骨髓间充质干细胞自体移植治疗心肌梗死的实验研究

Experimental Study on the Effect of Autologous Bone Marrow Mesenchymal Stem Cells Transplanting into Ischemic Myocardium in Rabbits

目的探讨兔骨髓间充质干细胞(MSCs)移植至缺血心肌后的增殖分化情况,对缺血心肌细胞的修复重建能力及心功能改善情况。方法将20只新西兰白兔随机分为骨髓间充质干细胞移植组(MSCs组,n=10)和对照组(n=10),采用结扎冠状动脉左前降支(LAD)制备心肌梗死模型,2周后分别将Dil标记的1×106个细胞悬液400μl或等量L-DMEM培养基用微量注射器注入梗死灶边缘,于建模前、建模后2周、细胞移植后2、4周采用多普勒超声心动图检测左心室收缩期末内径(LVESD)、左心室舒张期末内径(LVEDD),计算左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)评价心脏收缩功能,同时进行心肌声学造影评价心肌组织的血流灌注情况。细胞移植后8周处死所有动物,病理学检查移植细胞在梗死区的生长状况。结果多普勒超声心动图检测结果显示:两组动物建模前、建模后2周LVEF、LVFS差异无统计学意义(0.72±0.08vs.0.71±0.04,0.56±0.11vs.0.55±0.09;0.35±0.06vs.0.35±0.04,0.24±0.08vs.0.23±0.03,P>0.05),细胞移植后2、4周MSCs组LVEF、LVFS值均明显高于对照组(0.71±0.05vs.0.60±0.05,0.72±0.07vs.0.62±0.08;0.34±0.03vs.0.29±0.01,0.35±0.06vs.0.27±0.05,P<0.05);病理学检查见自体MSCs移植8周后存活于梗死心肌中,表达肌细胞特异性标志,并且能显著增加瘢痕区毛细血管密度(38.6±7.6/mm2vs.21.4±3.9/mm2,P<0.05),心肌声学造影亦显示梗死局部血流灌注MSCs组较对照组明显改善。结论自体MSCs移植缺血心肌中可向心肌细胞分化,增加心肌血流灌注,改善心脏收缩功能。

Objective To investigate the effect of autologous bone marrow mesenchymal stem cells （MSCs） transplantation on cardiac function and their proliferation and differentiation in the post-infarct myocardium in rabbits. Methods Twenty New Zealand rabbits were randomly divided into two groups, the autologous bone marrow mesenchymal stem cells group （MSCs group, n= 10） and control group （n= 10）. Myocardial infarct model was set up by ligation of the left anterior descending （LAD）, two weeks after establishment of the infarct model, either 400μl of cell suspension （total cells 1 × 10^6） labled by 1, 1＇-dioctadecyl-3,3,3＇, 3＇-tetramethyl indocarbocyanine perchlorate （Dil） or a comparable volume of L-DMEM medium were autologously transplanted into several different points of the periphery of the scar respectively. To evaluate the heart function, echocardiography were performed before modeling,two weeks after modeling, 2 and 4 weeks after the cells transplantation for measurements of left ventricular end systolic diameter （LVESD） and left ventricular end diastolic diameter （LVEDD）, to calculate left ventrieular eject fraction （LVEF） and left ventricular fractional shortening （LVFS）. Meanwhile the myocardial contrast echocardiography （MCE） were performed for evaluating the blood perfusion of the post-infarct myocardium. Eight weeks after the transplantation, the animals were undergoing euthanasia, specimens were acquired for pathology. Results Echocardiography indicated that： The LVEF and LVFS between two groups were fundamentally the same before modeling ,two weeks after modeling respectively （0.72± 0.08 vs. 0. 71±0. 04,0. 56± 0.11 vs. 0.55±0.09; 0.35：±0.06 vs. 0.35±0.04, 0.24±0.08 vs. 0.23±0.03, P〉0. 05）, but those were improved significantly in group MSCs when compared with control group at two weeks and four weeks after the cells transplantation（0.71±0.05 vs. 0.60±0.05,0.72±0.07 vs. 0.62±0.08 and 0.34±0.03 vs. 0.29±0.01, 0.35± 0.06 vs. 0. 27±0. 05 respectively, P〈0.05）. There were no differences in LVESD and LVEDD between two groups in any time points（P〉0.05）. MCE showed the blood perfusion of the infarct myocardium were improved two and four weeks after the cell transplantation. Pathology indicated that Dil positive cells were survived in MSCs transplanted hearts, stained positively for α-sarcomeric actin and desmin eight weeks after cell transplantation, HE slides indicated that the capillary density in all the cells transplanted hearts were much higher when compared with control group （38. 6±7. 6/mm^2 vs. 21. 4±3. 9/mm^2, P 〈 0. 05）. Conclusion MSCs can differentiate into cardiomyocytes, improve myocardial perfusion and cardiac function when transplanted into ischemie myocardium.