CCM3调控醋酸铅诱导人脐静脉内皮细胞迁移能力下降机制

CCM3 down-regulating the lead acetate-induced migration of human umbilical vein endothelial cells

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(P<0.05)。(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(P<0.05),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(P<0.05);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(P<0.05)。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(P<0.05),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(P<0.05)。(3)TUDCA组细胞迁移率低于铅染毒组(P<0.05)。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。

Objective To investigate the effects of knockout cerebral cavernous malformation （CCM） virulence gene CCM3 on the migration induced by lead acetate in immortalized human umbilical vein endothelial cells （HUVECs） and to explore the possible mechanism of endoplasmic reticulum stress （ERS）.Methods CCM3 wildtype （CCM3-WT） and CCM3 knockout （CCM3-KO） HUVECs were used as experimental cells.a） CCM3-WT and CCM3-KO HUVECs were treated with lead acetate at 0,10,50 and 200 μmol/L for 24 hours.The migration of these cells was observed by wound-healing assay.b） CCM3-WT and CCM3-KO HUVECs were treated with lead acetate at 0,10,50 and 200 μmol/L for 24 hours,and at 50 μmol/L for 0,6,12,24 and 48 hours,and the mRNA expression of genes of unfolded protein response pathway were detected by quantitative real-time polymerase chain reaction;the protein expression of glucose-regulated protein 78 （GRP78） was detected by Western blotting.c） CCM3-WT and CCM3-KO HUVECs were divided into lead exposure group and tauroursodeoxycholic acid （TUDCA） group.The former was treated with 50 μmol/L lead acetate for 24 hours,and the latter was pre-treated with ERS inhibitor TUDCA,followed by 50 μmol/L lead acetate.The migration of these cells was observed by wound-healing assay.Results a） The migration of CCM3-WT and CCM3-KO cells decreased and showed a dose-effect relationship with the increase of lead acetate concentration （P〈0.05）.b） The mRNA relative expression of the GRP78,protein kinase-like endoplasmic reticulum kinase （PERK）,transcription activator 4 （ATF4） and CCAAT enhancer binding homologous protein （CHOP） in CCM3-KO cells treated with 10,50 and 200 μmol/L lead acetate were higher than that in CCM3-WT cells at the same doses,except for the GRP78 in CCM3-KO cells treated with 10 μmol/L lead acetate （P〈0.05）.The mRNA expression of PERK and CHOP in CCM3-KO cells increased in a time-effect relationship with the increase of lead-exposure time （P〈0.05）.The mRNA relative expression of the four genes in CCM3-KO cells were higher than those in CCM3-WT cells at 48 hours （P〈0.05）.When cells were treated with 50 μmol/L lead acetate,the protein expression of GRP78 in CCM3-KO cells was higher than that in CCM3-WT cells （P〈0.05）,and the protein expression of GRP78 in CCM3-KO cells increased in a time-effect relationship with the increase of lead-exposure time （P〈0.05）.c） The cell migration of TUDCA group was lower than that of lead-exposure group （P〈0.05）.Conclusion Lead acetate may activate ERS by activating the PERK-ATF4-CHOP signaling pathway,thereby reducing the migration of HUVECs.CCM3 gene has a protective effect on cell migration.