鼠骨骺增殖区细胞的分离鉴定和克隆构建PTHrp基因真核表达载体.DOCVIP

临床医学论文-鼠骨骺增殖区细胞的分离鉴定和克隆构建PTHrp基因真核表达载体 ?????????? 作者? 黄仕龙　陈安民　郭风劲　张衣北 【关键词】? 殖区软骨细胞, ??? 摘要：［目的］分离、鉴定大鼠骨骺生长板增殖区软骨细胞，克隆甲状旁腺激素相关蛋白（PTHrp）基因并构建真核表达载体pEGFPIRES2PTHrp。［方法］Percoll不连续密度梯度离心法分离生长板增殖区软骨细胞，用X型胶原抗体和电镜鉴定，提取总RNA，RTPCR方法获得PTHrp基因的全长cDNA，插入pCR21 TA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pEGFPIRES2。［结果］分离出增殖区软骨细胞，经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒。［结论］准确分离增殖区软骨细胞并成功构建了PTHrp基因的真核表达载体，为进一步揭示PTHrp的生物学功能及其在在软骨细胞的分化和骨骼形态发生中的作用奠定了基础。 　　关键词：PTHrp基因；质粒；殖区软骨细胞；表达载体 　　Identification of epiphyseal proliferating zone cells and construction of eukaryotic expression vector containing PTHrp gene 　　Abstract：［Objective］To separates proliferating zone cells from the growth plate,then clone the parathroid hormonerelated peptide gene (PTHrp) and construct its eukaryotic expression vector［Method］After centrifugation through a discontinuous Percoll gradient,the third and fourth fractions cell populations of growth plate chondrocytes were identified with anticollagen type X and electron microscope imageThe total RNA was extracted from cells after identification and the full length eDNA encoding PTHrp gene was obtained by RTPCR method and inserted into pCR21 TA cloning vectorAfter the sequencing was confirmed,the gene was subcloned to pEGFPIRES2 to construct recombinant eukaryotic expression vector pEGFPIRES2PTHrp［Result］The proliferating zone cells were separated from growth plateEnzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant vector［Conclusion］The proliferating zone cells were identified accurately and the eukaryotic expression plasmid containing PTHrp gene was successfully constructed,which may be a promising for studying the biological function of the PTHrp gene and its role in chondrocyte differentiation and bone formation 　　Key words：PTHrp gene; Plasmid;Growth chondrocyte;? Expression vector???????????????????????????????????????????????????? 　　软骨缺损临床极为常见。软骨受损后自我修复能力极为有限，且对软骨细胞的增殖、分化规律和调控机理缺乏认识，目前对软骨缺损尚无很好的治疗方法。 　　甲状旁腺激素相关蛋白(PTHrp)在研究恶性肿瘤高钙血症时被发