同种异体骨髓间充质干细胞穴位注射零天与七天治疗急性心肌梗死兔的对比研究

【作者】 罗智博；

【导师】 杨关林；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:探讨兔骨髓间充质干细胞(MSCs)穴位注射对缺血心肌细胞的修复重建能力,同时进行兔骨髓间充质干细胞(MSCs)穴位注射零天与七天对梗死区心肌细胞凋亡的对比研究。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。方法:1.将已建立心肌梗死模型的日本大耳白兔随机分为3组,模型组、穴位注射干细胞零天组、穴位注射干细胞七天组。用开胸结扎冠状动脉左室支的方法建立急性心肌梗死模型,对梗死后的兔分两组分别在零天与七天穴位注射相同数量的已经分离、培养、扩增、标记后的MSCs悬液。在梗死后五周分别对各组兔心肌梗死区及周边区进行Brdu免疫组化染色以观察穴位注射MSCs是否迁移至梗死区,对Brdu染色阳性的细胞复染心肌特异性肌钙蛋白T染色以观察迁移的细胞是否分化及分化的性质。2.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.穴位注射MSCs各组梗死区组织免疫组化Brdu染色阳性;心肌特异性肌钙蛋白T染色阳性。2.免疫组化示:梗死区凋亡细胞的表达,模型组高于正常组、穴位注射干细胞零天组和穴位注射干细胞七天组(P＜0.05),穴位注射干细胞七天组低于穴位注射干细胞零天组,但无统计学差异(P＞0.05);Fas蛋白的表达,模型组高于正常组、穴位注射干细胞零天组和穴位注射干细胞七天组,穴位注射干细胞七天组低于穴位注射干细胞零天组(P＜0.05);FasL蛋白的表达,穴位注射干细胞零天组高于正常组、模型组、穴位注射干细胞七天组(P＜0.05);Bcl-2蛋白的表达,模型组低于正常组、穴位注射干细胞零天组和穴位注射干细胞七天组(P＜0.05),穴位注射干细胞七天组高于穴位注射干细胞零天组,但无统计学差异(P＞0.05)。3.RT-PCR与Western Blot显示:与正常组比较,Fas mRNA水平:模型组和穴位注射干细胞的两组明显升高(P＜0.05);FasL mRNA水平:模型组和穴位注射干细胞的两组明显升高(P＜0.05);Bcl-2 mRNA水平:模型组明显下降(P＜0.05),穴位注射干细胞的两组有所升高,但升高程度不同,穴位注射零天组明显升高(P＜0.05),穴位注射七天组虽有升高但无统计学意义(P＞0.05)。与模型组比较,Fas mRNA水平:穴位注射干细胞的两组明显下降(P＜0.05);FasL mRNA水平:穴位注射干细胞的两组明显下降(P＜0.01);Bcl-2 mRNA:穴位注射干细胞的两组明显升高(P＜0.05)。与穴位注射干细胞零天比较,Fas mRNA水平:穴位注射干细胞七天组明显下降(P＜0.05);FASL mRNA水平:穴位注射干细胞七天组明显下降(P＜0.05);Bcl-2 mRNA水平:穴位注射干细胞七天组也明显下降(P＜0.05)。与正常组比较,Fas与FasL蛋白水平:模型组和穴位注射干细胞的两组明显升高(P＜0.05);Bcl-2蛋白水平:模型组明显下降(P＜0.05),穴位注射干细胞的两组有所升高,但升高程度不同,穴位注射零天组明显升高(P＜0.05),穴位注射七天组明显升高(P＜0.05)。与模型组比较,Fas蛋白:穴位注射干细胞的两组明显下降(P＜0.05);FasL蛋白:穴位注射干细胞的两组明显下降(P＜0.05);Bcl-2蛋白:穴位注射干细胞的两组明显升高(P＜0.05)。与穴位注射干细胞零天比较,Fas蛋白:穴位注射干细胞七天组明显下降(P＜0.05);FASL蛋白:穴位注射干细胞七天组明显下降(P＜0.05);Bcl-2蛋白:穴位注射干细胞七天组也明显下降(P＜0.05)。结论:1.穴位注射兔骨髓间充质干细胞可迁移至梗死区,并可以在梗死区分化为类心肌样细胞。2.穴位注射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。3.穴位注射骨髓间充质干细胞七天较穴位注射骨髓间充质干细胞零天更能减少凋亡细胞的数量。更多还原

【Abstract】 Objective: To investigate the reconstruction capacity of rabbit bone marrow-derived mesenchymal stem cells (MSCs) Point-cell transplantation for ischemic myocyte, at the same time to make a comparative research on cardiomyocyte apoptosis in infarct area between rabbit bone marrow mesenchymal stem cells (MSCs) transplantation Point of zero-day and seven days. To determine the number of cell apoptosis by TUNEL, and to observe the expression of Fas, FasL, Bcl-2 protein and mRNA in myocardial cells by immunohistochemistry SABC methods, RT-PCR and Western blot.Methods: (1) The Japan rabbits that have been established model of myocardial infarction were randomly divided into 3 groups: the model group, the group with point transplantation of stem cell (0 day) , the group with point transplantation of stem cell (7 days). By the method of thoracotomy with anterior descending coronary artery ligation to establish acute myocardial infarction model, the infarction rabbits were injected with the same volume of separated, cultured, amplified, marked MSCs suspension apartly on 0 day and 7th day. In the five weeks after infarction, immunohistochemical staining Brdu is carried out in myocardial infarction area in rabbits of each group respectively to observe whether the migration of MSCs transplantats to the infarct area. The positive cells in Brdu staining were stained again by cardiac-specific troponin T in order to observe whether the migration of cells differentiate and the nature of differentiation. (2) With the method of TUNEL to determine the number of apoptosis cells, and with the method of SABC, RT-PCR and Western blot to observe the myocardial cells expressing in Fas, FasL, Bcl-2 protein and mRNA. Results: (1) There were Brdu positive cells and cTnT positive protein in Point injection MSCs group in infarct area. (2) Immunohistochemistry showed: The expression of apoptotic cells in infracted area in model group were more than normal group, the point of stem cell transplantation group(0 day)and the point of stem cell transplantation group (7 days) (P<0. 05), the expression of apoptotic cells in the point of stem cell transplantation group(7 days) was less than the point of stem cell transplantation group(0 day) (but no significant difference P>0. 05); the expression of Fas protein:the model group was more than the normal group, the point of stem cell transplantation group(0 day) and the point of stem cell transplantation group(7 days), the point of stem cell transplantation group(7 days) was less than the point of stem cell transplantation group(0 day) ; the expression of FasL protein: the point of stem cell transplantation group(0 day)more than the normal group, the model group and the point of stem cell transplantation group(7 days) (P<0. 05) .The expression of Bcl-2 protein: the model group was less than the normal group, the point of stem cell transplantation group(0 day) and the point of stem cell transplantation group(7 days)(P<0. 05) ,the point of stem cell transplantation group(7 days)more than the point of stem cell transplantation group(0 day) (but no significant difference P>0. 05). (3) RT-PCR and Western Blotting showed: Compared with the normal group, Fas mRNA levels: the model group and the two groups with point injection of stem cells was significantly increased (P <0. 05); FasL mRNA levels: the model group and the two groups with point injection of stem cells was significantly increased (P <0.05); Bcl-2 mRNA levels: Model group decreased significantly (P <0.05), the two groups with point injection of stem cells has increased, but increased in varying degrees, the group with point transplantation of stem cell (0 day) was significantly higher (P <0. 05), the group with point transplantation of stem cell(7days) also increased , but statistical significance. Compared with model group, Fas mRNA levels: the two groups with point injection of stem cells decreased significantly (P <0. 05); FasL mRNA levels: the two groups with point injection of stem cells decreased significantly (P <0. 01); Bcl-2 mRNA: the two groups with point injection of stem cells increased significantly (P <0. 05) Compared with the group with point transplantation of stem cell(0 day), Fas mRNA levels: stem cells, the group with point transplantation of stem cell (7 days) decreased significantly (P <0. 05); FASL mRNA levels: the group with point transplantation of stem cell (7 days) decreased significantly (P <0. 05); Bcl-2 mRNA levels: the group with point transplantation of stem cell (7 days) was also significantly decreased (P <0. 05). Compared with the normal group, Fas and FasL protein levels: the two groups with point injection of stem cells was significantly increased (P <0. 05); Bcl-2 protein levels: the model group decreased significantly (P <0. 05), The two groups of acupoint Injection Stem Cells have increased but increased in varying degrees, the group with point transplantation of stem cell (0 day) was significantly higher (P <0. 05), the group with point transplantation of stem cell (7 days) was significantly higher(P <0. 05). Compared with model group, Fas protein: the two groups with point injection of stem cells decreased significantly (P <0. 05); FasL protein: the two groups with point injection of stem cells decreased significantly (P <0. 05); Bcl-2 protein: the two groups with point injection of stem cells increased significantly (P <0. 05).Compared with the group with point transplantation of stem cell (0 day), Fas protein: stem cells, the group with point transplantation of stem cell (7days) decreased significantly (P <0. 05) ; FASL protein: the group with point transplantation of stem cell (7 days) decreased significantly (P <0.05) ; Bcl-2 protein: the group with point transplantation of stem cell (7 days) was also significantly decreased (P <0.05).Conclusion: (1) Point transplantation of bone marrow mesenchymal stem cells, can moved to the infarct zone, and can differentiate into myocardial cells; (2) Point transplantation of bone marrow mesenchymal stem cells can reduce the number of cardiomyocyte apoptosis in the infracted and surrounding zone, which may be caused by the reduction of Fas, FasL and the increasing of Bcl-2 to adjustments. (3) Bone marrow-derived mesenchymal stem cells Point-cell transplantation for 7 days can reduce more apoptosis cells than bone marrow-derived mesenchymal stem cells Point-cell transplantation for 0 day.更多还原