摘要
背景:钙黏蛋白11对骨髓间充质干细胞进行基因修饰,可促进细胞的黏附性能。利用核心结合因子α1对骨髓间充质干细胞进行基因修饰,有望实现多能干细胞的成骨定向分化,并在诱导成骨分化的级联效应中发挥重要的信号作用。目的:拟采用腺病毒为载体,介导钙黏蛋白11、核心结合因子α1基因修饰骨髓间充质干细胞,观察两基因的表达情况。设计、时间及地点:细胞-基因学实验,于2008-08在珠江医院中心实验室完成。材料:人骨髓间充质干细胞来源于1例胸椎骨折男性患者,由珠江医院提供,患者对实验知情同意。钙黏蛋白11全长cDNA质粒由日本神户理研Riken分子生物中心Takeichi教授惠赠,全长核心结合因子α1基因质粒由美国Baylor医学院Ducy博士提供,pAdeasy腺病毒系统由美国约翰霍普金斯遗传所Bert博士馈赠。方法:Percoll密度梯度离心+贴壁法分离培养人骨髓间充质干细胞。利用PCR技术获得钙黏蛋白11和核心结合因子α1基因,分别构建到腺病毒穿梭载体上,然后分别通过同源重组获得重组腺病毒质粒,再将两病毒质粒包装获得重组病毒液,最后通过病毒将两基因导入骨髓间充质干细胞。主要观察指标:Westernblot法检测钙黏蛋白11、核心结合因子α1基因在人骨髓间充质干细胞中的表达。结果:获得携带钙黏蛋白11和核心结合因子α1基因的病毒液,将其感染人骨髓间充质干细胞后,以Westernblot检测两种基因同时获得高表达。结论:实验成功构建钙黏蛋白11和核心结合因子α1基因腺病毒载体,通过病毒转染人骨髓间充质干细胞,二者获得高效表达。
BACKGROUND： Cadherin-11 which was used to modify bone marrow mesenchymal stem cells aimed to promote cell adhesion ability; while, core binding factor α1 （cbfα1） which was used to modify bone marrow mesenchymal stem cells aimed to realize ossification of multipotential stem cells and play a signalization role in cascade effect.
OBJECTIVE： To construct cadherin-11 and cbfal gene adenovirus expression vectors so as to modify bone marrow mesenchymal stem cells, and observe the expressions of the two genes.
DESIGN, TIME AND SETTING： Cell-genetics experiment was performed at Central Laboratory of Zhujiang Hospital in August 2008.
MATERIALS： Bone marrow mesenchymal stem cells were extracted from a male patient with thoracic vertebral fracture from Zhujiang Hospital. The patient provided the informed consent. Full length cadherin-11 cDNA plasmid was provided by Professor Takeichi, Riken Molecular Organism Center, Japan; full length cbfal gene plasmid was provided by Professor Ducy, Baylor Medical College, USA; pAdeasy adenovirus system was provided by Professor Bert, McKusick-Nathans Institute of Genetic Medicine, USA.
METHODS： Human bone marrow mesenchymal stem cells were separated using Percoll density gradient centrifugation combined with adherence method. Cadherin-11 and cbfα1 genes were obtained by PCR, and then they were inserted into shuttle vector of adenovirus; thereafter, the recombinant adenovirus plasmids were gained by homologous recombination. Finally, the two plasmids were re-packed to obtain recombinant adenovirus venom, and cadhedn-11 and cbfal genes were transfected in bone marrow mesenchymal stem cells by adenovirus.
MAIN OUTCOME MEASURES： The expressions of cadherin-11 and cbfal genes were detected by Western blotting.
RESULTS： The adenovirus venom carrying the cadherin-11 and cbfal gene was successfully obtained. Western blotting showed that the expressions of the cadherin-11 and cbfα1 genes in bone marrow mesenchymal stem cells were remarkably increased by infection viral.
CONCLUSION： The bone marrow mesenchymal stem cells may express high level of cadherin-11 and cbfα1 by gone modification of adenovirus.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第45期8843-8848,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30570517)~~
