载体，通过菌落PCR和基因测序鉴定；将慢病毒表达载体与包装载体pMD2.G和pSPAX在293T细胞中包装，收集病毒液，转染大鼠脂肪基质干细胞（ADSCs）。首先，从细胞形态学和MTT来初步分析诱导剂多西环素（Dox）不同浓度对ADSCs活性的影响；其次，缩小Dox浓度范围，在荧光显微镜下通过荧光表达强度判断Dox对ADSCs中基因表达的诱导效率，相同条件下，qRT-PCR分析BMP-2的mRNA的相对表达量，进一步优化Dox的诱导浓度；最后，通过western blot检测不同Dox浓度下，转染目的基因的ADSCs中BMP-2表达，Image J软件进行灰度值分析判断二者之间的剂量依赖关系。 结果：1%琼脂糖凝胶电泳证实pUC57-BMP-2经双酶切后可获得大小约1 100 bp的目的基因片段，菌落PCR和基因测序证实含目的基因的慢病毒表达载体pLVCT-BMP-2构建成功。ADSCs对Dox最大耐受浓度为6 μg/mL，Dox对ADSCs中目的基因表达的最大诱导浓度为5 μg/mL，western blot证实ADSCs中BMP-2表达与Dox浓度（0~5 μg/mL）存在剂量依赖关系。 结论：成功构建可调控BMP-2表达的单质粒慢病毒表达载体，转染ADSCs后，BMP-2的表达与Dox浓度存在剂量依赖关系。

Abstract:

Objective: To construct a single vector in BMP-2 expression in a controlled manner and investigate its expression in adipose derived stromal cells（ADSCs）. Methods: The lentiviral vector containing BMP-2 gene was constructed through genetic recombination and identified by bacterial PCR and sequence analysis. ADSCs were transfected by lenvirus collected from 293T cell. First, cell viability was analyzed through cell morphology and MTT assay at different induced concentrations of doxycyline (Dox). Second, to estimate the efficiency of the Tet-ON and determine the optimal induction concentration of Dox, the strength of fluorescence was detected. The mRNA of BMP-2 was also analyzed through qrT-PCR. Lastly, the capacity of inducing BMP-2 expression in ADSCs by Dox was investigated by western blot and densitometric analysis. Results: PLVCT-BMP-2 was successfully generated in this research by bacterial PCR and sequence analysis. The results indicated ADSCs from rat could stand up to 6 μg/mL Dox, and Dox of 5 μg/mL was finally determined as the optimal induction concentration. The densitometric analysis of BMP-2 expression showed that BMP-2 expression in ADSCs was in a dose-dependent manner. Conclusion: Lentiviral vector containing BMP-2 could be successfully generated. The conditional BMP-2 expression is regulated by Dox in a dose-dependent manner, providing the opportunity to explore BMP-2 expression and guide the bone healing process in vivo.

参考文献/References:

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相似文献/References:

备注/Memo

备注/Memo:

基金项目? 天津市自然科学基金资助项目（12JCYBJC16400，14JCTPJC00487）
作者简介 王鹏，男，硕士在读，?通信作者：胡永成，E-mail:yongchenghu@126.com。

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更新日期/Last Update: 2016-05-24