经冠状动脉自体骨髓单个核细胞移植对扩张型心肌病兔心肌组织病理学的影响

Cardiac pathological changes following autologous intracoronary bone marrow mononuclear cells transplantation in rabbits with dilative cardiomyopathy

目的:骨髓干细胞在扩张型心肌病动物模型中具有向心肌样细胞和血管内皮细胞分化的潜能。评价经冠脉自体骨髓单个核细胞移植治疗对扩张型心肌病兔心肌组织病理学影响及心功能的变化。方法:实验于2006-09/2007-03在复旦大学附属华山医院心血管研究室完成。①实验材料:3月龄雄性新西兰兔25只,随机数字表法分为正常组5只、模型组10只、冠脉移植组10只。②实验方法:冠脉移植组、模型组兔经耳缘静脉注射盐酸阿霉素建立扩张型心肌病模型。冠脉移植组10只兔麻醉后以髂骨为穿刺点,共采集15mL混有肝素生理盐水的骨髓液,密度梯度离心得到自体骨髓单个核细胞悬液2mL,其中5只兔的骨髓单个核细胞悬液行DAPI标记,经塑性的4F造影导管,模拟临床冠脉内输注细胞悬液。③实验评估:术后4周通过检测血流动力学指标评价心功能的改变;苏木精-伊红、天狼猩红染色观察心肌组织形态学特点,透射电镜观察心肌组织超微结构的改变;TnT间接免疫荧光检测心肌组织DAPI标记情况,以评价骨髓单个核细胞的分化和转归。结果:①血流动力学指标的测定:与基础值比较,细胞移植4周后模型组兔血流动力学指标无明显改变(P>0.05),而冠脉移植组心功能显著改善(P<0.05)。②心肌组织形态学观察:扩张型心肌病兔心肌纤维排列紊乱,坏死区大量淋巴细胞浸润,细胞间隙增宽,心肌间质面积增大。③心肌组织超微结构的变化:造模结束后,模型组心肌细胞线粒体肿胀,肌丝断裂,心肌细胞核呈锯齿状,心肌间质中淋巴细胞浸润,胶原纤维增多。细胞移植4周后,冠脉移植组出现线粒体堆积现象,亦存在巨噬细胞浸润和胶原纤维轻度增生,未发现心肌组织中存在植入的特殊类型细胞。④心肌组织TnT间接免疫荧光检测:DAPI标记的骨髓单个核细胞在心肌组织中富集,血管腔内不存在。结论:①经冠脉自体骨髓单个核细胞移植治疗阿霉素扩张型心肌病兔,利于植入细胞在心肌组织中富集,改善心功能。②透射电镜尚无法证实心肌组织中存在外来的移植细胞。

AIM： Bone marrow-derived stem cells have the multipotency of differentiation into cardiac-like cells and vascular endothelial cells in animal models of dilative cardiomyopathy （DCM）. In this study, the cardiac pathological and function changes in dilative cardiomyopathic rabbits after autologous intracoronary bone marrow mononuclear cells （BMMNCs） transplantation. METHODS： The experiment was performed in the Cardiovascular Laboratory of Huashan Hospital, Fudan University from September 2006 to March 2007. （1）Twenty-five 3-month-old male New Zealand rabbits were randomly divided into normal group （n =5）, DCM group （n =10） and intracoronary transplantation group （n =10）. （2）Adriamycin was injected in intracoronary transplantation group and DCM group through ear-border vein to establish DCM models. After the rabbits of intracoronary transplantation group were anesthetized, ilium was punctured. BMMNCs that ware separated from 15 mL bone marrow mixed with normal saline by density gradient centrifugation ware resuspended into 2 mL. Of them, BMMNCs of 5 rabbits were labeled with DAPI and infused into left coronary artery through plastic 4F catheter, which was similar to intracoronary BMMNCs transplantation in clinical trials. （3） Four weeks after transplantation, indexes of hemodynamics were measured to evaluate the cardiac function again. Histomorphological changes ware evaluated by myocardium tissues hematoxylin ＆ eosin staining and Sirius red staining. Cardiac ultrastructure characteristics ware investigated by transmission electron microscopy, and the differentiation and attribution of BMMNCs labeled with DAPI ware detected by indirect immunofluorescence staining for troponin T （TnT）. RESULTS： （1）Compared with baseline characteristics, after 4 weeks of intracoronary transplantation, the examination of hemodynamic indexes indicated there was no statistical significance in DCM group （P 〉 0.05）, while cardiac function was improved significantly in intracoronary transplant group （P 〈 0.05）. （2）Cardic muscle fibers distributed disorderly with massive lymphoid cell infiltration in necrotic area and cardiac matrix area was increased significantly in DCM rabbits. （3） Mitochondrial swelling, filaments breakage, massive lymphoid cell infiltration and collagen hyperplasia ware found by transmission electron microscopy in adriamycin-induced DCM model. After 4 weeks of intracoronary transplantation, the number of mitochondria was found with infiltration of macrophages and mild hyperplasis in the collagen of myocardium, while no evidence indicated the existence of specific transplanted cells in myocardial tissues. （4） Indirect immunofluorescence staining for TnT showed that DAPI labeling BMMNCs were enriched in damaged myocardial tissue, while no contribution of immune fluorescent staining was observed in vascular lumen. CONCLUSION： （1）Autologous intracoronary BMMNCs transplantation significantly improves the cardiac function of DCM rabbits by enrichment of BMMNCs in myocardium. （2）No evidence indicates there are specific transplanted cells by transmission electron microscopy.