课题背景：课题为安徽省教育厅自然科学重点项目计划，项目批准号2006KJ088A，研究目的在于观察心肌梗死后骨髓间充质干细胞移植后对心肌功能的影响。目前课题初步成果包括：骨髓间充质干细胞的体外分离、培养、诱导分化为心肌样细胞；体外标记骨髓间充质干细胞体内移植存活及其对心功能的改善；移植的骨髓间充质干细胞体内细胞因子分泌及其对梗死区残存心肌的保护作用。

应用要点：①实验选择骨髓间充质干细胞作为种子细胞，是因其体外分离方法简单、增殖能力强、体外标记效果好且易于体内移植示踪。②实验观察骨髓间充质干细胞移植后在体内分泌的细胞因子对梗死区新生微血管的作用和对心肌细胞凋亡的抑制作用，能够从侧面说明骨髓间充质干细胞移植治疗对心功能改善的作用机制。

偏倚或不足：实验存在需要改进和完善之处包括增加动物样本量和观察时间点、增加动态心功能的监测及动物标本的垂直对照等。

摘要
目的：经冠状动脉途径行自体骨髓间充质干细胞移植治疗急性心肌梗死已有较多改善心功能的肯定结论。实验拟验证骨髓间充质干细胞自体移植心肌梗死犬的细胞因子分泌情况，及其对血管新生和心肌细胞凋亡的作用。
方法：实验于2005-12/2007-05在安徽医科大学第一附属医院动物实验室和中心实验室完成。选择健康杂种犬36只，无菌抽取犬骨髓液10 mL，经Percoll液密度梯度离心后贴壁法体外培养骨髓间充质干细胞，传2代后行5-氮胞苷诱导，5-溴脱氧尿苷标记备用。结扎冠状动脉途左前降支建立急性心肌梗死模型，术后随机分为细胞移植组、模型对照组，12只/组。细胞移植组分别在梗死区边缘4个不同部位点局部注射植入骨髓间充质干细胞悬液2 mL，模型对照组在相同对应部位注射等量DMEM培养基。术后取心肌梗死组织进行指标检测。
结果：①经流式细胞仪检测，传至第3代后的骨髓间充质干细胞CD34和CD11b阳性率小于5%，CD44和CD105阳性率高达90%以上。②原位末端标记结果示，细胞移植组心肌细胞凋亡指数显著低于模型对照组（P < 0.01）。③免疫组织化学检测显示，移植后2周细胞移植组梗死区微血管密度明显高于模型对照组（P < 0.05）；随移植时间的延长，细胞移植组梗死区新生微血管密度逐渐增加，趋势检验差异有显著性意义（F=439.588，P < 0.01）。移植后1，2，4周与模型对照组比较，细胞移植组心肌梗死区血管内皮生长因子、碱性成纤维细胞生长因子的表达均显著升高（P均 < 0.01）。
结论：自体骨髓间充质干细胞移植心肌梗死犬后，能分泌血管内皮生长因子、碱性成纤维细胞生长因子，诱导血管新生，同时抑制心肌细胞凋亡。
关键词：骨髓间质干细胞；移植；心肌梗死；细胞因子；血管再生；细胞凋亡

余其贵，王永，吴继雄.自体骨髓间充质干细胞移植心肌梗死区后细胞因子分泌和血管新生及心肌细胞凋亡的变化[J].中国组织工程研究与临床康复，2008，12(16):3001-3005 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3001(ps).pdf]

Cytokine secretion, angiogenesis and cardiomyocyte apoptosis after transplantation of autologous bone marrow mesenchymal stem cells into myocardial infarction regions

Abstract

AIM：The autologous bone marrow mesenchymal stem cell (BMSC) transplantation for treating acute myocardial infarction (AMI) via coronary artery in clinic has made remarkable and encouraging progress. The experiment aimed to study cytokine secretion in autologous BMSC transplantation for myocardial infarction dogs and its influence on angiogenesis and cardiomyocyte apoptosis.
METHODS: Experiments were performed at the Animal Laboratory and the Central Laboratory, First Affiliated Hospital of Anhui Medical University between December 2005 and May 2007. 10 mL of bone marrow was sterilely aspirated from the posterosuperior iliac spine of 36 healthy mongrel dogs. BMSCs were isolated and purified by Percoll density gradient centrifugation and cultured in vitro by adherence method. After being co-cultured with 5-azacytidine for two passages, these cells were labeled by 5-bromodeoxyuridine (Brdu) for preparation. Dog models of AMI were established in a model control group and a cell transplantation group randomly. 2 mL of cell suspension of autologous BMSCs were implanted into four different regions in the acute myocardial site via topical injection in the cell transplantation group, while the same dose of DMEM was injected into the corresponding regions in the model control group. Myocardial infraction tissues were measured after transplantation.
RESULTS: Flow cytometry demonstrated that the positive rate of CD34 and CD11b on third passage of BMSC cell surface was below 5%, while the positive rate of CD44 and CD105 was above 90%. In situ end labeling showed that apoptotic index of cardiomyocytes was lower in the cell transplantation group than in the model control group (P < 0.01). Immunohistochemistry demonstrated that microvessel density in infarcted site was much higher in the cell transplantation group than in the model control group 2 week after transplantation (P < 0.05). With time prolonging, it increased gradually, and the distinction of tendency analysis had significance (F=439.588, P < 0.01). Compared to the model control group, expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in infarcted regions were much higher in the cell transplantation group at 1, 2 and 4 weeks after implantation (P < 0.01).
CONCLUSION: After implanted into the infarcted site, autologous BMSCs can secrete VEGF and bFGF, and induce angiogenesis and reduce the numbers of the cardiomyocyte apoptosis.

Yu QG, Wang Y, Wu JX.Cytokine secretion, angiogenesis and cardiomyocyte apoptosis after transplantation of autologous bone marrow mesenchymal stem cells into myocardial infarction regions.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3001-3005 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3001(ps).pdf]