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背景:脐血间充质干细胞诱导分化为心肌细胞后移植入心肌缺血组织,可以通过与宿主细胞建立联系来修复取代缺血心肌组织。目的:对犬脐血干细胞进行肌源性诱导,观察其植入后与宿主细胞间的连接情况。设计、时间及地点:随机对照动物实验,于2006-07/2007-10在上海交通大学医学院附属新华医院动物实验中心完成。材料:接近足月的妊娠杂种犬2只,用于分离培养脐血间充质干细胞。成年杂种犬36只,随机数字表法分为细胞移植组、模型对照组,18只/组。方法:取传至第4代的犬脐血间充质干细胞,加入含lacZ基因的重组腺相关病毒载体进行基因转染,继续培养3d后,行10μmol/L5-氮杂胞苷肌源性诱导,常规培养3周。两组犬均建立心肌梗死模型,造模后细胞移植组经冠状动脉和局部注射将2mL细胞悬液(约107个脐血间充质干细胞)移植到梗死区,模型对照组同法注射等量生理盐水。分别于移植后2,4,8周取材制备心肌组织切片,免疫组化检测细胞间连接情况。主要观察指标:脐血间充质干细胞的基因转染、肌源性诱导分化情况,移植细胞与宿主心肌细胞的细胞间连接情况。结果:转染72h后大部分细胞表达LacZ基因,并合成半乳糖苷酶,X-gal染色呈蓝色。5-aza诱导3周后,脐血干细胞α-Actin(+),Desmin(+),Connexin43(+),而诱导前均呈阴性。移植后第8周,心肌切片中移植细胞和宿主心肌相连处可形成连接,连接处可见cadherin和connexin43绿色荧光阳性表达;模型对照组仅表达cadherin和connexin43,未见带红色荧光的移植细胞。结论:脐血间充质干细胞可在体外诱导为心肌样细胞,移植后能与宿主心肌可能形成有效连接,从而具有通讯联系。
BACKGROUND: Umbilical cord blood mesenchymal stem cells (BMSCs) are induced to differentiate into cardiomyocytes and then transplanted into myocardial ischemic tissue. The ischemic myocardium can be repaired by establishing contact with host cells. OBJECTIVE: To induce myogenic cord blood stem cells and observe the connection between them and host cells after implantation. DESIGN, TIME AND SETTING: The randomized controlled animal experiment was performed at the Animal Experimental Center, Xinhua Hospital, Shanghai Jiaotong University School of Medicine from July 2006 to October 2007. MATERIALS: Two full-term pregnant mongrel dogs were used for isolation and culture of umbilical cord blood mesenchymal stem cells. Thirty-six adult dogs were randomly divided into cell transplantation group, model control group and 18 rats / group. Methods: The umbilical cord blood mesenchymal stem cells (MSCs) were passaged into passage 4 and transfected with the recombinant adeno-associated virus vector containing lacZ gene for further gene transfer. After 3 days of further culture, myogenic induction of 10 μmol / L 5-azacytidine , Routine training for 3 weeks. Two groups of dogs were established myocardial infarction model, cell transplantation group after transplantation by coronary artery and local injection of 2mL cell suspension (about 107 umbilical cord blood mesenchymal stem cells) into the infarct area, the model control group with the same method of injection Physiological saline. Myocardial tissue sections were prepared at 2, 4, and 8 weeks after transplantation, and the intercellular junctions were detected by immunohistochemistry. MAIN OUTCOME MEASURES: Gene transfection of umbilical cord blood mesenchymal stem cells, differentiation induced by myogenic origin, and cell-cell junctions between transplanted cells and host cardiomyocytes. Results: Most cells expressed LacZ gene at 72h after transfection, and galactosidase was synthesized. X-gal staining was blue. After 3-week induction of 5-aza, umbilical cord blood stem cells showed a-Actin (+), Desmin (+) and Connexin43 (+ At the 8th week after transplantation, the connections between the transplanted cells and the host myocardium in the myocardial slices could be formed. The positive expression of cadherin and connexin43 were observed in the junction. The model control group only expressed cadherin and connexin43, and no transplanted cells with red fluorescence were found. CONCLUSION: Umbilical cord blood mesenchymal stem cells can be induced into cardiomyocyte in vitro, which may form an effective connection with the host myocardium after transplantation and thus have communication links.