全骨髓法培养SD大鼠骨髓间充质干细胞及其生物学特性

The biological characteristics of SD rat bone marrow-derived mesenchymal stem cells isolated and cultured with attachment method

目的建立一种体外纯化及培养扩增SD大鼠骨髓间充质干细胞(MSCs)的简便有效方法。方法全骨髓法分离培养SD大鼠MSCs,通过体外培养和连续传代达到纯化和扩增MSCs目的。倒置显微镜下观察细胞形态变化情况;利用四唑盐比色(MTT)法测定MSCs的生长曲线;流式细胞仪(FCM)鉴定MSCs膜抗原。结果 SD大鼠胫骨全骨髓细胞在分离后与含10%优质胎牛血清DMEM培养基混合,约5～10min后即可见大量有核细胞贴壁,细胞形态为圆形,24～48h后可见贴壁细胞转变为椭圆形、多角形及短梭形,7～12d时细胞呈长梭形并达到90%单层融合。通过传代细胞进一步纯化及扩增。细胞传代后2d内处于潜伏期,3d后进入生长期,5d后进入平台期。FCM检测第4代MSCs膜表面抗原CD45、CD90、CD29阳性率分别为1.50%、99.18%、97.70%。结论 SD大鼠全骨髓贴壁法能有效得到分离纯化的MSCs,用此方法培养的SD大鼠MSCs生长稳定,增殖能力活跃,具有MSCs的一般生物学特性,可以为组织工程提供理想的种子细胞。为本实验研究打下基础。

objective To establish a simple and effective method of isolation, purification and cultivation of SD rat bone marrow-derived mesenchymal stem cells （MSCs） in vitro, and explore their biological characteristics. Methods MSCs were isolated and purified for culture in vitro and serial passage by the attachment culture method. The morphological changes of the MSCs were continuously observed under the inverted microscope. The cell growth curve was measured by MTT method, and membrane antigens were detected with flow cytometry （FCM）. Results The MSCs in primary culture which were adhered to plastic surface within 48 h after displacement took the oval, asteroid, short and fusiform shapes. 7 to 12 days after culture in DMEM medium containing 10% FBS, the cells reached 90% confluence in single layers, taking a long and fusiform shape. Further purification was achieved by expansion at serial passages. MSCs were in latency for 2 days, converted into growth period on the 3rd day and entered the stationary phase on the 5th day. FCM results indicated that the positive rate ofCD45, CD90 and CD29 was 1.50%, 99.18%, 97.70%, respectively. Conclusions The attachment culture method can be effectively used to isolate and purify SD rat MSCs. The cultured MSCs are stable in growth with active pro-liferation and share the general biological characteristics of MSCs, which will further make them ideal seed cells for tissue engineering.