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目的研究丝裂原活化蛋白激酶(MAPKs)活化对热打击致小鼠肺微血管内皮细胞(PMVECs)凋亡的影响。方法建立重症中暑小鼠模型,采用TUNEL染色及免疫组化检测肺组织损伤情况。二次磁珠分选法分离乳鼠PMVECs,TUNEL染色检测PMVECs凋亡情况,Western blotting检测热打击恢复期(0、2、6h)MAPKs家族活化情况。通过检测单层内皮细胞跨膜电阻(TEER)及辣根过氧化物酶(HRP)值观察不同热打击温度对单层细胞通透性的影响,同时使用MAPKs家族抑制剂检测热打击对单层细胞通透性及凋亡的影响。结果在重症中暑小鼠恢复期肺组织中可观察到PMVECs发生凋亡。TUNEL染色发现随着恢复期时间的延长,PMVECs凋亡数目增多,热打击可使PMVECs MAPKs家族活化且微血管通透性增加,给予p38活化抑制剂SB203580及ERK活化抑制剂PD98059预处理后细胞通透性增加,凋亡数目增多,而给予JNK抑制剂SP600125预处理后细胞则出现相反的变化。结论重症中暑小鼠PMVECs可发生凋亡,p38及ERK起着抗凋亡的作用,JNK起着促凋亡的作用。
Objective To investigate the effects of mitogen-activated protein kinases (MAPKs) activation on the apoptosis of mouse pulmonary microvascular endothelial cells (PMVECs) induced by thermal shock. Methods Severe heat stroke mice model was established. TUNEL staining and immunohistochemistry were used to detect lung tissue injury. The PMVECs were isolated by secondary magnetic bead sorting, the apoptosis of PMVECs was detected by TUNEL staining, and the activation of MAPKs family during recovery from heat shock (0, 2, 6h) was detected by Western blotting. The effects of different thermal shock temperatures on the permeability of monolayer cells were observed by measuring the transmembrane resistance (TEER) and horseradish peroxidase (HRP) values of monolayer endothelial cells. Meanwhile, MAPKs family inhibitors were used to detect the effects of thermal shock on monolayer Cell Permeability and Apoptosis. Results The apoptosis of PMVECs was observed in the lung tissue of convalescent severe heat stroke mice. TUNEL staining showed that the number of apoptotic PMVECs increased with the prolongation of recovery time. The thermal shock could activate the MAPKs family of PMVECs and increase the microvascular permeability. After pretreatment with p38 activation inhibitor SB203580 and ERK activation inhibitor PD98059, Increased sexuality, the number of apoptosis increased, while given JNK inhibitor SP600125 pretreatment cells appear the opposite change. Conclusion Severe heat stroke may induce apoptosis in PMVECs, p38 and ERK play an anti-apoptotic role, JNK plays a role in promoting apoptosis.