体外培养大鼠心肌微血管内皮细胞缺氧模型的建立

Establishment of Hypoxic Model of Culturing Cardiac Microvascular Endothelial Cells in Vitro

目的建立大鼠心肌微血管内皮细胞(CMECs)缺氧模型。方法植块法培养CMECs,免疫细胞化学鉴定微血管内皮细胞特异性标志。将原代培养的CMECs在1%O2-94%N2-5%CO2低氧气体环境中培养24h制备低氧损伤模型(L组),以无血清常氧培养作为对照组(N组)。倒置相差显微镜观察缺氧前后细胞形态变化,MTT法测定细胞活力,Annexin V-FITC和PI双标记法测定细胞凋亡率。结果植块法培养的大鼠CMECs具备典型微血管内皮细胞特征;Ⅷ因子、CD31相关抗原免疫染色鉴定均阳性。缺氧前后细胞形态无明显变化;与N组比较,L组细胞活力明显增加(P<0.05);细胞凋亡率明显降低(P<0.05),其中早期凋亡降低最明显(P<0.01)。结论本方法可以成功建立简便、易行的CMECs细胞缺氧模型。

Objective To establish a hypoxic model of culturing cardiac microvascular endothelial celis（CMECs） in vitro. Methods The CMECs were cultured by the method of planting myocardium tissue. Its specific antigen were observed by immunocytochemistry. The original generation of cultivating CMECs in 1% O2 -94% N2 and 94% CO2 24 h hypoxic gas environment to develop the preparation of low oxygen damage model（L） ,with normal serum free oxygen training as the control group（N）. Inverted phase contrast microscope observation of cell morphological changes before and after hypoxia, MTT determinate cell vitality, Annexin V- FITC and PI double marking method to determine cell apoptosis rate. Results Cultured rat CMECs has the typical characteristics of microvaseular endothelial ceils. VIII factor,CD31 related antigen immune staining were positive identification. No obvious change in morphology before and after cells anoxic. Compared to the N group,cell vitality in L group was obviously increased（P〈0.05）. Cell apoptosis rate significantly reduced（P〈0.05）. The early apoptosis reduced the most obvious（P〈0.01）. Conclusion This method can successfully establish an simple and easy CMECs cell hypoxia model.