尖吻蝮蛇毒PCA对HUVEC活性的影响

发布时间：2019-03-30 15:31

【摘要】：目的:观察皖南尖吻蝮蛇毒蛋白C激活剂(PCA)对人脐静脉血管内皮细胞(HUVEC)的超微结构及其合成组织因子(TF)、血管性血友病因子(vWF)和内皮素-1(ET-1)水平的影响,并探讨PCA的抗血栓机制。方法:实验分为对照组(DMEM液)、脂多糖(LPS)组(LPS 0.1μg/ml)、PCA组(PCA 1μg/ml)、PCA+LPS组(1μg/ml PCA+0.1μg/ml LPS),加药作用12 h后在透射电镜下观察各组细胞的内质网、线粒体形态及自噬体数量变化;ELISA检测各组培养上清中TF、vWF、ET-1含量;RT-PCR检测细胞内vWF、ET-1基因表达水平;Western blot检测细胞内TF蛋白表达水平。结果:LPS组的HUVEC与对照组相比,细胞膜不光滑、出现凸起样改变,并发生线粒体、内质网肿胀,自噬体数量增多等超微结构的改变,PCA组HUVEC的超微结构与对照组比无明显变化;与LPS组相比,PCA+LPS组的HUVEC线粒体、内质网肿胀消失,自噬体数量明显减少。与对照组相比,LPS组细胞培养上清中TF、vWF、ET-1的含量及细胞内vWF、ET-1基因、TF蛋白的表达水平显著增高(P0.05);PCA组细胞培养上清及细胞内3种物质的表达量较对照组无显著变化(P0.05);与LPS组相比,LPS+PCA组HUVEC培养上清及细胞内TF、vWF、ET-1的表达水平显著降低(P0.05)。结论:1μg/ml的PCA可减轻LPS引起的HUVEC超微结构的改变,也可抑制LPS对HUVEC的TF、vWF、ET-1分泌量的增加作用。
[Abstract]:Objective: to observe the ultrastructure of human umbilical vein endothelial cells (HUVEC) and its synthetic tissue factor (TF), (TF),) induced by protein C activator (PCA) from Agkistrodon acutus venom in southern Anhui. The effects of von Willebrand factor (vWF) and endothelin-1 (ET-1) levels and the antithrombotic mechanism of PCA were discussed. Methods: the experiment was divided into control group (DMEM solution), lipopolysaccharide (LPS) group (LPS 0.1 渭 g / ml), PCA group) and PCA 1 渭 g / ml), PCA LPS group (1 渭 g / ml PCA 0.1 渭 g / ml LPS),). The changes of endoplasmic reticulum (ER), mitochondrial morphology and autophagy were observed under transmission electron microscope (TEM) after 12 h treatment. ELISA was used to detect the content of TF,vWF,ET-1 in the supernatant, and RT-PCR was used to detect the expression level of vWF,ET-1 gene in cells.; Western blot was used to detect the expression level of intracellular TF protein. Results: compared with the control group, the membrane of HUVEC in LPS group was not smooth, there were bulge-like changes, mitochondria, endoplasmic reticulum swelling and autophagy increased. The ultrastructure of HUVEC in PCA group had no obvious change compared with control group. Compared with LPS group, the endoplasmic reticulum swelling disappeared and the number of autophagy decreased significantly in, PCA LPS group. Compared with the control group, the content of TF,vWF,ET-1 and the expression of vWF,ET-1 gene and TF protein in the supernatant of LPS group were significantly higher than those in the control group (P0.05). Compared with the control group, the expression levels of the three substances in the supernatant and intracellular cells of PCA group had no significant change (P0.05), but the expression level of HUVEC supernatant and intracellular TF,vWF,ET-1 in, LPS PCA group was significantly lower than that in, LPS PCA group (P0.05). Conclusion: (1) PCA (渭 g / ml) can reduce the ultrastructural changes of HUVEC induced by LPS and inhibit the increase of TF,vWF,ET-1 secretion of HUVEC induced by LPS.
【作者单位】：皖南医学院病理生理学教研室;皖南医学院蛇毒研究室;皖南医学院活性生物大分子研究安徽省重点实验室;
【基金】：安徽省教育厅自然科学研究基金重点项目(KJ2011A266)
【分类号】：R96

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