摘要
CXC趋化因子受体2(the CXC chemokine receptor 2,CXCR2)介导的中性粒细胞趋化在细菌感染、慢性炎症、肿瘤发生发展过程中发挥着至关重要的作用,但是目前对于CXCR2受体及下游信号的调控并未被完全揭示。该研究首先以Flag-CXCR2-Tango质粒为模板设计Flag-CXCR2的特异性引物,PCR扩增后将其连接至pCDH-MCS-T2A-Neo-MSCV慢病毒表达载体上,随后对阳性克隆进行测序及表达验证。接着在HEK-293T细胞中包装过表达Flag-CXCR2的慢病毒颗粒并感染HEK-293细胞,使用遗传霉素筛选后,蛋白质免疫印迹和免疫荧光检测发现,Flag-CXCR2稳定表达在HEK-293细胞膜上。最后利用白细胞介素-8(interleukin-8,IL-8)刺激过表达Flag-CXCR2的细胞株,检测发现CXCR2下游信号通路活化及F-actin组装。
CXCR2 (the CXC chemokine receptor 2)-mediated neutrophil chemotaxis plays an important role in the process of bacterial infection,chronic inflammation and tumor development,but the regulation of the activation of CXCR2 receptor and downstream signals are not fully understood at present.In this study,Flag-CXCR2 was firstly amplified from Flag-CXCR2-Tango plasmid,then inserted it into pCDH-MCS-T2A-Neo-MSCV expression vector.The construction was confirmed by sequencing and the expression of Flag-CXCR2 was detected by Western blot.Then,the lentivirus particles expressing Flag-CXCR2 were generated in HEK-293T cells and transduced into HEK-293 cells.After screening with neomycin,Western blot and immunofluorescence detection showed that Flag-CXCR2 stably expressed on HEK-293 cell membrane.Finally,the overexpression of Flag-CXCR2 cell line was stimulated with IL-8 (interleukin-8).The activation of signal pathways downstream of CXCR2 and F-actin assembly were detected.
作者
张洁
任广明
马文兵
葛志强
詹轶群
尹荣华
杨晓明
ZHANG Jie;REN Guangming;MA Wenbing;GE Zhiqiang;ZHAN Yiqun;YIN Ronghua;YANG Xiaoming(Deparment of Pharmaceutical Engineering,Tianjin Universit,Tianjin 300072,China;Bejing Institute of Lifeomics,Academy of Military Medical Sciences,Academy of Military Sciences,Bejing 102206,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2020年第8期1325-1331,共7页
Chinese Journal of Cell Biology
基金
国家自然科学基金面上项目(批准号:81870412)资助的课题。
