Observation and Analysis of Coronary Microcirculation by Establishing Rat Myocardial Ischemia and in Vitro CMECs Hypoxia Model
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摘要:
目的 建立在体大鼠心肌缺血与体外心肌微血管内皮细胞（CMECs）缺氧模型，通过其结构及生物学特性变化，探讨冠脉微循环的微血管生成基础。 方法 应用1/3结扎冠脉前降支法建立大鼠心肌缺血模型，利用HE、Masson染色、透射电镜分别检测心肌组织结构及超微结构。采用低氧培养箱建立大鼠CMECs时间梯度缺氧模型（缺氧时间分别设置为0 h，4 h，8 h，12 h，24 h，48 h，72 h），倒置相差显微镜观察CMECs形态特征及生长特点，CCK-8法测定增殖率，计数法测定存活率。ELISA法检测炎症因子（IL-1β、IL-6、TNF-α）以及血管生成因子（VEGF、Ang-2）表达水平。 结果 在冠脉结扎72 h后，HE和MASSON染色提示成功建立大鼠心肌缺血缺氧模型；透射电镜发现细胞超微结构存在缺血缺氧性改变。CMECs具有鲜明的形态特征。随缺氧时间延长，48 h后增殖速率显著下降（P = 0.0426 ）；24 h后存活率显著下降（72.8%）。长期缺氧导致IL-1β（24～72 h，P分别=0.0007 ，0.0007 ，0.001）、IL-6（24～72 h，P分别=0.0015 ，0.0005 ，0.0007 ）和TNF-α（24～72 h，P分别=0.0015 ，0.0063 ，0.0008 ）释放水平显著高于短期缺氧IL-1β（4～12 h，P = 0.007，0.0034 ，0.0009 ）、IL-6（4～12 h，P分别=0.0026 ，0.0013 ，0.0045 ） 和TNF-α（12 h，P =0.0087 ）释放水平。血管生成因子VEGF在缺氧8 h后表达开始升高（P <0.0001 ），在12～24 h（P均<0.0001 ）下降后随即迅速升高（P < 0.01）；Ang-2的表达自4～12 h起表达降低（P < 0.05），自24 h起逐渐增高（P < 0.01）。结论 不同缺血缺氧时间心肌组织和CMECs出现的生物学变化各异，炎症反应在早期即开始出现，血管生成反应在晚期出现。有助于阐明缺血性心肌损伤的关键细胞及分子机制。 Abstract:Objective To establish in vivo rat ischemic myocardial injury and in vitro cardiac microvascular endothelial cell （CMEC） hypoxia models so as to investigate the structural and biological changes and probe the angiogenesis basis of coronary microcirculation. Methods In vivo rat myocardial ischemia model was established using the 1/3 ligation of the left anterior descending coronary artery and myocardial tissue structure and ultrastructure were detected using HE, Masson staining, and transmission electron microscopy, respectively. In vitro time-gradient hypoxia model of rat CMECs （hypoxia times set at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h） was established using a hypoxic incubator. An inverted phase-contrast microscope was used to observe the morphological and growth characteristics of CMECs. The proliferation rate was determined by CCK-8 method, and the survival rate was determined by counting method. The expression of inflammatory factors （IL-1β, IL-6, TNF-α） and angiogenic factors （VEGF, Ang-2） were detected using ELISA method. Results After 72 hours of coronary artery ligation, HE and MASS staining indicated the successful establishment of a rat model of myocardial ischemia and hypoxia. The transmission electron microscopy revealed the ischemic and hypoxic changes in the ultrastructure of cells. . CMECs exhibited the distinct morphological characteristics and adhered to the surface. With the prolonged hypoxia time, the proliferation rate significantly decreased after 48 h （P = 0.0426 ）, and the survival rate significantly decreased after 24 h （72.8%）. Long-term hypoxia led to significantly higher release levels of IL-1β （24～72 h, P =0.0007 ,0.0007 , 0.001）, IL-6 （24～72 h, P =0.0015 ,0.0005 ,0.0007 ）, and TNF-α （24～72 h, P =0.0015 ,0.0063 ,0.0008 respectively） compared to short-term hypoxia IL-1β （4～12 h, P = 0.007,0.0034 ,0.0009 respectively）, IL-6 （4～12 h, P =0.0026 ,0.0013 ,0.0045 respectively）, and TNF-α （12 h, P =0.0087 ）. In addition, the expression of angiogenic factor VEGF began to increase 8 hours after hypoxia （P <0.0001 ）, decreased at 12-24 hours （P <0.0001 respectively）, and then increased rapidly （P < 0.01）; The expression of Ang-2 decreased from 4-12 hours （P < 0.05）, and gradually increased from 24 hours （P < 0.01）.Conclusions Myocardial tissues and CMECs exhibit the different biological changes at different ischemia-hypoxia time points with inflammatory reactions beginning in the early stages and angiogenesis reactions occurring in the late stages. These findings contribute to elucidating the key cellular and molecular mechanisms underlying ischemic myocardial injury. -
图 1 心肌组织HE染色
A：对照组（200×）；B：对照组（400×）；C：模型组（200×）；D：模型组（400×）。
Figure 1. HE staining of myocardial tissue
图 2 心肌组织Masson染色
A：对照组（200×）；B：对照组（400×）；C：模型组（200×）；D：模型组（400×）。
Figure 2. Masson staining of myocardial tissue
图 3 透射电镜观察心肌组织超微结构
A：对照组；B：模型组。
Figure 3. The ultrastructure of myocardial tissue under transmitting electron microscope
图 4 CMECs显微镜普通视野下形态特征（100×）
A：对照组0 h；B：模型组0 h；C：对照组24 h；D：模型组24；E:对照组48 h；F:模型组48 h；G:对照组72 h；H:模型组72 h。
Figure 4. The morphological characteristics of CMECS under general microscope（100×）
图 5 CCK8法检测CMECs增殖状态
注：黑线代表对照组，红线代表缺氧组；*P < 0.05，**P < 0.01。
Figure 5. Proliferation of CMECs detected by CCK8
图 7 ELISA法检测炎性因子IL-1β的浓度
与0 h组比较，**P < 0.01。
Figure 7. The concentration of inflammatory factor IL-1β detected by ELISA
图 9 ELISA法检测炎性因子TNF-α的浓度
与0 h组比较，**P < 0.01。
Figure 9. The concentration of inflammatory factor TNF-α detected by ELISA
图 8 ELISA法检测炎性因子IL-6的浓度
与0 h组比较，**P < 0.01。
Figure 8. The concentration of inflammatory factor IL-6 detected by ELISA
图 10 ELISA法检测血管生成因子VEGF的浓度
与0 h组比较，**P < 0.01。
Figure 10. The concentration of angiogenesis factor VEGF detected by ELISA
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