兔骨髓单个核细胞自体移植对缺血心肌修复重建及心功能的改善

Effect of autoplastic transplantation of rabbit bone marrow mononuclear cells on the restoration and rebuilding of ischemic myocardium and the amelioration of heart function

目的:观察兔骨髓单个核细胞自体移植至缺血心肌后的修复重建能力,以及心功能的改善状况。方法:实验于2005-07/2006-04在安徽省干细胞研究和治疗中心完成。①选取清洁级健康雄性新西兰白兔20只,随机数字表法分为心肌梗死模型组、骨髓单个核细胞组,10只/组。②两组兔均于左心耳下缘结扎前降支冠状动脉左前降支建立心肌梗死模型,心电图出现S-T段弓背样抬高为结扎成功。③2周后于股骨处行骨髓穿刺取5mL骨髓,分离纯化骨髓单个核细胞。骨髓单个核细胞组将Dil标记的含2.5×107细胞数目的细胞悬液400μL注射至心肌梗死周边区域,心肌梗死模型组仅注射等量L-DMEM培养基。③两组分别于造模前、细胞移植前及细胞移植后2,4周行多普勒心脏超声心动图检查,计算左室射血分数、左室短轴短缩率,进行心功能评价。④细胞移植8周后处死全部动物,制备组织冰冻切片,病理学检查移植细胞在梗死区的生长状况,镜下见有Dil标记阳性的细胞认为是存活的移植细胞。普通光学显微镜200倍视野下计数毛细血管数量。结果:骨髓单个核细胞组10只均进入结果分析;心肌梗死模型组因造模死亡4只,6只兔完成整个实验过程。①不同时相点心功能指标超声心动图检查结果:两组造模前、细胞移植前、细胞移植后2周左室射血分数、左室短轴短缩率均基本相似(P>0.05);移植后4周,骨髓单个核细胞组左室射血分数、左室短轴短缩率均明显高于心肌梗死模型组犤(69.1±5.3)%,(62.4±7.8)%;(34.3±8.4)%,(27.2±5.3)%;P均<0.05犦。②骨髓单个核细胞在梗死心肌中的存活情况:荧光显微镜下,骨髓单个核细胞组梗死灶边缘有Dil标记的细胞存活,免疫组化结果显示Dil阳性细胞表达心肌细胞特异性标志α-sarcomericactin。两组均未见炎性细胞浸润,但骨髓单个核细胞组新生毛细血管密度明显高于心肌梗死模型组犤(38.5±2.0)mm-2,(21.4±3.9)mm-2,P<0.05犦。结论:兔骨髓单个核细胞自体移植8周后存活于梗死心肌中,并表达心肌细胞特异性标志,移植后可明显改善左室收缩功能,增加毛细血管密度,但短期内未减轻心室重构的进程。

AIM： To observe the restoration and rebuilding capacities of rabbit bone marrow mononuclear cells （BMMNCs） from autoplastic transplantation to post-myocardial infarction as well as its effect on the amelioration of heart function. METHODS： The experiment was conducted in Anhui Provincial Central Laboratory of Stem Cells from July 2005 to April 2006, ①Twenty healthy male clean New Zealand rabbits were randomly divided into myocardial infarction model group （model group） and the BMMNCs group with 10 rabbits in each group, ②Rabbits in both groups were ligated of the left anterior descending coronary artery below the ＂left auricular appendage to establish myocardial infarction models, The successful myocardial infarction models were recognized by arc-like elevation of the S-T segments in electrocardiogram （EKG）, ③Two weeks later, 5 mL away from the bone marrow was aspirated from the thighbone of the rabbits, The BMMNCs were separated and purified, Rabbits in the BMMNCs group were injected with 400 μL of cell suspension （total 2.5×10^7） labeled by Dil into several different points in the periphery of myocardial infarction, and rabbits in the model group were injected with LoDMEM at the same volume. ④Rabbits in both groups were detected by Doppler echocardiography respectively before modeling, before transplantation and at 2 and 4 weeks after transplantation, The left ventricular ejection fraction （LVEF） and left ventricular fractional shortening （LVFS） were calculated to evaluate the heart function, ⑤All rabbits were executed at eight weeks after cell transplantation to make frozen sections for pathology study. The growth of transplanted cells in infarction was pathologically inspected. Dil-labeled positive cells found under the microscope were considered to be live transplanted cells. The capillary density was counted under 200 magnified visual field with ordinary microscope. RESUILTS： Ten rabbits in the BMMNCs group were involved in the analysis of results, Four rabbits in the model group died for modeling and 6 rabbits accomplished all the tests. ①Detection of the indexes of heart function at different time-points by echocardiography： The LVEF and LVFS between the two groups were fundamentally the same respectively before modeling, before cell transplantation and at 2 weeks after cell transplantation （P 〉 0.05）, while those were obviously higher in the BMMNCs group at 4 weeks after cell transplantation than those in the model group [（69.1±5.3）%, （62.4±7.8）%; （34.3±8.4）%, （27.2±5.3）%;P all 〈 0.05], ②The survival of BMMNCs in infarct myocardium： it could be seen under the fluorescent microscope that Dil-labeled positive cells survived in the border of the infarct myocardium, and immunohistochemical study indicated that the Dil positive cells expressed the specific mark of myocardium α-sarcomeric actin. There was no inflammatory cell infiltrate （ICI） in both groups, but the capillary density was much higher in the BMMNCs group than that in the model group [（38.5±2.0） mm^-2, （21.4±3.9） mm^-2,P 〈 0,05]. CONCLUSION; BMMNCs can survive in the infarct myocardium and express myocardium specific mark at 8 weeks after autoplastic transplantation, which can significantly improve the contractile function of left ventricle and elevate the capillary density, but can not attenuate the process of ventricular remodeling in short-term after transplantation.