休克淋巴液对大鼠肺微血管内皮细胞的损伤作用

无菌条件下复制大鼠重症失血性休克模型,引流肠系膜淋巴液或收集门静脉血,同时,引流正常淋巴液、正常门静脉血。以不同处理因素与原代培养的第三代肺微血管内皮细胞(PMVEC)共同孵育,通过光镜、透射电镜、扫描电镜观察细胞形态及超微结构,MTT法检测不同终浓度的休克淋巴液及正常淋巴液对PMVEC增殖的影响;流式细胞仪检测PMVEC周期变化;同时进行细胞核DNA电泳分析。结果表明,休克淋巴液对PMVEC具有损伤作用,表现为细胞收缩、核固缩等,扫描电镜可观察到凋亡小体;随着休克淋巴液终浓度增加,PMVEC的增殖活力逐渐降低,显著低于正常淋巴液组;4%终浓度的休克淋巴液作用PMVEC 4h后,G0-G1期细胞比值增大,S G2-M期细胞比值下降,其他处理因素无明显变化,同时细胞核DNA电泳形成典型的阶梯状电泳图谱(DNA ladder)。结果提示,休克淋巴液可导致PMVEC形态学及超微结构损伤,同时抑制细胞增殖、影响细胞周期、诱导细胞凋亡。

DAMAGE EFFECTS OF SHOCK LYMPH ON THE PULMONARY MICRO-VASCULAR ENDOTHELIAL CELLS OF RATS

The model of serious hemorrhagic shock was established under the condition of asepsis and mesentery lymph was taken out. As control,normal mesentery lymph fluid,normal portal vein blood,and shock portal vein blood of rats were taken out. The primary pulmonary micro-vascular endothelial cells (PMVECs) of passages 3 were treated by different treatment factors,respectively. The morphology and ultrastructure changes of PMVECs were observed under optics microscope, transmit electronic microscope and scan electronic microscope. The cells proliferation under different final concentrations of shock lymph and normal lymph were measured with MTT method. The cell cycle arrest was analyzed by flow cytometry and the DNA of cell nucleus was analyzed by electrophoresis. The results showed that shock lymph played a role in damaging PMVECs. The contracted cells and condensed nucleus were found,and apoptosic bodies were observed under a transmission electron microscope. The proliferation of PMVECs was decreased when the concentration of shock lymph increased to some extent and showed statistic significance compared with normal lymph group. The cells cocultured with shocking lymph fluid at 4% final concentration showed that G0-G1 cell population was higher and the proportion of S G2-M cell population was lower than that of other groups,and the DNA ladder was observed in electrophoresis of cell nucleus DNA at same time. The results demonstrated that shock lymph could damage the morphology and ultrastructure of PMVECs, reduce the cells proliferation,interfere with cell cycle, and induce the apoptosis.