Edaravone体外诱导rBMSCs分化为神经元样细胞

Edaravone induce in vitro differentiation of rat bone marrow stromal cells into neuron-like cells

目的:探讨新型氧自由基清除剂依达拉奉诱导大鼠骨髓基质干细胞(rat bone marrow stromalcells,rBMSCs)分化为神经元样细胞的可行性。方法:采用密度梯度离心法和贴壁法分离纯化1月龄SD rBMSCs,体外培养扩增与传代,实验分依达拉奉处理组和空白对照组,每组各10例。处理组用终浓度0.02g/L依达拉奉的无血清L-DMEM诱导6h,对照组继续用10%胎牛血清、L-DMEM培养。观察细胞形态变化,免疫细胞化学鉴定神经元烯醇化酶、胶质纤维酸性蛋白、巢蛋白、微管相关蛋白2的表达。结果:分化的rBMSCs胞体收缩,突起伸出;诱导后细胞神经元烯醇化酶阳性表达率(81.6±3.2)%,微管相关蛋白2阳性表达率(26.0±2.5)%,胶质纤维酸性蛋白阳性表达率(5.6±1.9)%,巢蛋白阳性表达率(9.0±2.8)%。对照组均阴性表达。两组NSE、GFAP、Nestin比较差异均有统计学意义(P<0.001),处理组NSE阳性表达明显高于MAP2、GFAP(P<0.005)。结论:依达拉奉可以诱导rBMSCs分化为神经元样细胞,从而为脊髓损伤的治疗提供种子细胞和理论基础。

Objective： To explore the feasibility of inducing differentiation of rat bone marrow-de- rived stromal cells（rat bone marrow stromal cells, rBMSCs）to neuron like cells by edaravone. Methods： rBMSCs were separated and purified from one-month-old SD rat using density gradient centrifugation combining with attachment culture method, then cultured for passage and proliferation. The treatment group （10 samples） was treated with treated with edaravone at a final concentration of 0.02g / L in serum-free L-DMEM for 6 h; while the control group （10 samples） was cultured with 10 % fetal calf serum and L DMEM. Morphologic changes were observed and expression of NSE, GFAP, MAP2 and nestin were analyzed by imrnunocytochemieal method. Results. Body contraction and long procession were shown in differentiated stromal cells of the treatment group. The immunocytochemical detection showed that the percentage of NSE, MAP2, GFAP and nestin positive ceils were （81. 6±3. 2）%, （26±2. 5）%, （5. 64±1.9）% and （9.0±2.8） %, in the treatment group, while in the control group they were all absent showing significant difference between the two groups （P〈0. 001）. In the treatment group, expression level of NSE was significant higher than that of siMAP2, GFAP and Nestin （P〈0. 005）. Conclusions： Edaravone shows in vitro promotion effects on the differentiation of rBMSCs into neuron-like cells, which provides theoretical foundations for its clinical application in spinal cord injury.