羧甲基壳聚糖温敏凝胶诱导骨髓间充质干细胞向神经细胞分化

摘要

背景:壳聚糖生物材料能够诱导骨髓间充质干细胞向神经细胞分化,壳聚糖衍生物羧甲基壳聚糖是否也能够诱导骨髓间充质干细胞向神经细胞分化尚不清楚.目的:探讨羧甲基壳聚糖温敏凝胶对骨髓间充质干细胞向神经细胞分化的影响及其可能的机制.方法:取传至3代的大鼠骨髓间充质干细胞用不同质量浓度(0,50,100,150,200,500 g/L)羧甲基壳聚糖温敏凝胶浸提液培养,对照组不添加羧甲基壳聚糖温敏凝胶浸提液.MTT法检测不同质量浓度羧甲基壳聚糖温敏凝胶浸提液对骨髓间充质干细胞增殖的影响,Western blot检测150 g/L羧甲基壳聚糖温敏凝胶浸提液对骨髓间充质干细胞NSE、Nestin、GAFP、Vimentin、NF-M、MAP-2、β3-tubulin以及Notch通路信号分于Notch1,Jag1表达水平的影响.结果与结论:①羧甲基壳聚糖温敏凝胶浸提液促进骨髓间充质干细胞增殖,在质量浓度为150 g/L时,增殖率达到最高水平.②150 g/L羧甲基壳聚糖温敏凝胶组NSE、Nestin、GAFP、Vimentin、NF-M、MAP-2、β3-tubulin的蛋白表达水平明显高于对照组,表明羧甲基壳聚糖温敏凝胶能够诱导骨髓间充质干细胞向神经细胞分化.③羧甲基壳聚糖温敏凝胶组Notch1和Jag1的蛋白表达水平明显低于对照组,表明其在诱导分化过程中能抑制Notch信号通路的活性.%BACKGROUND:Chitosan biological materials can induce bone marrow mesenchymal stem cells to differentiate toward neurons. As a derivative of chitosan, carboxymethyl chitosan has a series of excelent properties. However, whether carboxymethyl chitosan can induce the neuronal differentiation of bone marrow mesenchymal stem cells remains unclear.OBJECTIVE:To investigate the effect of carboxymethyl chitosan thermosensitive hydrogel on the differentiation of bone marrow mesenchymal stem cells into neurons and the possible mechanism.METHODS:Passage 3 bone marrow mesenchymal stem cells from rats were selected and cultured in carboxymethyl chitosan thermosensitive hydrogel extracts in different concentrations (0, 50, 100, 150, 200, 500 g/L). Control cells were cultured in culture medium with no addition of carboxymethyl chitosan thermosensitive hydrogel extracts. MTT assay was performed to investigate the effects of different concentrations of carboxymethyl chitosan thermosensitive hydrogel extracts on bone marrow mesenchymal stem cell proliferation. Western blot assay was used to explore the effect of 150 g/L carboxymethyl chitosan thermosensitive hydrogel extracts on the expression of neuron-specific enolase, Nestin, Vimentin, NF-M, microtubule associated protein 2, glial fibrillary acidic protein, β3-tubulin, Notch1 and Jag1 protein.RESULTS AND CONCLUSION:MTT assay showed that carboxymethyl chitosan thermosensitive hydrogel promoted the cell proliferation, and the proliferation rate reached the peak at the concentration of 150 g/L. Western blot assay showed that the cells induced by 150 g/L carboxymethyl chitosan thermosensitive hydrogel extract had significant increases in neuron-specific enolase, Nestin, Vimentin, NF-M, microtubule associated protein 2, glial fibrillary acidic protein, and β3-tubulin protein expression, and obvious decreases in Notch1 and Jag1 protein expression in comparison with the control group. These results indicate that the carboxymethyl chitosan thermosensitive hydrogel induces rat bone marrow mesenchymal stem cells to differentiate toward neurons, and suppresses the activity of Notch signal pathway in the process of differentiation.