还原型谷胱苷肽对骨髓基质干细胞增殖及向神经样细胞分化的影响

Reduced glutathione effect on the proliferation and neuronal differentiation of bone marrow stromal stem cells

背景:骨髓基质干细胞具有易获取及多向分化潜能,通过诱导骨髓基质干细胞分化获得神经干细胞是治疗脊髓损伤的重要途径和方法。目的:观察不同浓度还原型谷胱苷肽对骨髓基质干细胞增殖及向神经样细胞分化的影响。方法:配置5组不同浓度的还原型谷胱苷肽(0,5,10,20,40 mmol/L)培养液,干预骨髓基质干细胞72 h后,采用MTT法分析细胞增殖活性,倒置显微镜下连续观察细胞形态变化,免疫组织荧光染色法测定MAP-2和GFAP表达。结果与结论:(1)低浓度5 mmol/L组还原型谷胱苷肽培养液能够促进骨髓基质干细胞增殖(P<0.05),20 mmol/L组和40 mmol/L组还原型谷胱苷肽培养液抑制细胞增殖(P<0.05),10 mmol/L还原型谷胱苷肽组细胞集落的数量和大小与对照组未见明显差别;(2)10 mmol/L组还原型谷胱苷肽培养液诱导骨髓基质干细胞向神经细胞分化作用较明显,荧光显微镜下可见大部分细胞表达MAP-2和GFAP,随机选择5个视野观察并计算骨髓基质干细胞分化为神经样细胞的比率为(62.0±4.8)%;(3)结果证实,低浓度(5 mmol/L)还原型谷胱苷肽可以促进骨髓基质干细胞增殖,10 mmol/L还原型谷胱苷肽有较强的诱导骨髓基质干细胞向神经样细胞分化的作用。

BACKGROUND：As bone marrow stromal stem cel s can be easily obtained and have multi-directional differentiation potential, it is an important approach to obtain neural stem cel s for treatment of spinal cord injury through induced differentiation of bone marrow stromal stem cel s. 〈br〉 OBJECTIVE：To observe the effects of different concentrations of reduced glutathione on proliferation and neuronal differentiation of bone marrow stromal stem cel s. 〈br〉 METHODS：Bone marrow stromal cel s were isolated and cultured by limited dilution method. The cloned bone marrow stromal cel s were stimulated with reduced glutathione at different concentrations （0, 5, 10, 20, 40 mmol/L） respectively. After 72 hours of culture, proliferation of bone marrow stromal cel s was examined by MTT assay. The morphology of bone marrow stromal cel s was observed under inverted microscope and the cel s were identified by immunofluorescence staining of microtubule associated protein-2 and glial fibril ary acidic protein. 〈br〉 RESULTS AND CONCLUSION：We found that 5 mmol/L reduced glutathione medium could promote the proliferation of bone marrow stromal cel s （P〈0.05）, but reduced glutathione at 20 and 40 mmol/L inhibited the cel proliferation （P〈0.05）. The number and size of cel colonies in the 10 mmol/L reduced glutathione group had no obvious difference from the control group. Reduced glutathione at 10 mmol/L was powerful to induce the neuronal differentiation of bone marrow stromal stem cel s, and most cel s expressed microtubule associated protein-2 and glial fibril ary acidic protein. A random selection of 5 fields of view was conducted and percentage of bone marrow stromal cel s differentiating into neuron-like cel s was calculated as （62.0±4.8）%. These results confirmed that low-concentration （5 mmol/L） reduced glutathione can promote the proliferation of bone marrow stromal cel s, while 10 mmol/L reduced glutathione has a strong induction of bone marrow stromal cel s differentiating into neuron-like cel s.