体外纯化后和诱导后骨髓间充质干细胞与骨髓单个核细胞移植治疗心肌梗死的效果比较

In vitro purified and induced bone mesenchymal stem cells transplantation versus bone marrow mononuclear cells transplantation in treating myocardial infarction

目的:将骨髓单个核细胞、体外培养纯化后及诱导后的骨髓间充质干细胞注射至大鼠心肌梗死区域,观察移植细胞的增殖及分化情况,分析其对缺血心肌细胞修复重建能力与心功能改善的影响。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。取清洁级健康雄性新西兰白兔40只,按随机数字表法分为4组:骨髓间充质干细胞组、5-杂氮胞苷诱导组、骨髓单个核细胞组、对照组,10只/组。均于左心耳下缘结扎冠状动脉左前降支建立心肌梗死模型,心电图出现S-T段弓背样抬高认为结扎成功。2周后骨髓间充质干细胞组注射第3代自体骨髓间充质干细胞1×106个,5-杂氮胞苷诱导组注射经5-杂氮胞苷诱导24h的第3代自体骨髓间充质干细胞1×106个,骨髓单个核细胞组注射骨髓单个核细胞2.5×107个/400μL,对照组仅注射400μL L-DMEM培养基。分别在术前、移植前、移植后2,4周应用超声心动图评价心脏功能,同时利用心肌声学造影观察缺血区的血流灌注情况。细胞移植后8周采用免疫荧光检测移植细胞在梗死区的生长状况,苏木精-伊红染色于普通显微镜下记数梗死区毛细血管密度。结果:骨髓间充质干细胞组9只、5-杂氮胞苷诱导组9只、骨髓单个核细胞组10只、对照组6只进入结果分析,其余动物因造模死亡未能完成整个实验。①超声心动检查发现术前、细胞移植前4组左室射血分数、左室短轴短缩率组间比较差异不显著;与对照组相比,细胞移植后2,4周骨髓间充质干细胞组和5-杂氮胞苷诱导组左室射血分数、左室短轴短缩率明显升高[对照组:(60.4±5.1)%,(62.4±7.8)%;(28.8±1.4)%,(27.2±5.3)%;骨髓间充质干细胞组:(70.8±4.6)%,(70.4±6.1)%;(33.8±3.4)%,(33.9±3.5)%;5-杂氮胞苷诱导组:(71.7±6.8)%,(70.3±5.8)%;(34.7±5.7)%,(35.5±6.3)%,P<0.05];左室收缩末内径、左室舒张末内径细胞移植组与对照组相比,差异不显著;心肌声学造影见细胞移植后心肌梗死区血流灌注改善明显。②细胞移植术后8周,所有细胞移植组均见Dil阳性移植细胞存活,并表达α横纹肌肌动蛋白和结蛋白。5-杂氮胞苷诱导组Dil阳性细胞出现具有典型横纹和肌小节样结构的心肌样细胞,各细胞移植组毛细血管密度均较对照组明显增多[(38.6±7.6)/mm2,(32.9±5.7)/mm2,(38.5±2.0)/mm2,(21.4±3.9)/mm2,P<0.05],各移植组间差异不显著。结论:①体外纯化培养与经5-杂氮胞苷诱导24h的骨髓间充质干细胞,以及新鲜分离的骨髓单个核细胞自体移植8周后均存活于梗死心肌中,并表达心肌细胞特异性标志α横纹肌肌动蛋白和结蛋白,移植后可明显改善左室收缩功能,增加毛细血管密度,改善局部血流灌注。②5-杂氮胞苷有助于促进骨髓间充质干细胞在体内向心肌细胞的分化成熟。

AIM： To observe the proliferation and differentiation of bone marrow-derived mesenchymal stem cells （MSCs）, and analyze the cytothesis and reconstruction ability of ischemic myocardial cells as wall as its influence on heart function after injecting MSCs （following the mononuclear cell, in vitro culture, purification and induction） into post-infarct myocardium. METHODS： The experiment was conducted in Anhui Provincial Stem Cells Research and Treatment Center from July 2005 to April 2006. Forty healthy male clean New Zealand rabbits ware randomly divided into four groups： MSCs group, 5-aza-treated bone marrow-derived mesenchymal stem cells （5-aza-treated MSCs group）, fresh bone marrow mononuclear cells （BMMNCs group） and control group, 10 in each group. Rabbits in each group ware ligated of the left anterior descending coronary artery （LAD） below the left auricular appendage to establish the myocardial infarction models. The successful myocardial infarct models ware recognized by arc-like elevation of S-T segments in Electrocardiogram （EKG）. Two weeks later,1×10^6 the third generation of MSCs ware injected into MSCs group, 1×10^6 the third generation of MSCs after inducing 5-aza for 24 hours ware injected into the 5-aza-treated MSCs group. 2.5×10^7 / 400 μL mononuclear cells ware injected into the BMMNCs group. 400 μL L-DMEM medium was injected into the control group. Cardiac function was evaluated by ultrasound cardiotachograph at weeks 2 and 4 before operation, before and after transplantation, respectively. The blood perfusion in ischemic myocardium of the rabbits was observed by myocardial acoustic contrast. Cell growth in infarcted region was measured by immunofluorescence test at week 8 after cell transplantation. The capillary densities ware counted under ordinary microscope by hemotoxylin-eosin staining （HE staining）. RESULTS： Nine rabbits in MSCs group, nine in 5-aza-treated MSCs group, ten in BMMNCs group and six in control group ware involved in the analysis of the results （other rabbits died for modeling）. ①Detection of heart function by Echocardiography： The left ventricular ejection fraction （LVEF） and left ventricular fractional shortening （LVFS） ware fundamentally the same in each group before modeling, before tansplantation. As compared with the control group, the LVEF and LVFS ware significantly increased in the MSCs group and 5-aza-treated MSCs group [control group： （60.4± 5.1 ）%, （62.4±7.8）% ; （28.8±1.4）%, （27.2±5.3）% ;MSCs group： （70.8±4.6）%, （70.4±6.1）% ; （33.8±3.4）%, （33.9±3.5）% ; 5-aza-treated MSCs group： （71.7±6.8）%, （70.3±5.8）%; （34.7±5.7）%, （35.5±6.3）%; P 〈 0.05]. There was no signifcant difference in left ventricular end-diastolic dimension and end-systolic dimension between the cell transplantation group and control group. Myocardial contrast echocardiography （MCE） showed that the blood perfusion of the infarcted myocardium was obviously improved after the cells transplantation.②Dil-positive cells could be seen in all three bone marrow cells transplanted hearts group 8 weeks after cell transplantation, and Dil-positive cells expressed α-sarcomeric actin and desmin. The Dil positive cells appeared relative mature sarcemeric and sarcomere structure only in 5-aza-treated MSCs group. The capillary density in the cells transplantation group was much higher as compared with control group [（38.6±7.6）/mm^2, （32.9±5.7）/mm^2, （38.5±2.0）/mm^2, （21.4±3.9）/mm^2,P 〈 0.05]. There was no significant difference in each cell transplantation group. CONCLUSION： ①Either the purified MSCs, 5-aza-treated MSCs or fresh separated bone marrow cells can survive in the infracted myocardium, expressing myocardium specific marks α-sarcomeric actin and desmin 8 weeks after transplantation. Left ventricle systolic function is improved markedly, capillary density is increased, and local blood perfusion is improved after transplantation. ②-aza facilitate the maturation of MSCs into myocardial cells in vivo.