休克淋巴液诱导大鼠肠系膜微淋巴管内皮细胞凋亡

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,另取动物引流正常淋巴液、正常门静脉血。以不同处理因素与第3代原代培养的肠系膜微淋巴管内皮细胞(MMLEC)共同孵育,MTT法检测不同终浓度的休克淋巴液及正常淋巴液对MMLEC增殖的影响;流式细胞仪检测MMIEC增殖周期变化、并进行细胞核DNA电泳分析;RT-PCR检测凋亡相关基因bcl-2、bax及fas、fas L的表达。结果表明,随着休克淋巴液终浓度增加,MMLEC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MMLEC 4h后,MMLEC凋亡率为(35.4±1.6)%,C_0-G_1期细胞比值增大,S G_2-M期细胞比值下降,其它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)。4%终浓度的休克淋巴液作用4h后,显著高于其它各组(P<0.01);4%终浓度的休克淋巴液作用6h后,MMLEC的fas、fas L、bax mR- NA表达高于其它组、bcl-2 mRNA表达低于其它组(P<0.01)。结果提示,休克淋巴液可抑制MM- LEC增殖、干扰细胞周期、上调凋亡促进基因表达.从而诱导细胞凋亡。

EFFECTS OF SHOCK LYMPH INDUCING THE MESENTERY MICRO-LYMPHATIC ENDOTHELIAL CELLS APOPTOSIS OF RATS

The model of serious hemorrhagic shock was established in 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 mesentery micro-lymphatic endothelial cells (MMLECs) of passages 3 were treated by different treatment factors, respectively. The cells proliferation by shock lymph and normal lymph of different final concentrations was measured using MTT method. The cell cycle arrest was analyzed by flow cytometry, and the electrophoresis analyze on DNA of cell nucleus was observed. The expressions of relative genes of apoptosis such as fas, fasL, bcl-2 and bax of MMLECs were detected by RT-PCR. The results showed that the MMLECs proliferation was decreased when the concentration of shock lymph increased to some extent and showed statisticly significant difference compared with normal lymph group. The cells cocultured with shocking lymph fluid at 4% final concentration showed that apoptosis rate of MMLECs was (35.4 +/- 1.6)%, and 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. And the expressions level of fas, fasL and bax mRNA were higher and bcl-2 mRNA was lower in shock lymph group than that of control group. The results demonstrated that shock lymph could reduce the cells proliferation, interfere with cell cycle, and accelerate high expression of apoptosis accelerative genes. As a result, shock lymph could induce the MMLECs apoptosis.