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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 effect of CCM3 gene knockdown on the migration of immortalized human umbilical vein endothelial cells (HUVECs) induced by lead acetate and explore the possible mechanism of endoplasmic reticulum stress (ERS). Methods Wild type CCM3 (CCM3-WT) and CCM3-KO HUVECs were used as experimental cells. (1) Two kinds of genotypes were treated with 0, 10, 50 and 200 μmol / L lead acetate for 24 h respectively, and the cell migration was observed by scratch test. (2) Two genotypes of cells were treated with different concentrations of lead acetate (0, 10, 50, 200μmol / L) for 24 h and at a concentration of 50μmol / L for 0, 6, 12, Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect mRNA relative expression level of unfolded protein pathway, and the relative expression level of GRP78 was detected by Western blotting. (3) Two genotypes of cells were divided into lead-exposed group and TUDCA group. The former was treated with lead acetate at a concentration of 50μmol / L for 24 h. The latter was exposed to lead acetate at the same concentration Before pretreatment of endoplasmic reticulum stress inhibitor TUDCA, the cell migration was observed by scratch test. Results (1) The migration rates of CCM3-WT and CCM3-KO cells showed a dose-response relationship (P <0.05) with the increase of lead acetate exposure. (2) The expression of GRP78, protein kinase-like endoplasmic reticulum kinase (ERK), activator of transcription 4 (ATF4) and CCAAT in CCM3-KO cells in 10, 50 and 200 μmol / (P <0.05). The mRNA relative expression levels of PERK and CHOP in CCM3-KO cells increased with the increase of the concentration of lead acetate (P <0.05). The relative mRNA expression levels of these four genes in CCM3-KO cells in 48 h group were significantly higher than those in CCM3-WT cells at the same time point (P <0.05). The relative expression level of GRP78 protein in CCM3-KO cells was higher than that in CCM3-WT cells (P <0.05) at lead acetate dose of 50μmol / L, and the relative expression level of GRP78 protein in CCM3- (P <0.05) increased with time. (3) The cell migration rate in TUDCA group was lower than that in lead poisoning group (P <0.05). Conclusion Lead acetate may activate ERS by activating PERK-ATF4-CHOP signaling pathway, thereby reducing the migration ability of HUVECs. CCM3 may have a protective effect on the migration ability of HUVECs.