饥饿状态下Lewis细胞HMGB1的表达、定位与细胞自噬发生的相关性研究  

Effects of the expression and localization of HMGB1 on starvation-induced autophagy

作　　者：汪汀[1] 高云涛[2] 王承琳[3] 徐鑫鑫[1] 尹晶平[1] 田莎莎[1] 邢丽丹[1] 王胜军[1] 张盼[1] 纪晓昀[1] 沈慧玲[4] 许化溪[1] 苏兆亮[1] 

机构地区：[1]江苏大学免疫学研究所,镇江212013 [2]第四军医大学航空航天学院医学系,西安710032 [3]天津市血液中心,300110 [4]江苏大学附属四院肿瘤中心,镇江212001

出　　处：《免疫学杂志》2013年第12期1013-1017,共5页Immunological Journal

基　　金：国家自然基金(81001319;31270947);中国博士后基金(2012M511705);江苏省博士后基金(1102129C);江苏大学高级人才启动基金(11JDG128);大学生课外学术科研项目(11A386)

摘　　要：目的探讨饥饿状态下小鼠肺癌细胞(Lewis)自噬的发生与高迁移率组蛋白B1(HMGB1)的相关性。方法分别用HBSS诱导Lewis自噬0、3、6及9 h,免疫荧光检测HMGB1在胞内表达水平及迁移情况;收集细胞及培养上清,Western blot检测微管相关蛋白轻链3(LC3)及其磷脂酰乙醇胺共轭物(LC3-Ⅱ)、泛素结合蛋白p62与高迁移率组蛋白(HMGB1)的表达水平;PCR检测Lewis细胞表面HMGB1配体RAGE、TLR2及TLR4的表达。结果免疫荧光显示,Lewis细胞饥饿6 h后HMGB1表达量增加并伴随从胞核向胞浆的迁移;Lewis细胞饥饿3 h后,Western blot结果显示,HMGB1及LC3-Ⅱ表达水平逐渐增加;Lewis细胞表面表达HMGB1的配体RAGE、TLR2及TLR4;利用重组的HMGB1蛋白刺激Lewis后细胞自噬明显增加。结论 HMGB1可以促进Lewis细胞自噬的发生。This study was designed to investigate the correlation between starvation-induced autophagy and the expression and localization of high-mobility group box 1 （HMGB1）. Firstly, Lewis cells and cell supernatants were harvested after culturing in HBSS 0, 3, 6, 9 h. Then expression of cytosolic light chain 3 （LC3; LC3-I）/LC3- phosphatidylethanolamine conjugate （LC3- Ⅱ ） and HMGB1 in Lewis were detected by Western blot; localization of HMGB1 and HMGB1 receptors in HBSS-induced Lewis cells was detected by immunofluorescence and PCR, respectively; moreover, the expression of HMGB1 and LC3-I/LC3- ]l in Lewis stimulated by 10 μg/ml rHMGB1 for 0 h, 12 h, 24 h, 36 h was also detected by Western blot. We found that HMGB1 was transferred from nucleus to cytoplasm at 6 h, and also detectable in cell supernatants; LC3-II and HMGB1 increased at 3 h and 6 h after being cultured by HBSS, but LC3- I level decreased. Furthermore, PCR demonstrated that HMGB1 receptors RAGE, TLR2, and TLR4 were expressed on Lewis cells. In conclusion, all the result suggested that HMGB 1 could transfer from nucleus to cytoplasm and then secrete to the outside of Lewis and combine with receptors on the cells surface to promote autophagy.

关 键 词：自噬 肿瘤 高迁移率组蛋白B1 LC3-Ι/LC3-Ⅱ P62 

分 类 号：R392.1[医药卫生—免疫学]