灌注培养对骨髓基质干细胞在大段材料上增殖与分布的影响  

The role of perfusion bioreactor in proliferation and distribution of bone marrow stromal cells in a large-scale scaffold

作　　者：王磊[1] 李方国[1] 张鑫鑫[1] 金丹[1] 江汕[1] 郭小磊[1] 姜晓锐[2] 裴国献[3] 

机构地区：[1]南方医科大学南方医院创伤骨科,广州510515 [2]山东省烟台市毓璜顶医院手足骨科 [3]第四军医大学西京医院骨科

出　　处：《中华创伤骨科杂志》2011年第5期448-453,共6页Chinese Journal of Orthopaedic Trauma

基　　金：国家重点基础研究发展计划（973）资助项目（2009CB930003）;罔家自然科学基金项目（31070866）

摘　　要：目的 探讨灌注培养对骨髓基质干细胞（BMSCs）在大段材料上增殖与分布的影响。方法在大段多孔磷酸三钙材料上种植转染了增强型绿色荧光蛋白基因的大鼠BMSCs（eGFP—BMSCs），分别采用灌注式生物反应器（实验组）和静止培养法（对照组）进行培养。培养7、28d行扫描电镜观察；培养7、14d行荧光显微镜观察；动态监测葡萄糖Ft耗量；培养7、14、28d测定支架上各层细胞的数量，观察eGFP—BMSCs在支架上的增殖与分布情况。结果 扫描电镜和荧光显微镜下观察发现各时间点实验组eGFP—BMSCs均可在支架边缘和内部分布与增殖，而对照组细胞仅能在支架边缘分布与增殖。两组葡萄糖日耗量均随着培养时间的延长而增加，培养28d时实验组葡萄糖日耗量【（36．33±3．14）mg／d】是对照组【（9．82±1．33）mg／d]的3．7倍，两组分别于20、15d进入平台期。实验组培养7、28d时支架各层细胞数量差异无统计学意义（P〉0．05），14d时各层细胞数量的差异有统计学意义（P〈0．05）；而对照组各时间点支架各层细胞的数量差异均有统计学意义（P〈0．05）。结论灌注培养法能促进BMSCs在大段多孔材料上增殖，并能使其在大段材料上均匀分布。Objective To study the role of perfusion bioreactor in proliferation and distribution of rat bone marrow stromal cells （BMSCs） in a large-scale scaffold. Methods SD rat BMSCs transfected with enhanced green fluoreseent protein （eGFP） （eGFP-BMSCs） were planted in large-scale porous [3-tricalcium phosphate（[3-TCP） scaffolds. In the dynamic perfusion culture group, the scaffold with eGFP-BMSCs was continuously cultured in our self-designed three-dimensional perfusion bioreactor for 7, 14 and 28 days. In the static culture group, the scaffold was put into a medium reservoir without perfusion for 7, 14 and 28 days. Proliferation and distribution of the cells in the scaffold were examined by scanning electronic microscopy （SEM）, fluorescence microscopy （FM）, measuring daily glucose consumption, and counting eGFP-BMSCs in each layer. Results SEM and FM showed that eGFP-BMSCs distributed and proliferated throughout the scaffold in dynamic peffusion culture, but distributed and proliferated only in the peripheral pores of the scaffold in static culture. The daily glucose consumption in both groups increased with time. Cell proliferation reached the plateau phase after culture for 14 days in the static culture group, but after culture for 21 days in the perfusion culture group. The rate and margin of increase were much more evident in the peffusion culture group. On day 28, glucose consumption in the perfusion culture group was 36.33±3.14 mg/d, 3.7 times as large as that in the static culture group （9.82±1.33 mg/d）. The eGFP-BMSCs counting revealed there were no significant differences in cell number between layers of the scaffold on days 7 and 28 d （ P 〉 0.05）, but there were significant differences on day 14 in the perfusion culture group （ P 〈 0.05）; while in the static culture group, there were significant differences in cell number between layers of the scaffold （ P 〈 0.05）, with most of the cells assembled at the bottom of the scaffold. Conclusion Our self-designed thre

关 键 词：骨髓细胞 生物反应器 细胞培养技术 组织工程 

分 类 号：R3[医药卫生—基础医学]