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医学论文范文:重组腺病毒载体Ad-LMP-1的构建及其转染MSC后成骨活性
【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。
【关键词】 LIM-1矿化蛋白; 间充质干细胞; 成骨活性; 基因转染
中图分类号: R329.2+8 文献标志码: A 文章编号: 1672-3554(2010)02-0199-07
Osteogenic Activity of MSC Infected by Recombinant Adenovirus Vector Ad-LMP-1
CHENG Zhi-an1, LIU Dong-bin3, WU Yan-feng2, HUANG Lin2, SHEN Hui-yong2, LIU Shang-li2
1. Department of Orthopaedices, The Second Affiliated Hospital of Guangzhou Chinese Medicine University Guangdong Provincial Hospital of Traditional Chinese Medicine), Guangzhou 510120, China;2. Department of Orthopaedics, The Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, China;3. Department of Orthopadics, The People’s Hospital of Nanhai District, Foshan 528200, China
Abstract: 【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 LIM 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.
Key words: LIM mineralization protein-1; mesenchymal stem cells; osteogenic activity; gene transfect
[J SUN Yat-sen Univ(Med Sci),2010,31(2):199-206]
矿化蛋白-1(LIM mineralization protein-1, LMP-1)是直接参与成骨分化的细胞内因子,它的成骨作用可能表现在参与合成和分泌其他的成骨诱导蛋白如骨形态发生蛋白(bone morphogenetic proteins,BMP)以及转化生长因子子β1(transforming growth factor β1,TGF-β1),同时招募附近的细胞分化直接参与骨膜或软骨内成骨。在动物或体外实验中,小剂量的含LMP-1基因的腺病毒或质粒载体转染骨髓基质细胞,可持续稳定地诱导骨形成[1]。LMP-1的高效骨诱导特性为治疗骨质疏松性骨折提供了新的选择。本研究用Gateway技术构建LMP-1重组腺病毒载体,以腺病毒为载体,将LMP-1基因导入间充质干细胞(mesenchymal stem cells,MSC),通过比较实验组与对照组中MSC成骨活性的差异,来进一步验证LMP-1的骨诱导作用,为后续的LMP-1基因治疗骨质疏松及其骨折研究奠定基础。
