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目的探讨压力增高对血管内皮细胞分泌血管活性物质功能的影响及其在门静脉高压症发病机制中的作用。方法用RT-PCR方法半定量研究人脐静脉内皮细胞ET-1、eNOS、i-NOS mRNA的表达;用硝酸还原酶法检测各组细胞培养液中NO代谢产物NO2-/NO3-的含量,免疫荧光标记激光扫描共聚焦显微镜检测培养细胞的ET-1蛋白表达。结果ET-1和eNOS mR-NA在正常培养的内皮细胞中即有表达,iNOS mRNA则几乎无表达。生理水平压力刺激下各组各待测基因mRNA表达与对照组无显著性差异。超生理范围压力刺激后,ET-1 mRNA有显著性升高。eNOS mRNA在40 mm Hg 24 h后才有显著性差异。iNOS mRNA在不同条件下均无显著变化。在细胞培养液中分别加入细胞外钙螯合剂EGTA、PKC抑制剂后,则见ET-1、eNOS mRNA表达下降。结论压力增高可刺激血管内皮细胞合成ET-1、NO,其作用在转录水平,其作用机制分别与PKC信号通路和细胞外钙浓度有关。门静脉压力增高与血管内皮细胞合成ET-1、NO增多之间存在相互作用。
Objective To investigate the effect of increased pressure on the function of vascular endothelial cells secreting vasoactive substances and its role in the pathogenesis of portal hypertension. Methods The mRNA expression of ET-1, eNOS and i-NOS in human umbilical vein endothelial cells were semi-quantitatively determined by RT-PCR. The content of NO metabolites NO2- / NO3- was determined by nitrate reductase method. Immunofluorescence labeling laser scanning confocal microscopy of cultured cells ET-1 protein expression. Results The expression of ET-1 and eNOS mR-NA in normal cultured endothelial cells was almost no expression of iNOS mRNA. Physiological level pressure stimulation of each group of test gene mRNA expression and the control group no significant difference. ET-1 mRNA was significantly increased after hypertonic stress stimulation. There was a significant difference in eNOS mRNA at 40 mm Hg for 24 h. iNOS mRNA showed no significant change under different conditions. After adding extracellular calcium chelator EGTA and PKC inhibitor into cell culture medium, ET-1 was observed, and eNOS mRNA expression decreased. Conclusions The increased pressure stimulates the synthesis of ET-1 and NO by vascular endothelial cells. Its action is at the transcriptional level. The mechanism is related to PKC signaling pathway and extracellular calcium concentration. Increased portal pressure and endothelial cell synthesis of ET-1, there is an interaction between increased NO.