高血压病和2型糖尿病患者血清损伤ECV-304细胞的比较研究

目的:以高血压病和2型糖尿病为例进行血瘀证血管内皮细胞损伤模型的比较研究,为中医学"异病同证"理论提供实验依据。方法:取对数生长期ECV-304细胞,分组如下:空白对照组(无血清的DMEM组)、糖尿病血瘀证组(糖尿病血瘀证患者血清)和高血压病血瘀证组(高血压病血瘀证患者血清)。采用噻唑蓝比色法(MTT法)观察细胞活性;在倒置相差显微镜、扫描电镜和透射电镜下观察细胞形态的变化;应用放免法测定内皮素(ET)含量;硝酸还原酶法测定一氧化氮(NO)含量;利用双抗夹心酶联免疫法(ELISA)检测各组细胞培养上清液中内皮细胞蛋白C受体(EPCR)、血管内假性血友病因子(vWF)和血栓调节蛋白(sTM)的含量;应用激光扫描共聚焦显微镜,采用Fluo-3/AM作为荧光指示剂观察细胞内游离钙([Ca2+]i)浓度的变化;并采用荧光探针标记的鬼笔环肽染色法观察细胞肌动蛋白微丝分布的差异。结果:(1)MTT结果显示,10%血清作用下,高血压病血瘀证组的细胞活力低于对照组(P0.05),糖尿病血瘀证组的细胞活力也低于对照组(P0.05),但高血压病血瘀证组的细胞活力与糖尿病血瘀证组差异无显著(P0.05);(2)高血压病血瘀证组的ET水平高于对照组(P0.05),糖尿病血瘀证组也高于对照组(P0.05),且高血压病血瘀证组的ET水平低于糖尿病血瘀证组(P0.05);高血压病血瘀证组的NO水平低于对照组(P0.05),糖尿病血瘀证组也低于对照组(P0.05),且高血压病血瘀证组的NO水平高于糖尿病血瘀证组(P0.05);(3)高血压病血瘀证组的EPCR、vWF和sTM含量高于对照组(P0.05),糖尿病血瘀证组也高于对照组(P0.05),且高血压病血瘀证组的EPCR水平低于糖尿病血瘀证组(P0.05);而高血压病血瘀证组的vWF和sTM含量与糖尿病血瘀证组之间差异无显著(P0.05);(4)高血压病血瘀证组[Ca2+]i高于对照组(P0.05),糖尿病血瘀证组[Ca2+]i高于对照组(P0.05),高血压病血瘀证组胞浆内荧光分布不均匀,[Ca2+]i高于糖尿病血瘀证组(P0.05);(5)高血压病血瘀证组可见细胞骨架微丝减少但排列较为规则,糖尿病血瘀证组的微丝断裂且排列紊乱,二者微丝分布差异显著。结论:高血压病患者血清和糖尿病患者血清对ECV-304细胞骨架、[Ca2+]i及ET、NO和EPCR表达的影响不同,但均能造成ECV-304细胞损伤,这可能是中医学"异病同证"的病理学基础。

Comparison of ECV-304 cells injured by the sera from patients with hypertension and type 2 diabetes mellitus

AIM: To compare the cellular injury model of ECV-304 induced by the sera from patients with the same blood stasis syndrome (BSS) in hypertension and type 2 diabetes mellitus (T2DM). METHODS: Studies were conducted in ECV-304 cell line, which was treated with different sera during growing, named as sera from patients with T2DM (with the sera from patients with BSS associated with T2DM), sera from patients with hypertension (with the sera from patients with BSS associated with hypertension) and control groups. The cell viability was measured by MTT colorimetry, and the morphological changes were identified by light microscopy and electron microscopy. The level of nitric oxide (NO) and endothelin (ET) in the cells was detected by nitric acid deoxidizing enzyme and non-balance method,respectively. Enzyme-linked immunosorbent assay (ELISA) was applied to assay von Willabrand factor (vWF), thrombomodulin (sTM) and endothelial cell protein C (EPCR) content in the cell culture supernatants. Intracellular free calcium ( _i) and cytoskeleton of the model cells were measured by laser scanning confocal microscopy (LSCM). RESULTS: The cells were damaged when treated with 100 mL/L sera for 24h. Compared with the control group, the level of NO, vWF, sTM, EPCR and intracellular calcium concentration in the damaged cells markedly increase while cell viability and the level of ET significantly decreased (P<0.05). Compared with the sera from patients with T2DM group, the excretion of NO and intracellular calcium concentration markedly increased while ET level, EPCR and cytoskeleton significantly decreased (P<0.05) in the cells induced by the sera from patients with hypertension. CONCLUSION: These results suggest that the sera of patients with BSS associated with T2DM or hypertension reduce cell viability and the level of ET, increase the level of NO, vWF, sTM, EPCR and intracellular free calcium ( _i) and damage cell shape and cytoskeleton. The results also clearly show that the changes of ET, NO, EPCR, _i and cytoskeleton in the cells between the sera from patients with the same BSS syndrome associate with T2DM and hypertension, which may partially explain the pathophysiology mechanism of "different disease and identical syndrome" in traditional Chinese medicine.