| LMP-1基因慢病毒重组体对大鼠骨髓间充质干细胞的增殖影响及其表达 |
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Received:April 25, 2013
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| 作者 | Author | 单位 | Unit | E-Mail |
| 梁长生 |
LIANG Chang-sheng |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital Affiliated to 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 Affiliated to Shanxi Medical University, Taiyuan 030001, Shanxi, China |
xcml7275@163.com |
| 魏增永 |
WEI Zeng-yong |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital Affiliated to Shanxi Medical University, Taiyuan 030001, Shanxi, China |
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| 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 Affiliated to Shanxi Medical University, Taiyuan 030001, Shanxi, China |
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| 秦迎泽 |
QIN Ying-ze |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital Affiliated to Shanxi Medical University, Taiyuan 030001, Shanxi, China |
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| 卫小春 |
WEI Xiao-chun |
山西医科大学第二医院骨科, 山西 太原 030001 |
Department of Orthopaedics, the Second Hospital Affiliated to Shanxi Medical University, Taiyuan 030001, Shanxi, China |
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| 期刊信息：《中国骨伤》2013年26卷，第12期，第1023-1027页 |
| DOI：10.3969/j.issn.1003-0034.2013.12.012 |
| 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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目的：探讨LMP-1重组慢病毒载体体外转染大鼠骨髓间充质干细胞（BMSC）的方法，并检测LMP-1基因对BMSC增殖能力的影响及表达.
方法：选取清洁级4周龄SD大鼠6只（雌雄不限），无菌条件下提取骨髓间充质干细胞并培养至第3代，设立空白对照组（未经特殊处理，只有第3代骨髓间充质干细胞）、慢病毒载体转染组（在未经特殊处理的第3代骨髓间充质干细胞中加入PGC-FU-GFP及转染试剂Polybrene）和重组基因转染组（在未经特殊处理的第3代骨髓间充质干细胞中加入PGC-FU-LMP-1-GFP及转染试剂Polybrene）进行转染.转染48 h后，通过免疫荧光显微镜观察荧光表达，流式细胞仪检测慢病毒的转染效率，采用MTT 法评价慢病毒转染对 BMSC 增殖的影响；Western Blot检测转染后基因的表达情况.
结果：①成功培养出第3代SD大鼠BMSC，以100的感染复数（MOI）转染，48 h后免疫荧光显微镜下可见大量绿色荧光蛋白表达，转染效率达67%;② 不同时间点慢病毒载体转染组和重组基因转染组，与空白对照组细胞增殖比较差异无统计学意义；③Western Blot 检测示重组基因转染组72 kDa处有条特征带，其大小与LMP-1融合蛋白（～50 kDa+28 kDa=78 kDa）基本吻合.
结论：经LMP-1基因慢病毒重组体转染大鼠骨髓间充质干细胞对其增殖活力没有影响并可有效表达LMP-1. |
| [关键词]:基因组,病毒 骨髓细胞 细胞增殖 转染 |
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| Effects of recombinant gene lentivirus containing LIM mineralization protein-1 on proliferation effect and expression of bone marrow mesenchymal stem cells in rats |
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Abstract:
Objective: To explore method of recombinant gene lentivirus containing LIM mineralization protein-1(LMP-1) in transfecting bone marrow mesenchymal stem cells(BMSC),and to observe the effect of gene LMP-1 on proliferation effect and expression of BMSC.
Methods: Six clean SD rats aged 4 weeks were selected,bone marrow mesenchymal stem cells were extracted under sterile conditions and cultured to the third generation,then divided into three groups:control group(the third generation of BMSC),lentiviral vector transfection group (PGC-FU-GFP and Polybrene were injected into the third generation of BMSC) and recombinant gene transfection group (PGC-FU-LMP-1-GFP and Polybrene transfection were injected into the third generation of BMSC). After 48 hours'transfection,fluorescent expression were detected under immuno-fluorescence microscopy;lentiviral transfection efficiency were detected by flow cytometry;effect of lentiviral transfection on BMSC were evaluated by MTT;gene expression of transfected cells were determined by Western Blot.
Results: ①The third generation of BMSC was cultured successfully,and transfected with MOI:100. After 48 hours,green fluorescent expression were detected and transfection efficiency was 67% under immuno-fluorescence microscopy;②Compared to control group,there were no statistical differences between control group and other two groups;③Western blot teast showed that 72KDa specific band was observed in recombinant gene transfection group and its sizewas similar to LMP-1 fusion protein(50 kDa+28 kDa=78 kDa).
Conclusion: There is no effect of recombinant gene lentivirus containing LIM on BMSC,and can effectively influence the expression of LMP-1. |
| KEYWORDS:Genome, viral Bone marrow cells Cell proliferation Transfection |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 梁长生,向川,魏增永,侯慧铭,秦迎泽,卫小春.LMP-1基因慢病毒重组体对大鼠骨髓间充质干细胞的增殖影响及其表达[J].中国骨伤,2013,26(12):1023~1027 |
| 英文格式: | LIANG Chang-sheng,XIANG Chuan,WEI Zeng-yong,HOU Hui-ming,QIN Ying-ze,WEI Xiao-chun.Effects of recombinant gene lentivirus containing LIM mineralization protein-1 on proliferation effect and expression of bone marrow mesenchymal stem cells in rats[J].zhongguo gu shang / China J Orthop Trauma ,2013,26(12):1023~1027 |
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