摘要： 研究背景 壳聚糖作为组织工程中的常用支架材料，具有生物降解性强、抗原性低、生物相容性佳和无热原反应等优势。尝试将经分离纯化的SD 大鼠骨髓间充质干细胞与自制的可吸收壳聚糖多孔支架共培养，研究其生物相容性，以为今后应用细胞-支架复合体植入中枢神经系统行替代治疗，实现神经细胞再生、神经功能修复奠定基础。方法 采用全骨髓贴壁培养法分离3 周龄SD 大鼠骨髓间充质干细胞，选择第3 代细胞行流式细胞术纯化鉴定并与壳聚糖多孔支架于体外三维立体环境共培养。乙醇替代法检测壳聚糖多孔支架孔隙率；扫描电子显微镜观察支架内部结构、测量孔径大小，以及细胞在支架内部生长状态及其与生物支架融合情况；MTT 法测定细胞在支架内增殖状况。结果 经体外培养的大鼠骨髓间充质干细胞形态均匀、呈纤维状排列，混杂细胞比例明显减少；CD29 和CD45RA 阳性表达率分别为98.49%和0.85%，达高纯度表达；支架孔隙率为90%，支架内部为孔径均匀、相通的三维立体结构，细胞紧密贴附于支架微孔内壁上、黏附牢固，可见伪足伸出，与支架融合较为理想。支架对骨髓间充质干细胞增殖无明显影响，细胞生长状况良好。结论 壳聚糖多孔支架具备良好的孔径和孔隙率等性状，与骨髓间充质干细胞生物相容性良好，可作为干细胞移植的支架载体为今后细胞替代治疗中枢神经系统疾病的转化医学研究奠定基础。

关键词: 骨髓细胞, 间质干细胞, 壳聚糖, 生物相容性材料, 细胞, 培养的, 流式细胞术, 显微镜检查, 电子, 扫描

Abstract: Background  As commonly used scaffold material in tissue engineering, chitosan has many advantages, such as strong biodegradability, low antigenicity, good biocompatibility and no pyrogen reaction. This study aims to isolate, cultivate and purify Sprague-Dawley (SD) rat bone marrow-derived mesenchymal stem cells (BMSCs), and to observe the growth of BMSCs when co-cultured with self-made chitosan porous scaffold in vitro and to test the biocompatibility of this tissue engineering scaffold, so as to lay the foundation for promoting nerve regeneration of transplant treatment.  Methods  Three-week-old healthy male SD rats were used in this study, and BMSCs were isolated and purified through bone marrow adherent culture method. The surface markers of BMSCs at Passage 3 were detected and identified by flow cytometry (FCM) and the BMSCs were three?dimensionally cultured in vitro on chitosan porous scaffolds produced by freeze-drying method. Ethanol alternative method was used to detect the chitosan scaffold porosity. Scanning electron microscope was used to explore the internal structure of the scaffold, measure the size of its aperture, and observe the morphology and development of the cells within the scaffold. Methyl thiazolyl tetrazolium (MTT) method was used to determine the cells' proliferation.  Results  The cultured BMSCs were uniform and similiar to fibrous arrangement, and mixed cells reduced obviously. The identification result of FCM showed the CD29 positive rate was 98.49% and CD45RA positive rate was only 0.85%. The chitosan scaffold had an interlinked, uniform similar three-dimensional porous structure and its aperture porosity was 90%. Some cells stretched out pseudopod and infiltrated into the porous structure of scaffold, even fusing with them. The BMSCs were seeded in the scaffold successfully. The chitosan scaffold had no obvious effect on BMSCs' proliferation. Conclusions  Chitosan porous scaffolds have good structural character and biocompatibility, and can be used as an alternative cell-carrier for later cell replace treatment of central nervous system diseases.

Key words: Bone marrow cells, Mesenchymal stem cells, Chitosan, Biocompatible materials, Cells, cultured, Flow cytometry, Microscopy, electron, scanning