尿毒症血清蛋白对血管内皮细胞的损伤作用

Damage effect of uremic serum protein on vascular endothelial cells

目的研究尿毒症血清蛋白对人血管内皮细胞的损伤作用,探讨蛋白结合尿毒症毒素对人血管内皮细胞的损伤作用。方法采集维持性血液透析尿毒症患者和正常健康人的静脉血,经过血清分离、透析、低温真空冻干等处理提取血清蛋白,以不同浓度的尿毒症血清蛋白溶液或正常血清蛋白溶液(0.4%、1%、2%、4%)刺激人脐静脉内皮细胞(HUVEC)不同时间(24、48、72 h),设置空白对照组。倒置相差显微镜观察细胞形态,CCK-8检测细胞增殖抑制率,一氧化氮(NO)试剂盒、一氧化氮合酶(NOS)试剂盒分别检测细胞培养上清液NO浓度和细胞NOS活性,流式细胞仪检测内皮细胞膜微粒(EMPs)产生率、细胞凋亡率。结果与正常血清蛋白溶液组、空白对照组比较,尿毒症血清蛋白溶液组细胞折光性差,细胞间隙增宽,细胞膜表面毛糙,细胞突起明显增多,细胞形态各异,胞浆颗粒明显增加。不同浓度的尿毒症血清蛋白溶液处理HUVEC的细胞增殖率均显著低于相应正常血清组和空白对照组(P<0.05)。与正常蛋白溶液组、空白对照组比较,尿毒症蛋白溶液组细胞培养上清液中NO浓度降低、细胞NOS活性明显降低(P<0.05)。尿毒症蛋白溶液组EMPs产生率为(62.57±1.59)%,显著高于空白对照组(7.3±0.57)%和正常蛋白溶液组(13±0.94)%(P<0.05)。尿毒症蛋白溶液对HUVEC细胞凋亡无明显影响。结论尿毒症血清蛋白溶液能使内皮细胞形态发生变化,抑制内皮细胞增殖,损伤内皮细胞分泌功能,导致内皮细胞功能受损。

Objective To investigate the damage effect of protein-bound uremic toxins on human umbilical vein endothelial cells（HUVECs）.Methods HUVECs were cultured in growth medium with the addition of different concentrations（0.4%,1%,2% and 4%） of proteins collected from the uremic patients or the normal persons for different time（24,48 and 72 h）,and the blank control group was established.The cell morphology was observed with an inverted phase contrast microscope.The cell proliferation rate,nitric oxide（NO） concentration and nitric oxide synthase（NOS） activity were detected by respective assay kits.The cell apoptotic rate and the level of endothelial microparticles（EMPs） were detected by flow cytometry（FCM）.Results Compared with the normal group and the blank control group,the uremic group showed irregular cell morphology,lower cell proliferation rate（P 〈 0.05）,less NO in culture supernatant（P 〈 0.05）,and lower NOS activity（P 〈 0.05）.The level [（62.57 ± 1.59） %] of the EMPs in the uremic group was significantly higher than that [（7.3 ± 0.57） %] in the blank control group and that [（13 ± 0.94） %]in the normal group（P 〈 0.05）.However,the uremic protein solution had no effect on cell apoptosis.Conclusion The serum protein solution from the uremic patients may change the HUVEC morphology,inhibit the HUVEC proliferation and NO secretion,decrease the NOS activity,and promote the released EMPs from the HUVECs,which may lead to the dysfunction of the vascular endothelial cells.