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目的构建CXCR1-pTREhyg可调控性真核表达载体,检测其在NIH3T3细胞内的表达情况。方法通过RT-PCR的方法从原代培养的类风湿性关节炎患者滑膜成纤维细胞中克隆得到CXCR1的全长基因,测序正确后将其亚克隆入pTREhyg载体中。用脂质体将pTet-on质粒和CXCR1-pTRE-hyg共转染入NIH3T3细胞,以不同浓度的强力霉素诱导,用Westernblot检测CXCR1蛋白表达水平。结果Westernblot显示,我们所构建的可调控性表达载体能在NIH3T3细胞内表达,不同浓度药物诱导的CXCR1表达水平明显不同;同时还观察到IL-8刺激后,NIH3T3细胞中Erk-1/2磷酸化水平升高。结论成功构建了CXCR1-pTREhyg真核表达载体,并可在NIH3T3细胞内表达。经IL-8刺激的转染NIH3T3细胞中Erk-1/2活性发生变化。为进一步观察CXCR1与类风湿性关节炎损伤之间的关系奠定了基础。
Objective To construct a CXCR1-pTREhyg regulatable eukaryotic expression vector and detect its expression in NIH3T3 cells. Methods The full length gene of CXCR1 was cloned from synovial fibroblasts from primary cultured patients with rheumatoid arthritis by RT-PCR. After sequencing, the full-length CXCR1 gene was subcloned into pTREhyg vector. The pTet-on plasmid and CXCR1-pTRE-hyg were co-transfected into NIH3T3 cells with liposomes and induced with different concentrations of doxycycline. The expression of CXCR1 protein was detected by Western blot. Results Westernblot showed that the expression of CXCR1 was significantly up-regulated in NIH3T3 cells. The expression of CXCR1 in NIH3T3 cells induced by IL-8 was significantly lower than that in NIH3T3 cells The level of increase. Conclusion The eukaryotic expression vector CXCR1-pTREhyg was successfully constructed and expressed in NIH3T3 cells. Erk-1/2 activity changes in IL-8-transfected NIH3T3 cells. Which laid the foundation for the further observation of the relationship between CXCR1 and the injury of rheumatoid arthritis.