大鼠骨骺干细胞免疫纯化和Sox9基因真核表达载体的克隆构建  

Immunological purification of rat precartilaginous stem cells and construction of Sox9 gene eukaryotic expression vector

作　　者：张伟凯[1] 陈安民[1] 郭风劲[1] 黄晖[1] 祁军[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,湖北省武汉市430032

出　　处：《中国组织工程研究与临床康复》2008年第12期2321-2325,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30650006)~~

摘　　要：目的:Sox9基因是一种重要的早期胚胎发育相关基因,是前软骨细胞浓聚所必需的因子,其促进成骨但同时可以抑制骨骺细胞的终末分化,以延长软骨发育成熟过程,延迟软骨发育,抑制软骨细胞凋亡,有利于骨骼在生长发育阶段形成复杂的形状和结构。从骨骺干骨细胞中克隆得Sox9基因,并构建其真核表达载体。方法:实验于2007-03/07在同济医学院矫形外科实验室完成。①实验材料:出生<24h纯系清洁级新生SD大白鼠,由华中科技大学同济医学院实验动物中心提供,实验过程中对动物处置符合动物伦理学标准。②实验方法:参考游洪波等方法,采用免疫磁珠技术分离纯化具有FGFR-3特异性表面标志的大鼠骨骺干细胞,以FGFR-3抗体对骨骺干细胞的细胞爬片进行免疫细胞化学鉴定。提取骨骺干细胞总RNA,以RT-PCR方法获得Sox9基因的全长,插入pGEM○R-TEasyVectorSystem克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pEGFP-IRE2构建重组复合质粒。结果:免疫组织化学证实,显微取材分离并纯化的细胞为骨骺干细胞。从骨骺干细胞中提取的总RNA经电泳可得28s、18s和5s共3条带,测吸光度值为0.2635,A260/A280为1.8741,说明提取的总RNA完整性较好,纯度高,符合RT-PCR的要求。PCR产物经电泳可得到约2000bp的特异性条带,与预期大小一致。经限制性内切酶酶切图谱分析DNA序列测定证实的目的基因已经插入重组质粒,成功地构建了Sox9质粒。结论:成功地分离纯化了骨骺干细胞,克隆出Sox9基因并构建了Sox9基因的真核表达载体,为进一步研究Sox9的生物学功能及其在成软骨和成骨分化过程中的作用奠定了基础。AIM： The Sox9 （SRY-related high mobility group-box gene9） gene is an important early embryo growth related gene. It is a necessary cell factor for precartilaginous cell concentrated poly. It can promote osteogenesis, and at the same time inhibite the epiphyseal plate cells terminal differentiation. It can extend the cartilage development and maturation process, delay cartilage development and inhibit cartilage cells apoptosis. It is advantageous to form complex shape and structure in the growth developmental stage of skeleton. We clone the Sox9 gene from precartilaginous stem cells （PSCs） and construct its eukaryotic expression vector. METHODS： Experiments were performed at the Laboratory of Department of Orthopaedic Surgery of Tongji Medical College from March to July 2007.（1） SPF grade SD rats （less than 24 hours） were offered by Animal Experimental Center of Tongji Medical College of Huazhong University of Science and Technology. It is conforms to the animal ethnics standards in the experimental process of animal handling. （2）Refer to the method made by You et al. The PSCs labeled with fibroblast growth factor receptor-3 （FGFR-3） was segregated by immunomagnetic separation, and they were identified by immunohistochemistry with anti-FGFR-3. The total RNA was extracted from PSCs after identification and the Sox9 gene was obtained by RT-PCR and be inserted into pGEM-T Easy Vector System cloning vector.After the sequencing was confirmed, the gene was subcloned to pEGFP-IRE2 to construct recombinant eukaryotic expression vector pEGFP-IRE2 - Sox9. RESULTS： The result of immunohistochemistry confirmed that the PSCs were obtained by the micro-segregation and purified by immunomagnetic technology. The total RNA extracted from PSCs through electrophoresis can get 28, 18, 5 s bands. The absorbance is 0.263 5 and the A 260/A280 is 1.874 1 which shows that the total RNA extracted has a good integrity and high purity, and accord with the request of RT-PCR. The PCR product through electrophoresi

关 键 词：骨骺干细胞 SOX9基因 质粒 表达载体 构建 

分 类 号：R394.2[医药卫生—医学遗传学]