蛋白结合尿毒症毒素对尿毒症患者血管内皮细胞的损伤作用及机制研究

Study on the Damage Effect and Mechanism of Protein Binding Uremic Toxin on Vascular Endothelial Cells in Uremic Patients

摘要: 目的：研究蛋白结合尿毒症毒素对尿毒症患者血管内皮细胞的损伤作用及机制。方法：选取我院透析室维持性血液透析的尿毒症患者42例和健康管理科健康志愿者31例，每例各抽取静脉全血10 ml，经过血清分离、透析、低温真空冻干等处理后提取血清蛋白，4℃保存备用。加入不同浓度的蛋白溶液尿毒症血清蛋白溶液为尿毒症组、正常血清蛋白溶液为正常组，完全培养基为对照组。采用CCK-8法对HUVEC细胞增殖能力进行检测；采用流式细胞仪分析HUVEC细胞凋亡能力；于倒置相差显微镜下观察3组培养72 h后的HUVEC细胞形态。使用NO试剂盒检测NO表达水平；采用rt-PCR检测IL-8 mRNA、ET-1 mRNA表达量。结果：同一时间点，3组相比，尿毒症组增殖率明显降低(P < 0.05)；同一时间点，随着浓度的增加，尿毒症组细胞受抑制的程度明显增加(P < 0.05)；同一浓度的血清蛋白溶液，随时间的增加，尿毒症组细胞增殖率明显下降(P < 0.05)。相较于对照组与正常组，尿毒症组不同时间点HUVEC细胞凋亡显著升高，但无差异(P > 0.05)。相较于对照组与正常组，尿毒症组NO分泌显著降低(P < 0.05)，随着浓度的增加NO分泌量显著减少(P < 0.05)。相较于对照组与正常组，尿毒症组IL-8 mRNA表达量显著升高(P < 0.05)，随着浓度的增加IL-8 mRNA表达量显著增加(P < 0.05)。相较于对照组与正常组，尿毒症组ET-1 mRNA表达量显著升高(P < 0.05)，随着浓度的增加ET-1 mRNA表达量显著增加(P < 0.05)。相较于对照组与正常组，尿毒症组eNOS mRNA表达量显著降低(P < 0.05)，随着浓度的增加eNOS mRNA表达量显著降低(P < 0.05)。尿毒症组EMPs表达较高(P < 0.05)。结论：蛋白结合尿毒症毒素能够抑制细胞增殖及NO的分泌，提高IL-8 mRNA、ET-1 mRNA的表达量，促进细胞损伤，提示其是心脑血管并发症的重要诱发因素之一。

Abstract: Objective: To study the damage effect and mechanism of protein binding uremic toxin on vascular endothelial cells in uremic patients. Methods: 42 uremic patients with maintenance hemodialysis in the dialysis room of our hospital and 31 healthy volunteers in the health management department were selected. 10 ml of venous whole blood was extracted from each patient. After serum separa-tion, dialysis, low-temperature vacuum freeze-drying and other treatments, serum protein was ex-tracted and stored at 4˚C for standby. Different concentrations of protein solution were added. Ure-mic serum protein solution was the uremic group, normal serum protein solution was the normal group, and complete culture medium was the control group. The proliferation of HUVEC cells was detected by CCK-8 method; The apoptotic ability of HUVEC cells was analyzed by flow cytometry; The morphology of HUVEC cells cultured for 72 h in three groups was observed under inverted phase contrast microscope. NO expression level was detected using NO kit; rt-PCR was used to detect the expression of IL-8 mRNA and ET-1 mRNA. Results: At the same time point, the proliferation rate of uremia group was significantly lower than that of the three groups (P < 0.05); At the same time point, with the increase of concentration, the degree of cell inhibition in uremia group increased significantly (P < 0.05); In the same concentration of serum protein solution, the cell proliferation rate of uremia group decreased significantly with the increase of time (P < 0.05). Compared with the control group and the normal group, the apoptosis of HUVEC cells in uremia group was significantly increased at different time points, but there was no difference (P > 0.05). Compared with the control group and the normal group, the NO secretion in the uremic group decreased significantly (P < 0.05), and the NO secretion decreased significantly with the increase of the concentration (P < 0.05). Compared with the control group and the normal group, the expression of IL-8 mRNA in the uremic group was significantly increased (P < 0.05), and the expression of IL-8 mRNA was significantly in-creased with the increase of the concentration (P < 0.05). Compared with the control group and the normal group, the expression of ET-1 mRNA in uremic group was significantly increased (P < 0.05), and the expression of ET-1 mRNA was significantly increased with the increase of concentration (P < 0.05). Compared with the control group and the normal group, the expression of eNOS mRNA in the uremic group decreased significantly (P < 0.05), and the expression of eNOS mRNA decreased sig-nificantly with the increase of concentration (P < 0.05). The expression of EMPs was higher in ure-mia group (P < 0.05). Conclusion: Protein binding uremic toxin can inhibit cell proliferation and NO secretion, increase the expression of IL-8 mRNA and ET-1 mRNA, and promote cell damage, sug-gesting that it is one of the important inducing factors of cardiovascular and cerebrovascular com-plications.