摘要目的 免疫纯化新生大鼠骨骺干细胞,克隆甲状旁腺相关蛋白的中间片段PTHrP(38-94)基因并构建四环素反应性元件调控的反应质粒pTRE-PTHrP(38-94).方法 采用免疫磁珠技术分离纯化具有FGFR-3特异性表面标志的骨骺干细胞,分别以FGFR-3抗体和PCNA抗体对骨骺干细胞的细胞爬片进行免疫细胞化学鉴定.提取骨骺干细胞的总RNA,以RT-PCR方法获得带有酶切位点PTHrP(38-94)基因片段,扩增的DNA片段与含潮霉素筛选标记的四环素反应性元件载体pTRE-2Hyg均双酶切后连接,转化、扩增后对重组质粒进行提取和酶切、测序鉴定.结果 免疫荧光证实所取材分离并纯化的细胞为骨骺干细胞;经限制性内切酶Barn H I、Not Ⅰ酶切图谱分析和DNA序列测定证实目的 基因已经插入重组质粒.结论 运用显微取材和免疫纯化技术可以成功分离纯化骨骺干细胞;成功克隆PTHrP(38-94)基因并构建了反应质粒pTRE-PTHrP(38-94),为进一步明确PTHrP(38-94)片段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.

abstractsObjective To establish the method of separating and purifying rat precartilaginous stem cells (PCSCs) by immunomagnetic technology, clone the mid-fragment of parathroid hormonerelated peptide gene PTHrP(38-94) and construct plasmids of tetracycline(Tet) responsive element which regulates and controls the expression of PTHrP(38-94). Methods Immunornagnetic separation was used to segregate PCSCs labeled with fibroblast growth factor receptor-3(FGFR-3). The PCSCs were identified by imrnunocytochemistry with anti-FGFR-3 and anti-PCNA. The total RNA was extracted from PCSCs after identification and the mid-fragment of PTHrP(38-94) gene was obtained by RT-PCR method. With the added endonuclease Sites, PTHrP(38-94) gene was double-enzyme digested by Bam H Ⅰ and Not Ⅰ. The gene was subcloned to plasmids of Tet-responsive element with the selection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic expressive plasmid pTRE-PTHrP(38-94). Then the recombinant plasmids were transferred into E. coli-DH5α and the clones were randomly selected. Then the recombinant plasmids were purified and identified by doubleenzyme digestion. Results The results of immunocytochemistry confirmed that the rat pre-cartilaginous stem cells was obtained by the micro-segregate and purified by immunomagnetic technology. Double enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant plasmids. Conclusions The purified PCSCs can be identified accurately and the responsive plasmids containing PTHrp(38-94) gene can be successfully constructed, which may give new strategy for investigation of the biological function of the PTHrp(38-94) gene in the differentiation of chondrocytes and osteocytes.