细胞膜微粒对血管内皮细胞功能的影响

目的 探讨细胞膜微粒(Mp)在激素相关性股骨头缺血坏死(ANFH)发生机制中所起的作用.方法 选择体外培养的第3代人脐静脉内皮细胞(HUVEC)作为研究对象.从健康人、股骨头坏死患者和使用大剂量激素治疗的非股骨头坏死患者血液中分离出的细胞膜微粒作为刺激因子.将不同来源的细胞膜微粒与HUVEC共同培养一段时间后观察细胞形态学变化,并用流式细胞仪检测细胞培养基中膜微粒种类和数量的改变,反转录聚合酶链反应(RT-PCR)检测内皮细胞凋亡基因的表达.结果 倒置相差显微镜观察细胞:发现各组内皮细胞的形状、密度、生长速度和细胞核的形态差异无统计学意义.流式细胞仪分析显示,CD62E+/CD31+代表细胞活跃程度.共培养48 h后,激素治疗的实验组CD62E+/CD31+明显低于对照组(P=0.035),其他各实验组和对照组差异无统计学意义(P＞0.05).RT-PCR分析显示,fas/β-肌动蛋白代表凋亡基因的表达.激素性ANFH的实验组fas/β-肌动蛋白明显高于对照组(0.776±0.230＞0.669±0.148,P=0.006).激素治疗的实验组高于对照组(0.914±0.226＞0.832±0.200,P=0.005).其余各实验组和对照组之间差异无统计学意义(如:酒精性ANFH 0.810±0.252＞0.781±0.194,P＞0.05).结论 使用激素患者的细胞膜微粒在体外有促进血管内皮细胞凋亡的作用.激素性股骨头坏死患者的细胞膜微粒在体外能够增加血管内皮细胞凋亡基因的表达,但是对内皮细胞释放膜微粒无明显影响.健康人和酒精性股骨头坏死患者的细胞膜微粒在体外对血管内皮细胞的凋亡无明显影响.

Impacts of microparticles on vascular endothelial cells

OBJECTIVE: To investigate the effects of microparticles (Mps) from different people on the vascular endothelial cell function. METHODS: Third generation human umbilical vein endothelial cells (HUVECs) were cultured with Mps-containing plasma samples from 5 systemic lupus erythromatosus (SLE) patients undergoing heavy steroid treatment, 5 patients with steroid-induced avascular necrosis of femoral head (ANFH), 4 patients with alcohol induced ANFH, and 4 healthy persons for 12 h, 24 h, and 48 h respectively. Plasma samples from the above persons with the Mps filtered were used as experimental controls. HUVECs cultured with blank culture fluid were used as blank controls. Inverse phase contrast microscopy was used to observe the morphology of the HUVECs. PE-CD31 and PEcy5-CD62E were added into the flow cytometric test tubes and flow cytometry (FC) was used to count the number of CD62E +/ CD31 + Mps in the medium. RT-PCR was used to measure the mRNA expression of the apoptotic gene fas (fas/beta-actin). RESULTS: Microscopy showed no distinct difference between the morphology of the HUVECs among the different groups. FC showed that the number of CD62E +/CD31 + Mps 48 hours after the 20% Mp stimulation of the steroid-treated SLE group was significantly lower than that of the control group (P = 0.035). RT-PCR showed that 48 hours after the stimulation the levels of mRNA fas/beta-actin of the steroid-treated SLE group and steroid induced ANFH group were 0.914 +/- 0.226 and 0.776 +/- 0.230 respectively, both significantly higher than those of the control groups (0.832 +/- 0. 200 and 0.669 +/- 0.148 respectively, P = 0.005 and P = 0.006). CONCLUSION: The Mps from the steroid treated patients aggravate the apoptosis of HUVECs, and the Mps from the steroid induced ANFH patients augment the production of apoptosis gene in HUVECs. The Mps from healthy people and alcohol induced ANFH patients have no relationship with HUVEC apoptosis.