大鼠缺血心肌微血管内皮细胞血管新生的生物学特征

Biological characteristics of angiogenesis of microvascular endothelial cells in rat with myocardial ischemia

关键词:  缺血心肌  微血管内皮细胞  血管新生  生物学特征

Key words:  ischemic myocardium  microvascular endothelial cell  angiogenesis  biological characteristics

摘要:

[目的] 观察大鼠缺血心肌微血管内皮细胞（CMECs）血管新生的生物学特征。[方法] 采用结扎法建立大鼠心肌缺血模型，植块法培养CMECs，倒置相差显微镜观察细胞形态学特征，免疫细胞化学法检测CMECs特异性抗原。实验分组将大鼠缺血CMECs作为缺血组（I组），正常大鼠CMECs作为对照组（N组），采用噻唑蓝（MTT）法绘制细胞生长曲线并检测细胞增殖能力，划痕法测定细胞迁移能力，倒置相差显微镜观察管腔结构形成情况。采用实时荧光定量聚合酶链式反应法（Real-time PCR）检测细胞增殖相关蛋白激酶（ERK）和细胞死亡相关分子（p53）mRNA的表达情况。[结果] 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为阳性。N组和I组迁移窗口期均为第1天，成管窗口期均为第2天，N组和I组的增殖窗口期分别为第3天、第6天。两组比较，I组迁移率、增殖率和成管率均低于N组，以迁移率降低最为明显（P<0.01）.I组ERK、p53 mRNA表达均高于N组（P<0.05）.[结论] 缺血CMECs的血管新生能力明显降低，血管新生增殖窗口期改变，可能与ERK和p53 mRNA表达的变化有关，为进一步研究其基因组学及药物干预提供实验依据。

Abstract:

[Objective] To observe the biological characteristics of angiogenesis of cardiac microvascular endothelial cells in rat with myocardial ischemia. [Methods] The model of myocardial ischemia was established with ligation method. The cardiac microvascular endothelial cells (CMECs)were cultured by the method of planting myocardium tissue. The morphology of CMECs were observed by inverted phase contrast microscope. Its specific antigen was observed by immunocytochemistry. CMECs of ischemic rats was served as ischemia group (group I), CMECs of normal rats was served as control group (group N); MTT were used to draw the cell growth curve and detect the proliferation. The cell migration ability were measured by wound healing assay, tube-like structure formation were observed by inverted phase contrast microscope; Real-time PCR were used to detect the mRNA expression of ERK and p53.[Results] The CMECs of rats had the typical characteristics of microvascular endothelial cells, factor Ⅷ,CD31 related antigen were all positive by immunocytochemical stain identification. The transfer window period were the first day, and the tube formation window period were the second day and the proliferation window period were respectively the third day, and the sixth day of group N and I. When compared two groups, rate of migration, proliferation and tube formation in the group I were lower than group N. The most obvious reducing was migration rate (P<0.01). The mRNA expression of ERK and p53 in the group I were higher than that in group N(P<0.05).[Conclusion] The angiogenesis capacity of ischemic CMECs is decreased obviously. The angiogenesis proliferation window period is changed, and this may be related to the change of mRNA expression of ERK and p53. This can provide some experimental basis for the further study of genomics and drug intervention.