摘要
目的:探究TLR5和NLRC4受体在不同乳腺癌细胞系MDA-MB-231、MCF-7和MDA-MB-435中的表达和定位情况,并探讨重组鞭毛素蛋白对乳腺癌细胞TLR5受体的激活情况。方法:利用Real-time PCR法检测MDA-MB-231、MCF-7和MDA-MB-435细胞TLR5和NLRC4 mRNA表达水平,用流式细胞仪检测MDA-MB-231、MCF-7细胞TLR5受体的表达和定位。纯化重组鞭毛素蛋白即全长鞭毛素蛋白Fli C(同时激活TLR5和NLRC4两条通路)、Fli CΔ90-97(不能激活TLR5通路)、FliCL3A(不能激活NLRC4通路)、Fli CΔ90-97:L3A(两条通路都不激活)。用1μg/ml重组鞭毛素蛋白刺激MCF-7细胞,12 h后,ELISA法检测IL-8的分泌。结果:MCF-7细胞TLR5 mRNA表达水平最高,约是MDA-MB-435细胞的1 700倍,MDA-MB-231细胞TLR5表达水平是MDA-MB-435的约200倍。TLR5在MCF-7细胞浆和胞膜上均有表达,而MDA-MB-231细胞只在胞浆中表达。激活TLR5通路的Fli C和Fli C-L3A能够刺激MCF-7细胞分泌IL-8,而不激活TLR5通路的Fli CΔ90-97和Fli CΔ90-97:L3A则不能。结论:不同乳腺癌细胞系都表达TLR5和NLRC4,但是表达部位和表达水平不同。MCF-7细胞TLR5和NLRC4表达高于其他乳腺癌细胞系。乳腺癌细胞系表面的TLR5受体可以被鞭毛素蛋白激活,为进一步探讨TLR5通路的激活在乳腺癌细胞增殖中的作用提供实验基础。
Objective： To explore the expression and location of TLR5 and NLRC4 on different breast cancer cell lines MDAMB-231,MCF-7 and MDA-MB-435 and TLR5 activation in breast cancer cell line by recombinant flagellin. Methods： The mRNA level of TLR5 and NLRC4 in MDA-MB-231,MCF-7 and MDA-MB-435 cell were detected with quantitative Real-time PCR and TLR5 expression and location in MDA-MB-231 and MCF-7 cell were detected with Flow cytometry. Induction,expression,purification and identification of recombiant flagellin,including Fli C（ activating both TLR5 and NLRC4）,Fli CΔ90-97（ unable to activate TLR5）,Fli C-L3A（ unable to activate NLRC4）,Fli CΔ90-97： L3A（ unable to activate both TLR5 and NLRC4）. 1 μg / ml recombinant flagellin were used to stimulate MCF-7 cell lines,12 h later,the supernate were collected,and ELISA was performed to assess the secretion of IL-8. Results：The mRNA level of TLR5 in MCF-7 cell was 1 700 folds higher than that of MDA-MB-435. TLR5 was expressed in MCF-7 cell surface and ctyosol,while expressed only in cytosol in MDA-MB-231 cell. Fli C and Fli C-L3 A,which were able to activate TLR5 pathway,stimualted MCF-7 cell line to secret IL-8,but Fli CΔ90-97 and Fli CΔ90-97： L3 A did not. Conclusion： TLR5 and NLRC4 have been expressed in different breast cancer lines,but there exists difference on the expression level and location of TLR5. Expression level of TLR5 and NLRC4 in MCF-7 cell were higher than other breast cancer lines. TLR5 receptor which is expressed on the surface of breast cancer cell can be activated by flagellin,and these work also provide us experimental basis to further understand the impact of TLR5 activation on breast cancer cell proliferation.
作者
李伟
卓召振
骆姝琳
任凌雁
陈琨
刘水和
袁军
LI Wei ZHUO Zhao-Zhen LUO Shu-Lin REN Ling-Yah CHEN Kun LIU Shui-He YUAN Jun(Central Laboratory of Guizhou Provincial People＇s Hospital, Guiyang 550002, Chin)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2016年第12期1761-1764,1768,共5页
Chinese Journal of Immunology
基金
贵州省卫计委科学技术基金(gzwjkj2015-1-020)
贵州省人民医院博士基金(GZSYBS[2015]11号)资助
