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目的:进行兔骨髓间充质干细胞(MSCs)穴位移植1天与7天对梗死区心肌细胞凋亡的对比研究。方法:将已建立心肌梗死模型的日本大耳白兔随机分为3组,包括模型组、穴位移植干细胞1天组、穴位移植干细胞7天组。用开胸结扎冠状动脉前降支的方法建立急性心肌梗死模型,将梗死后的兔分两组分别在1天与7天穴位注射相同数量的已经过分离、培养、扩增、标记后的MSCs悬液。采用脱氧核苷酸末端转移酶介导原位缺口标记(TUNEL法)确定细胞凋亡数,免疫组化SABC方法及RT-PCR和蛋白印迹法观察心肌细胞中Fas、FasL、Bcl-2蛋白及mRNA的表达。结果:(1)免疫组化示:梗死区凋亡细胞的表达,模型组高于正常组、穴位注射干细胞1天组和穴位注射干细胞7天组(P<0.05),穴位注射干细胞7天组低于穴位注射干细胞1天组(但无统计学差异P>0.05);Fas蛋白的表达,模型组高于正常组、穴位注射干细胞1天组和穴位注射干细胞7天组,穴位注射干细胞7天组低于穴位注射干细胞1天组(P<0.05);FasL蛋白的表达,穴位注射干细胞1天组高于正常组、模型组、穴位注射干细胞7天组(P<0.05);Bcl-2蛋白的表达,模型组低于正常组、穴位注射干细胞1天组和穴位注射干细胞7天组(P<0.05),穴位注射干细胞7天组高于穴位注射干细胞1天组(但无统计学差异P>0.05)。(2)RT-PCR与Western Blotting均显示:Fas、FasL、Bcl-2蛋白mRNA的表达穴位移植干细胞1天均高于穴位移植干细胞7天组。结论:(1)穴位移植骨髓间充质干细胞可使梗死及周围区心肌细胞凋亡数减少,其机制可能是通过Fas、FasL蛋白的减少、Bcl-2蛋白的增多来调节的。(2)穴位移植骨髓间充质干细胞7天较穴位移植骨髓间充质干细胞1天更能减少凋亡细胞的数量。
OBJECTIVE: To compare the apoptosis of cardiomyocytes in infarction zone induced by 1 and 7 days after MSCs transplantation in rabbits. Methods: The Japanese white rabbits with established myocardial infarction model were randomly divided into three groups, including model group, acupoint-grafted stem cells for 1 day and acupoint-derived stem cells for 7 days. Acute myocardial infarction model was established by thoracotomy for ligation of the anterior descending coronary artery. The infarcted rabbits were injected with the same number of isolated, cultured, expanded and labeled MSCs Suspension. The number of apoptotic cells was determined by TUNEL method. The expression of Fas, FasL and Bcl-2 protein in cardiomyocytes was detected by immunohistochemical SABC method and RT-PCR and Western blotting mRNA expression. Results: (1) The expression of apoptotic cells in the infarct area in the model group was significantly higher than that in the normal group (P <0.05); the number of the stem cells in the model group was 7 days (P <0.05). The expression of Fas protein in the model group was higher than that in the normal group, the acupoint injection of stem cells for 1 day and acupoint injection of stem cells for 7 days and the injection of stem cells for 7 days (P <0.05). The expression of FasL protein was higher in the 1 day group than that in the normal control group, model group and acupoint injection of stem cells for 7 days (P <0.05). The expression of Bcl-2 protein (P <0.05). The number of acupoints injected with stem cells for 7 days was higher than that of acupoints injected with stem cells for 1 day (there was no significant difference between the two groups > 0.05). (2) Both RT-PCR and Western Blotting showed that the expression of Fas, FasL and Bcl-2 mRNA in Apoptotic stem cells was higher than that in Apoptosis stem cells for 7 days. Conclusion: (1) Acupoint transplantation of bone marrow mesenchymal stem cells can reduce the number of myocardial infarction and myocardial cell apoptosis, the mechanism may be reduced by Fas, FasL protein, Bcl-2 protein to regulate. (2) Apoptotic bone marrow mesenchymal stem cells can reduce the number of apoptotic cells more than that of bone marrow mesenchymal stem cells one day after transplantation.