CCM3调控醋酸铅诱导人脐静脉内皮细胞迁移能力下降机制

发布时间：2018-05-23 17:40

本文选题：铅 + 细胞迁移　；参考：《中国职业医学》2017年02期

【摘要】：目的观察脑海绵状血管瘤(CCM)致病基因CCM3基因敲除对醋酸铅诱导的永生化人脐静脉内皮细胞(HUVECs)迁移能力的影响,并探讨可能的内质网应激(ERS)机制。方法以野生型CCM3(CCM3-WT)和基因敲除CCM3(CCM3-KO)HUVECs为实验细胞。(1)分别以浓度为0、10、50、200μmol/L醋酸铅处理2种基因型细胞24 h后,通过划痕实验观察细胞迁移情况。(2)取2种基因型细胞分别采用不同浓度(0、10、50、200μmol/L)醋酸铅处理24 h和以浓度为50μmol/L醋酸铅处理0、6、12、24、48 h后,以实时荧光定量聚合酶链式反应检测未折叠蛋白反应通路相关基因的mRNA相对表达水平,以蛋白免疫印迹法检测葡萄糖调节蛋白78(GRP78)相对表达水平。(3)取2种基因型细胞分为铅染毒组和牛磺熊去氧胆酸(TUDCA)组,前者仅予浓度为50μmol/L醋酸铅处理24 h,后者在采用相同浓度的醋酸铅染毒前予内质网应激抑制剂TUDCA预处理细胞,通过划痕实验观察细胞迁移功能。结果 (1)CCM3-WT和CCM3-KO细胞的迁移率均呈随着醋酸铅染毒浓度的增加而下降的剂量-效应关系(P0.05)。(2)除10μmol/L组CCM3-KO细胞GRP78外,10、50和200μmol/L组CCM3-KO细胞GRP78、蛋白激酶样内质网激酶(PERK)、转录激活因子4(ATF4)和CCAAT区/增强子结合蛋白同源蛋白(CHOP)的mRNA相对表达水平均分别高于同剂量组CCM3-WT细胞(P0.05),且CCM3-KO细胞PERK和CHOP的mRNA相对表达水平呈随着醋酸铅染毒剂量的增加而增加的时间-效应关系(P0.05);48 h组CCM3-KO细胞上述4种基因的mRNA相对表达水平均高于同时间点CCM3-WT细胞(P0.05)。在醋酸铅染毒剂量为50μmol/L时,CCM3-KO细胞GRP78蛋白相对表达水平高于CCM3-WT细胞(P0.05),且CCM3-KO细胞的GRP78蛋白相对表达水平呈随着铅染毒时间的增加而增加的时间-效应关系(P0.05)。(3)TUDCA组细胞迁移率低于铅染毒组(P0.05)。结论醋酸铅可能通过激活PERK-ATF4-CHOP信号通路启动ERS,从而降低HUVECs的迁移能力;CCM3对HUVECs迁移能力的降低具有保护作用。
[Abstract]:Objective to investigate the effect of CCM3 knockout on the migration of human umbilical vein endothelial cells (HUVECs) induced by lead acetate, and to explore the possible mechanism of endoplasmic reticulum stress (ERS). Methods the two genotypes were treated with wild type CCM3 CCM3-WT) and knockout CCM3(CCM3-KO)HUVECs as experimental cells. The two genotypic cells were treated with 0.10 渭 mol/L and 50200 渭 mol/L for 24 h, respectively. Cell migration was observed by scratch test.) two genotypic cells were treated with lead acetate at different concentrations for 24 h and 50 渭 mol / L (50 渭 mol / L), respectively, after 48 h treatment with 50 渭 mol/L lead acetate (50 渭 mol/L). Real-time quantitative polymerase chain reaction was used to detect the relative expression level of mRNA in unfolded protein reaction pathway. The relative expression level of glucose-regulated protein 78 (GRP78) was detected by Western blot. The two genotypes were divided into lead exposed group and TUDCA group. The former was treated with 50 渭 mol/L lead acetate for 24 h, and the latter was pretreated with endoplasmic reticulum stress inhibitor (TUDCA) before exposure to the same concentration of lead acetate. The migration function of the cells was observed by scratch test. Results the mobility of CCM3-WT and CCM3-KO cells decreased in a dose-effect relationship with the increase of lead acetate concentration (P 0.05). In addition to 10 渭 mol/L, 10 渭 mol/L and 200 渭 mol/L, CCM3-KO cells GRP78, protein kinase-like endoplasmic reticulum kinase PERK8, transcriptional activators were found to be responsible for transcriptional activation. The relative mRNA expression levels of ATF4) and CHOP4 (CCAAT region / enhancer protein homologous protein) were higher than those of CCM3-WT cells of the same dose group (P0.05), and the mRNA expression levels of PERK and CHOP in CCM3-KO cells increased with the increase of lead acetate exposure. The mRNA relative expression level of the four genes in CCM3-KO cells was higher than that in CCM3-WT cells at the same time. The relative expression of GRP78 protein in CCM3-KO cells was higher than that in CCM3-WT cells at a dose of 50 渭 mol/L, and the relative expression level of GRP78 protein in CCM3-KO cells increased with the increase of lead exposure time. The transfer rate was lower than that of lead exposure group (P 0.05). Conclusion lead acetate may initiate ERS by activating the PERK-ATF4-CHOP signaling pathway, thus reducing the migration ability of HUVECs. CCM3 has protective effect on the reduction of HUVECs migration.
【作者单位】：广州市环境污染与健康评价重点实验室中山大学公共卫生学院;
【基金】：国家自然科学基金(81273097,81472998)
【分类号】：R114

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