| LMP-1基因慢病毒载体构建及其在大鼠骨髓间充质干细胞的表达 |
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投稿时间：2013-01-17
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| 作者 | Author | 单位 | Address | E-Mail |
| 侯慧铭 |
HOU Hui-ming |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi, China |
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| 向川 |
XIANG Chuan |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi, China |
xcml7275@163.com |
| 郭丽 |
GUO Li |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi, China |
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| 张桦栋 |
ZHANG Hua-dong |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi, China |
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| 期刊信息：《中国骨伤》2013年，第26卷，第10期，第841-844页 |
| DOI：10.3969/j.issn.1003-0034.2013.10.012 |
| 基金项目:山西省自然科学基金资助项目(编号:2010011050-2) |
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中文摘要:
目的: 构建人LMP-1重组慢病毒载体,体外转染大鼠骨髓间充质干细胞,检测LMP-1基因在大鼠骨髓间充质干细胞的表达.
方法: 利用PCR法从cDNA文库中钓取LMP-1基因,将其与经AgeⅠ酶切线性化的慢病毒载体pGC-FU-EGFP相连接,转化感受态大肠杆菌,筛选出阳性克隆pGC-FU-LMP-1-EGFP,基因测序对其鉴定. 经293T细胞包装后,收集富含病毒颗粒LV-LMP-1-EGFP的细胞上清,浓缩并标定滴度,RT-PCR检测并鉴定. 以最佳MOI值体外转染大鼠骨髓间充质干细胞,荧光显微镜观察转染是否成功,流式细胞仪检测转染效率,RT-PCR和Western blot检测转染细胞LMP-1基因的表达.
结果: ①基因测序及RT-PCR检测证实携带人LMP-1基因的慢病毒载体构建成功,包装后获得滴度为2×108 TU/ml的LV-LMP-1-EGFP.②以MOI=100转染大鼠骨髓间充质干细胞,荧光显微镜下可见大量绿色荧光蛋白表达,转染效率可达93.5%,经RT-PCR与Western blot检测,被转染细胞内有LMP-1基因表达.
结论: 成功构建携带人LMP-1基因的慢病毒载体,可高效转染大鼠骨髓间充质干细胞,被转染细胞可高效表达LMP-1基因. |
| 【关键词】慢病毒 LMP-1 骨髓间充质干细胞 骨质疏松 |
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| Construction of lentivirus vector containing human LIM mineralization protein-1(LMP-1)and its expression in rat bone mesenchymal stem cells |
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ABSTRACT
Objective: To construct a recombiant lentivirus vector of human LMP-1 and detect the expression of LMP-1 in infected rat bone mesenchymal stem cells.
Methods: LMP-1 gene from the cDNA library were extracted by Polymerase Chain Reaction (PCR). The LMP-1 genes were connected into lentiviral vectors pGC-FU-EGFP which was linearized by AgeⅠenzyme to produce recombiant lentivirus vector called as pGC-FU-LMP-1-EGFP,then packaged by 293T cells. The virus supernant congtaining LV-LMP-1-EGFP was harvested,concentrated and titrated. The rat BMSCs were transfected with recombiant lentivirus LV-LMP-1-EGFP at the most appropriate MOI. The mRNA and protein expression of LMP-1 were detected by RT-PCR and Western blot.
Results: ①LV-LMP-1-EGFP was recombined successfully and the titer reached 2×108 TU/ml. ②The efficiency of infection was 93.5%,which was get after LV-LMP-1-EGFP infected rat BMSCs at the most appropriate MOI=100. The expression of LMP-1 gene was confirmed by RT-PCR and Western blot.
Conclusion: Lentivirus vector containing human LMP-1 gene is constructed successfully,which can transfected efficiently into rat BMSCs,and the infected rat BMSCs can effectively express LMP-1. |
| KEY WORDS Lentivirus LMP-1 Bone mesenehymal stem cells Osteoporosis |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 侯慧铭,向川,郭丽,张桦栋.LMP-1基因慢病毒载体构建及其在大鼠骨髓间充质干细胞的表达[J].中国骨伤,2013,26(10):841~844 |
| 英文格式: | HOU Hui-ming,XIANG Chuan,GUO Li,ZHANG Hua-dong.Construction of lentivirus vector containing human LIM mineralization protein-1(LMP-1)and its expression in rat bone mesenchymal stem cells[J].zhongguo gu shang / China J Orthop Trauma ,2013,26(10):841~844 |
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