门脉高压患者血浆刺激血管内皮细胞eNOS与Hsp90相互作用的实验研究

Plasma from patients with portal hypertension stimulates Hsp90 interacting with the eNOS in vascular endothelial cells

目的:用门脉高压患者血浆刺激血管内皮细胞研究内皮型一氧化氮合酶(eNOS)与热休克蛋白90(Hsp90)的相互作用。方法:免疫共沉淀技术是研究蛋白质相互作用的一个有力工具。用肝硬化门脉高压患者血浆处理培养的人脐静脉血管内皮细胞(human umbilical vascular endothelial cell,HUVEC)后,在0、15、30、60和120min时收集细胞,利用该技术检测对Hsp90和eNOS相互作用的影响。细胞裂解液分别用抗eNOS抗体和抗Hsp90抗体沉淀蛋白复合体,经SDS-PAGE电泳和转膜后,再分别用抗Hsp90抗体和抗eNOS抗体进行免疫识别。结果:本实验分别检测出了eNOS和Hsp90的表达,说明Hsp90和eNOS存在相互作用的关系。Hsp90和eNOS对刺激产生的反应有时相性,一般在刺激30min后Hsp90的量有明显增加,进一步说明Hsp90对eNOS调节NO的产生起关键的作用。结论:门脉高压患者血浆可以刺激血管内皮细胞Hsp90与eNOS结合,增强eNOS的生物活性,使NO释放增多。

Objective： To study the interaction between eNOS and Hsp90 after stimulating vascular endothelial cells with plasma from patients with portal hypertension. Methods： Immunoprecipitation technique was a powerful tool to study pro- tein-protein interactions. We used the plasma from portal hypertension in patients to deal with cultured human umbilical vascular endothelial cells （HUVEC）, and collected cells in 0, 15, 30, 60 and 120 min, then used this technique to detect the interaction between Hsp90 and eNOS. Cell lysate was used to deal with anti-eNOS antibody and anti-Hsp90 antibody protein complex precipitation, and then respectively performed immune recognition with anti-Hsp90 antibody and anti- eNOS antibody after the SDS-PAGE electrophoresis and transfer film. Results： The expressions of eNOS and Hsp90 were respectively detected in our study, suggesting there was an interaction between Hsp90 and eNOS. Hsp90 and eNOS had time phases on stimulation-induced response phase, and generally the amount of Hsp90 was significantly increased after 30 minutes, which further indicated that Hsp90 played a key role in the generation of NO regulated by eNOS. Conclusion： Plasma from portal hypertension patients can stimulate the eNOS binding to Hsp90 in vascular endothelial cells and en- hance the biological activity of eNOS, so that the release of NO is increased.