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目的研究exendin-4(Ex-4)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)内质网应激相关凋亡的拮抗作用。方法 RPMI1640培养基培养HUVECs,待细胞长到培养瓶底部80%左右后,分4组干预细胞,每组8瓶,共32瓶。实验分组:①正常对照组(n=8):不做任何处理;②Ex-4组(n=8):(Ex-4,8μg/mL,45 min);③EPBS组(n=8):(EPBS,100μL/mL,30 min);④双药组(n=8):Ex-4预处理(8μg/mL,45 min),后加EPBS(100μL/mL,30 min)。免疫荧光法检测确认HUVECs上存在胰高血糖素样肽-1受体(glucagon-like peptide-1 receptor,GLP-1R);内源性过氧化氢酶抑制物(endogenous peroxidase blocking solution;EPBS)诱导HUVECs的凋亡,流式细胞仪检测细胞凋亡率变化;加入Ex-4预处理后,观察EPBS诱导HUVECs凋亡率的变化。Western blot检测细胞Calnexin、(C/EBP homologous protein,CHOP)和Bax表达情况,探讨内质网应激相关凋亡的机制。结果①HUVECs上有GLP-1受体表达;②与正常对照组相比,EPBS组HUVECs的凋亡率显著增加[(52.27±3.66)%vs(7.25±0.62)%,P<0.01)];与EPBS组比较,Ex-4的预处理可明显降低EPBS诱导的HUVECs凋亡[(37.77±2.86)%vs(52.27±3.66)%,P<0.01)]。与正常对照组相比,EPBS能增加Calnexin[(1.76±0.16)vs(1.00±0.05),P<0.01]、CHOP[(4.58±0.32)vs(1.00±0.12),P<0.01]和Bax[(2.25±0.10)vs(1.00±0.06),P<0.01]的表达;与EPBS组比较,Ex-4的预处理可显著降低Calnexin[(0.42±0.05)vs(1.76±0.16),P<0.01]、CHOP[(0.64±0.10)vs(1.65±0.12),P<0.01]和Bax[(1.48±0.08)vs(2.25±0.10),P<0.01)]的表达。结论 Ex-4对EPBS诱导的HUVECs凋亡有明显抑制作用,其抑制作用可能与EX-4减少内质网应激相关蛋白Calnexin、CHOP和Bax相关。
Objective To investigate the antagonistic effect of exendin-4 (Ex-4) on endoplasmic reticulum stress-related apoptosis in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were cultured in RPMI1640 medium. After the cells grew to 80% of the bottom of the culture flask, the cells were divided into 4 groups with 8 bottles each for a total of 32 bottles. The experimental group: ① normal control group (n = 8): without any treatment; ②Ex-4 group (n = 8): Ex-4,8μg / mL, 45 min; EPBS 100 μL / mL for 30 min). ④ The double-drug group (n = 8): Ex-4 preconditioning (8 μg / mL, 45 min) followed by EPBS (100 μL / mL, 30 min). Immunofluorescence assay confirmed the presence of glucagon-like peptide-1 receptor (GLP-1R) on HUVECs; induction of endogenous peroxidase blocking solution (EPBS) The apoptosis of HUVECs was detected by flow cytometry. The apoptosis rate of HUVECs induced by EPBS was observed after Ex-4 pretreatment. Western blot was used to detect the expression of Calnexin (C / EBP homologous protein, CHOP) and Bax, and to explore the mechanism of endoplasmic reticulum stress-related apoptosis. Results ① The expression of GLP-1 receptor on HUVECs was observed. ② Compared with the normal control group, the apoptosis rate of HUVECs in EPBS group was significantly increased [(52.27 ± 3.66)% vs (7.25 ± 0.62)%, P <0.01) Compared with EPBS group, Ex-4 pretreatment significantly reduced the apoptosis of HUVECs induced by EPBS [(37.77 ± 2.86)% vs (52.27 ± 3.66)%, P <0.01)]. Compared with the normal control group, EPBS increased the expression of Calnexin [(1.76 ± 0.16) vs (1.00 ± 0.05), P <0.01], CHOP [(4.58 ± 0.32) vs (1.00 ± 0.12), P <0.01] (2.25 ± 0.10) vs (1.00 ± 0.06), P <0.01]. Ex-4 pretreatment significantly decreased Calnexin [(0.42 ± 0.05) vs (1.76 ± 0.16) ], CHOP [(0.64 ± 0.10) vs (1.65 ± 0.12), P <0.01] and Bax [(1.48 ± 0.08) vs (2.25 ± 0.10), P <0.01). Conclusion Ex-4 significantly inhibits the apoptosis of HUVECs induced by EPBS, and its inhibitory effect may be related to the decrease of endoplasmic reticulum stress-related proteins Calnexin, CHOP and Bax by EX-4.