摘要
【目的】快速构建大鼠LIM矿化蛋白-1基因重组腺病毒载体,转染MSC,探讨LMP-1基因对MSC成骨分化和成骨活性的影响。【方法】从大鼠成骨细胞内提取总RNA,并设计LMP-1特异性引物进行RT-PCR,获取LMP-1基因后利用Invitrogen公司的TOPO定向克隆技术创建入门克隆pENTR/D-LMP-1。转化TOP10细菌后,挑取阳性克隆摇菌提取质粒,入门克隆再与表达载体pAD/CMV/V5-DEST进行同源重组反应得到病毒载体pAD/CMV/V5-LMP-1,转化细菌后挑选阳性克隆摇菌提取重组病毒质粒,用PacI内切酶线性化后用转染293A细胞后得到重组腺病毒载体。以腺病毒为载体,将大鼠LMP-1基因体外转染于3代大鼠MSC细胞内,检测LMP-1基因在MSC细胞的表达,分别观察转染后实验组与对照组I型胶原、碱性磷酸酶、骨钙素表达变化以及骨钙结节的形成情况,评价LMP-1基因的成骨能力。【结果】成功获取大鼠LMP-1基因,入门质粒与病毒表达质粒经过酶切鉴定以及测序验证。LMP-1基因能在MSC中高效表达,转然后MSC的I型胶原,碱性磷酸酶、骨钙素的表达明显增强,骨钙结节形成增多。【结论】利用Gateway技术构建LMP-1重组腺病毒载体相对简单,可快捷获得的pAd-LMP-1。pAd-LMP-1转染MSC后,LMP-1基因能促进MSC向成骨细胞分化,增强其成骨作用。
【Objective】 This study was designed to construct a recombinant adenovirus vector contains LMP-1 gene,and investigate the osteoinductive activity of MSC which were transfected recombinated adenoviral vector carrying LMP-1 gene. 【Methods】 Total RNA was extracted from rat osteoblast and the LMP-1 gene was acquired by RT-PCR, the LMP-1 gene and entry vector pENTR / D-TOPO were used to create the entry clone with the directional TOPO clone technology, then the entry clone and the expression vector were used to create the expression clone through the LR recombination reaction. The adenovirus expression clone was linearized by PacI and transfected to the 293A cell line to harvest a high titer. Ad-LMP-1 was infected into the 3rd passage MSC, the expression of LMP-1 was detected by Western blot. The osteogenic activity of MSC was evaluated by the expression of collagen I, ALP, osteocalcin and the formation of bone nodule. 【Result】 The LMP-1 gene was successfully acquired and confirmed, the entry clone and the expression clone were both verified by enzymes digestion, and the expression clone was further confirmed by sequenced. The expression of LMP-1 was detected successfully in MSC. The increasing expression of collagen I, osteocalcin, ALP and bone nodule were observed by comparing to the control group. 【Conclusion】 Gateway technology not only make construction of the pAd-LMP-1 recombination adenovirus vector simple and fast, but also get a high transfection efficacy in MSC. LMP-1 gene can induce the osteoblast differention of MSCs, and improve its osteogenic activity. The adenovirus vector is reliable to be used in further gene therapy research.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2010年第2期199-206,共8页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学基金(5001761)
2005年国家教育部留学回国人员科研启动基金
关键词
LIM-1矿化蛋白
间充质干细胞
成骨活性
基因转染
LIM mineralization protein-1
mesenchymal stem cells
osteogenic activity
gene transfect
