LMP-1基因转染骨髓间充质干细胞的LIM矿化蛋白表达  

LIM mineralization protein expression in LIM mineralization protein-1 gene-transfected bone marrow mesenchymal stem cells

作　　者：金夏生[1] 王子江[1] 向川[2] 

机构地区：[1]山西医科大学第二医院骨科,山西省太原市030001 [2]山西医科大学第二医院骨关节科,山西省太原市030001

出　　处：《中国组织工程研究》2014年第23期3633-3638,共6页Chinese Journal of Tissue Engineering Research

摘　　要：背景:体外实验提示LMP-1基因可提高骨质疏松性骨髓间充质干细胞LMP-1蛋白的表达。目的:用含有LIM矿化蛋白1基因的反转录病毒载体(RV-LMP-1-GFP)转染骨质疏松性SD大鼠骨髓间充质干细胞,检测其对骨质疏松性骨髓间充质干细胞LIM矿化蛋白表达的影响。方法:随机取雌性SD大鼠12只,采用双侧卵巢切除法建立骨质疏松性大鼠模型,另取6只大鼠仅切除卵巢周围等量脂肪组织,保留卵巢为假手术组。喂养2个月后取双侧股骨、胫骨、肱骨分离培养其骨髓间充质干细胞。均取3代培养细胞随机分为去势组、LMP-1转染组(转染LMP-1基因)和假手术组。分别行RT-PCR及Western-Blot检测各组细胞LIM矿化蛋白1 mRNA和蛋白的表达。结果与结论:培养出骨质疏松性SD大鼠骨髓间充质干细胞,转染后倒置荧光显微镜下可见绿色荧光表达;3组细胞均可在基因及蛋白水平表达LIM矿化蛋白1。与假手术组及去势组比较,LMP-1基因转染组的LIM-1mRNA和蛋白的表达均升高显著(P<0.05),假手术组与去势组之间差异无显著性意义(P>0.05)。成功实现重组RV-LMP-1-GFP基因在骨质疏松性SD大鼠骨髓间充质干细胞中mRNA和蛋白水平的表达,且LMP-1的表达水平显著提高。结果表明重组LMP-1基因成功导入骨质疏松性大鼠骨髓间充质干细胞基因组中,并使得LMP-1 mRNA和蛋白的表达明显提高。BACKGROUND：In vitro experiments suggest that LIM mineralization protein-1 （LMP-1） gene can increase the expression of LMP-1 protein in osteoporotic bone marrow mesenchymal stem cells. OBJECTIVE：To investigate the LMP-1 expression in osteoporotic bone marrow mesenchymal stem cells transfected with RV-LMP-1-GFP in vitro. METHODS：Twelve SD female rats were selected and subjected to bilateral ovariectomy for establishment of osteoporosis models. After 2 months of feeding, bilateral femurs, tibiae, and humeri of rats were taken to isolate and culture bone marrow mesenchymal stem cells. Passage 3 cells were taken and randomly divided into ovariectomized group and LMP-1 transfection group. Another six rats only underwent removal of the same amount of fat tissue around the ovary, and passage 3 cells which were harvested as those in the former two groups served as sham group. RT-PCR and western blot assay were performed to determine the expression of LMP-1 protein and mRNA. RESULTS AND CONCLUSION：Under an inverted fluorescence microscope, transfected bone marrow mesenchymal stem cells from osteoporotic rats showed green fluorescent expression. The three groups al could express LMP-1 at the protein and mRNA levels. The expression levels of LMP-1 protein and mRNA in the LMP-1 transfection group were significantly higher than those in the ovariectomized group and sham group （P〈0.05）, but there was no statistical difference between the latter two groups （P〉0.05）. The successful expression of the RV-LMP-1-GFP gene in osteoporotic SD rat bone marrow mesenchymal stem cells was realized at the protein and mRNA levels;moreover, the expression of LMP-1 was increased dramatical y. These findings indicate that LMP-1 gene is successful y transferred into osteoporotic SD rat bone marrow mesenchymal stem cells, and significantly elevates the expression of LMP-1 mRNA and protein.

关 键 词：干细胞 骨髓干细胞 骨髓间充质干细胞 去势大鼠 LIM矿化蛋白1基因 反转录病毒 转染 骨质疏松 

分 类 号：R394.2[医药卫生—医学遗传学]