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目的 研究异基因CD8+T淋巴细胞诱导血管内皮细胞凋亡的分子机制.方法 磁珠分选正常人外周血CD8+T淋巴细胞,膜联蛋白V-异硫氰酸荧光素(FITC)试剂盒检测异基因CD8+T淋巴细胞作用后人脐静脉内皮细胞(HUVEC)和人皮肤微血管内皮细胞(HDMEC)凋亡率,RT-PCR检测蛋白酶激活受体1(PAR-1)mRNA表达,Western印迹检测内皮细胞PAR-1、丝裂原活化蛋白激酶(MAPK)和半胱氨酸蛋白水解酶(Caspage)-3表达.结果 HUVEC与HDMEC在基因和蛋白水平均有PAR-1表达.异基因CD8+T淋巴细胞作用24 h和48 h,HUVEC凋亡率分别为51.7%±4.1%和29.4%±3.3%,HDMEC凋亡率分别为28.9%±2.2%和15.2%±1.8%,均显著高于对照组(均P<0.01);SFLLRN(PAR-1激动剂)诱导两种内皮细胞凋亡的作用与异基因CD8+T淋巴细胞的差异无统计学意义(P>0.05).异基因CD8+T淋巴细胞作用后,HUVEC与HDMEC磷酸化p38MAPK表达均高于对照组(均P<0.05),30 min时分别为对照组的9.8、6.3倍,Caspase-3裂解增加,峰值分别为对照组的5.1、4.3倍;SFLLRN作用后,HUVEC与HDMEC磷酸化p38MAPK表达亦均高于对照组(均P<0.05),30 min时分别为对照组的9.8、6.3倍,Cagpase-3裂解增加,峰值分别为对照组的5.7、2.9倍.JNK磷酸化无明显变化.PAR-1抗体和p38MAPK抑制剂(SB203580)可显著降低异基因CD8+T淋巴细胞诱导的内皮细胞凋亡(P<0.01).结论 异基因CD8+T淋巴细胞通过PAR-1途径引起细胞内p38MAPK激活、Cagpage-3裂解,从而诱导血管内皮细胞凋亡.

Objective To investigate the molecular mechanism of allogeneic CD8+T cell-induced apoptoais of vascular endothelial cells. Methods Allogeneic CD8+ T cells were isolated from PBMC by positive selection using magnetic beads coated with anti-CD8 antibody. Apoptoais of human umbilical vein endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HDMEC) were detected by Annexin V-FITC labeling. Gene and protein expression of proteinase-aetivated receptor-1 (PAR-1) in vascular endothelial cells were tested by RT-PCR and Western blot. Western blotting was also used to detect the change of MAPK and Caspase-3 expression in vascular endothelial cells. The effects of SFLLRN (PAR-1 agonist), ATAP2 (PAR-1 antibody), SB203580 (inhibitor of p38MAPK), SP600125 (inhibitor of JNK) upon apoptosis were also examined. Results After co-culturing with allogeneic CD8+ T cells for 24 h and 48 h, the apoptotic rates of HUVEC were 51.7%± 4.1% and 29.4%±3.3% respectively (P<0.01, vs untreated HUVEC) and those of HDMECs 28.9%±2.2% and 15.2%+1.8% respectively (P<0.01, vs untreated HDMEC). The effect of PAR-1 agonist upon apoptosis of HUVEC and HDMEC similar to that of allogeneic CD8+T cells. These effects were largely prevented by ATAP2 and SB203580 (P<0.05). Allogeneic CD8+ T cells and PAR-1 agonist enhanced the cleavage of Caspase-3 and led to p38MAPK phosphorylation. Conclusion Allageneic CD8+ T cells induced the apoptoais of vascular endothelial cells through PAR-1 dependent modulation of intrinsic apoptotic pathway via the cleavage of Caspase-3 and the phosphorylation of p38MAPK.