摘要
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。
AIM： To construct tetracycline （ Tet ） -controlled inducible vector CXCR1-pTREhyg, and then detect the expression character of CXCR1 under the regulation of Dox in NIH3T3 cells. METHODS： A full length cDNA of human CXCR1 was cloned from sample of fibrablast like synovium （FLS） in rheumatoid arthritis （RA） patient by RTPCR. and then sub-cloned into the pTREhyg plasmid after sequence analysis. CXCR1-pTREhyg and pTet-on was cotransfected with Lipofect2000 to NIH3T3 cells, and the expression of IL-SRA was detected by Western blot after given different condense of Dox. RESULTS. Western blot showed that CXCR1 could be induced by Dox in NIH3T3 cells, and the phosphorylated Erk-1/2 level was significantly increased after IL-8 stimulation. CONCLUSION： Tet inducible recombinant vector of CXCR1-pTREhyg was successfully constructed, and it could be expressed in NIH3T3 cells . The stimulation of IL-8 obviously changed the activity of Erk-1/2 in the transfected NIH3T3 cells. This work has laid foundations for further study on the relationship between CXCR1 and RA disease.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第3期280-282,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展规划(973)资助(No.2002CB513007)
国家自然科学基金资助项目(No.30300319
30571734)
