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

[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pEGFP-IRES2-PTHrp.[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,RT-PCR方法获得PTHrp基因的全长cDNA,插入pCR2.1 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pEGFP-IRES2.[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.

Identification of epiphyseal proliferating zone cells and construction of eukaryotic expression vector containing PTHrp gene

Objective To separates proliferating zone cells from the growth plate,then clone the parathroid hormone-related 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 anti-collagen type X and electron microscope image.The total RNA was extracted from cells after identification and the full length eDNA encoding PTHrp gene was obtained by RT-PCR method and inserted into pCR2.1 TA cloning vector.After the sequencing was confirmed,the gene was subcloned to pEGFP-IRES_2 to construct recombinant eukaryotic expression vector pEGFP-IRES_2-PTHrp.Result The proliferating zone cells were separated from growth plate.Enzyme 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.