摘要
目的对大鼠的心肌微血管内皮细胞(myocardial microvascular endothelial cells,MMVECs)离体培养方法进行改良,建立该细胞的缺氧模型。方法取出7日龄左右的Wistar大鼠的心脏,用胰蛋白酶和胶原酶二次消化法获得MMVECs,密度梯度离心法纯化细胞,计算细胞纯度;用免疫细胞化学方法检测微血管内皮细胞特异性第Ⅷ因子、CD34及CD31相关抗原;将原代培养的细胞置于持续通入体积分数为1%O2、5%CO2和94%N2的自制缺氧装置中,并在37℃孵箱中分别缺氧处理4、6、12、18、24h,各时间段均设置正常对照组;用四甲基偶氮唑盐(MTT)比色实验检测缺氧对大鼠MMEVCs增值活力的影响,Hoechst33342染色观察缺氧诱导该细胞凋亡形态学变化。结果细胞形态特征及相关抗原检测证实:与仅用二次消化法培养细胞相比,密度梯度离心纯化后所得MMVECs纯度提高(P<0.01);MTT比色实验:随着培养时间的延长,与正常对照组相比,各缺氧组OD值均有所降低,其中缺氧12、18和24h的OD值降低显著(P均<0.01);从缺氧12h开始,各缺氧组的OD值逐渐降低,与12h相比均有显著差异(P<0.01或P<0.05);Hoechst33342染色:缺氧12h细胞开始出现核固缩、碎裂、蓝色浓染等典型的凋亡形态学变化,随着缺氧时间延长该变化更为明显。结论该原代离体培养方法可以获得纯度较高的MMVECs;缺氧对MMVECs的增殖有抑制作用,本方法可以简便、有效地建立MMVECs的缺氧模型。
Objective To improve the procedures of in vitro culture of the myocardial microvascular endothelial cells （MMVECs） of rats primarily and establish the hypoxie cell model of MMVECs. Methods The hearts of the Wistar（ about 7 days of age） rats were taken and the method of trypsin and eollagenase digestion was used to obtain cells, then the gradient centrifugation was applied to purify the cells and the purity of cells was measured. Factor Ⅷ, CD34 and CD31, the special related antigens of endothelial cells,were tested by immuno- cytochemistry. The cells were incubated in a closed container with continuous follow of 1% O2,5% CO2 and 94% N2 for 4,6,12,18,24h respectively and normal control groups of each time period were established. The viability of ceils were evaluated by MTT activity assay and hypoxia induced cell morphologic changes of apoptosis were assessed by Hoechst 33342 staining. Results The morphology and im- munocytochemistry results showed that the purity of MMVECs was improved by gradient centrifugation （P 〈 0.01 ）. MTT activity assay showed that compared to control groups, the OD value of hypoxic groups decreased, among which the group of 12,18 and 24h decreased significantly（ P 〈 0.01 ）. The OD value of hypoxic groups began to decrease gradually from 12h and differences were significant（ P 〈 0.01 or P 〈 0.05 ）. After 12h of hypoxia, cells began to show typical morphologic changes of apoptosis, such as condensation and fragmentation of nucleus and it became obvious with the increasing time of hypoxia. Conclusion The improved in vitro culture method of MMVECs can get ceils with relative high purity to perform hypoxic tests. The hypoxia inhibits the viability of MMVECs and the present method can estab- lish a successful hypoxic model of MMVECs.
出处
《医学研究杂志》
2013年第5期128-131,共4页
Journal of Medical Research
关键词
心肌微血管内皮细胞
缺氧
Myocardial microvascular endothelial cells
Hypoxia
