摘要
目的:观察心肌梗死后不同时间点移植骨髓间充质干细胞对梗死心肌修复的影响,寻找细胞治疗的有效移植时间窗。方法:实验于2006-04/10在郑州大学基础医学院完成。①实验动物:清洁级6周龄雄性SD大鼠81只,1只用于获取骨髓间充质干细胞,剩余80只随机数字表法分为细胞移植组、培养液对照组,再按心肌梗死后1d及1,2,3周进行移植的时间各分为4个亚组,10只/组。②实验方法:获取、培养大鼠骨髓间充质干细胞,培养至第3代接近70%融合时,加入5-溴脱氧尿嘧啶体外标记,调整细胞密度为4×1010L-1备用。两组大鼠均结扎左冠状动脉前降支建立心肌梗死模型。细胞移植组各亚组按对应时间点在心肌梗死中央及四周移植骨髓间充质干细胞悬液50μL/点(2×106个),培养液对照组各亚组于相同部位移植L-DMEM培养液,50μL/点。③实验评估:移植前和移植3周后检测心功能;苏木精-伊红、免疫组织化学染色观察心肌梗死区组织及细胞变化。结果:共79只大鼠进入结果分析。①骨髓间充质干细胞的生长特点:刚分离的细胞为圆形。5d后有集落形成,细胞呈梭形、纺锤形或多角形,胞核较大。传代后细胞体积较原代细胞增大,生长速度加快。②心肌梗死后不同时间移植骨髓间充质干细胞的心功能比较:与移植前比较,移植3周后细胞移植组各亚组左室射血分数、短轴缩短率均有所改善(P<0.05),以心肌梗死后2周细胞移植组最为明显(P<0.01);培养液对照组各亚组左室射血分数、短轴缩短率均下降(P<0.05)。③梗死心肌组织学观察:细胞移植组心肌梗死区及周边可见核蓝色深染的细胞,排列整齐,梗死区瘢痕化减轻。④骨髓间充质干细胞在心肌组织中的分化:细胞移植组梗死心肌内可见大量BrdU标记阳性的骨髓间充质干细胞,胞核呈棕黄色,对心肌特异性肌钙蛋白T呈阳性表达。结论:心肌梗死后早期移植骨髓间充质干细胞能够改善心功能,以2周内进行细胞移植的效果较佳。
TIM： To observe the effect of transplanted mesenchymal stem coils （MSCs） on functional restoration after myocardial infarction at different times and try to find the effective time for cell therapy. METHODS： The experiment was implemented from April to October 2006 at Basic Medical College of Zhengzhou University. ①Totally 81 SD male rats in clean grade aged 6 weeks were selected. One was used to acquire MSCs, the others ware randomly divided into MSCs transplanted group and control group, and according to the different transplanted times, these two groups further were divided into four subgroups, they were one day subgroup, one week subgroup, two weeks subgroup and three weeks subgroup, respectively, ten animals per subgroup. ②Using density grads centrifuge to acquire and culture rat MSCs to third generation that 70% cells ware fused, adding 5-BrdU to label cells in vitro and adjusting the density of cells to 4x10^10 L^-1. Left anterior descending coronary artery of all rats was ligated in these two groups to build the infarcted models. In every subgroup of MSCs transplanted group, MSCs 50 p.L （2xl0^6/spet） were transplanted into the sites of infarcted cardiac muscle at every corresponding time. Culture medium L-DMEM was injected into the corresponding sites of rats in control group, 50 p.L/spet. ③The state of cardiac function was examined at pre-transplantation and pest-transplantation after three weeks. The changes of cells and tissues in infarct sites ware observed by hematoxylin-eosin （HE） staining and immunochemical staining. RESULTS： Totally 79 rats were involved in the result analysis. ①Growth characterization of MSCs： The cells of just separation ware round. The cell clones began to form after 5 days, and they appeared in fusiform, spindle shape and polygon with big nuclei. Cells after transgeneration ware bigger than the original generation and grow rapidly. ②Comparison of cardiac function in transplanted MSCs groups at different times： Compared with pretransplantation, left ventricular ejection fractions （LVEF） and shortening fraction were improved after transplantation for 3 weeks （P 〈 0.05）, especially in two weeks subgroup （P〈 0.01 ）. LVEF and shortening fraction were decreased in control supgroups with culture medium L-DMEM（P 〈 0.05）. ③Histological observation of infarcted myocardium： In transplanted MSCs groups, cells with deeply stained nuclei were arranged in order at infarct site and its periphery, extent of scar in infarcted region was reduced. ④Differentiation of MSCs transplanted in cardiac tissue： Numerous MSCs with labeled positively with BrdU could be observed in infarct sites and nuclei appeared brown-yellow. These MSCs expressed cardiac cTnT for positively. CONCLUSION： Transplanted MSCs in early stage of myocardial infarction can improve the cardiac function, especially in two weeks after infarct occurred.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第33期6641-6644,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
