应用犬骨髓间充质干细胞体外构建组织工程心脏瓣膜的实验研究  

Experimental study of seeding acellularized porcine heart valve with canine bone marrow mesenchymal stem cells in vitro

作　　者：欧阳辉[1] 刘维永[1] 陈文生[1] 李悟[1] 卫向阳[1] 顾春虎[1] 宿学家[1] 郑奇军[1] 

机构地区：[1]第四军医大学西京医院心血管外科,陕西西安710032

出　　处：《心脏杂志》2005年第4期337-340,共4页Chinese Heart Journal

基　　金：国家自然科学基金资助项目(30271292)

摘　　要：目的:将犬骨髓间充质干细胞(BMSCs)分离、自然扩增后,体外与去细胞猪主动脉瓣复合构成组织工程瓣膜(TEHV),观察其生长情况,以评价其作为组织工程心脏瓣膜种子细胞的可行性.方法:采用密度梯度离心及贴壁筛选法分离犬的BMSCs,培养扩增,用免疫组化的方法进行鉴定;采用本实验室方法构建的生物材料--去细胞猪主动脉瓣为支架,种植犬的BMSCs,静态培养7～10 d;进行组织学切片及扫描、透射电镜观察,并进行机械力学性能检测.结果:分离的BMSCs细胞免疫组化显示SH2、Vimentin、α-SMA(+),CD34、Ⅷ因子、Laminin(-),生长扩增能力强,2周内经3代培养即可扩增到(5.57±0.34)×107细胞数量级;组织工程瓣组织学示活组织和细胞外基质(extracellularmatrix,ECM)形成;扫描电镜见细胞贴附均匀,透射电镜可见分泌活性的肌纤维细胞样细胞(含actin/myosin纤维、胶原纤维、弹性蛋白);机械力学性能得到保留.结论:使用本方法可以构建成功组织工程心脏瓣膜;BMSCs与去细胞猪主动脉瓣的相容性良好,可以作为种子细胞之一.AIM： To observe the growth status of seeding acellularized porcine heart valve with canine bone marrow mesenchymal stem cells （ BMSCs ） in vitro and evaluate the feasibility of constructing tissue engineering heart valve （TEHV）. METHODS： BMSCs were isolated and purified from the canine bone marrow by Percoll density gradient centrifugation and by adhering to the culture plastic. After successive culture and amplification, the growth curve was drawn. The morphology was observed under the phase contrast microscope, and the some antigens were examined by immunohistochemistry stain. The porcine aortic valves were acellularized by use of trypsin, Triton X-100, and hypertonic and hypotonic liquid treatments. Acellularized porcine heart valves were seeded with canine BMSCs in vitro, and then cultured for 7 days. RESULTS： SH2, Vimentin and σ-SMA were all positively expressed in naturally differentiated BMSCs while CD34, Ⅷ antigen and Laminin showed negative reaction. The acellularization procedure resulted in complete removal of the cellular components, with the construction of matrix maintained. The matrix could be successfully seeded with in vitro expanded BMSCs. Electron microscope demonstrated cell elements typical of viable, secretionally active myofibroblasts （ actin/myosin filaments, collagen fibills, elastin） and confluent, homogenous tissue surfaces. Mechanical properties of the TEHV were comparable to native tissues （ P 〉 0.05 ）. CONCLUSION ： The acellularied porcine aortic valves can be used as a scaffold to develop tissue engineering heart valves. Canine BMSCs have the biocompatibility and characteristics of natural myofibroblast differentiation.

关 键 词：干细胞 种子细胞 组织工程 心脏瓣膜 去细胞 犬 

分 类 号：R654[医药卫生—外科学]