氯化锂通过调节自噬通路促进大鼠骨髓间充质干细胞向神经细胞分化

Lithium chloride promotes neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy

目的:研究氯化锂(Li Cl)对大鼠骨髓间充质干细胞(MSCs)神经分化的影响,并探讨自噬通路在其中的作用。方法:体外分离培养大鼠骨髓间充质干细胞,细胞分为Li Cl组和对照组,采用β-巯基乙醇诱导MSCs向神经细胞分化,免疫荧光法和Western blot法检测诱导后神经元标志蛋白神经元特异性烯醇化酶(NSE)和微管相关蛋白2(MAP-2)以及自噬标志蛋白微管相关蛋白1轻链3(LC3)的表达变化。进一步采用自噬激活剂雷帕霉素(rapamycin)及自噬抑制剂3-甲基腺嘌呤(3-MA)干预自噬,Western blot法观察NSE和MAP-2的表达变化。结果:诱导分化后,Li Cl组及对照组细胞均有NSE和MAP-2表达,Li Cl组细胞NSE和MAP-2蛋白阳性表达率及表达量均大于对照组(P<0.05);此外,Li Cl组细胞LC3阳性荧光点以及LC3-Ⅱ表达量亦多于对照组(P<0.05)。加用雷帕霉素可进一步促进Li Cl处理的细胞NSE和MAP-2蛋白的表达(P<0.05),而3-MA则抑制Li Cl处理的细胞NSE和MAP-2蛋白的表达(P<0.05)。结论:氯化锂可能通过调节自噬通路促进大鼠骨髓骨髓间充质干细胞向神经细胞分化。

AIM： To study the influence of lithium chloride （LiCl） on the neuronal differentiation of rat bone marrow mesenchymal stem cells （MSCs） , and to explore whether autophagy was involved in this process. METHODS ： MSCs were isolated and cultured in vitro. The cells were divided into LiCl group and control group. MSCs were treated with p-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons. The expression of neuronal markers- neuron specific enolase （NSE） and microtubule-associated protein-2 （MAP-2） , and autophagic marker-microtubule-associ-ated protein 1 light chain 3 （ LC3） were measured by immunofluorescence method and Western blot. An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine （ 3-MA） were applied to modulate the autophagy in the LiCl treated- cells. The protein expression of NSE and MAP-2 were determined by Western blot. RESULTS ： After induction, the ex-pression of NSE and MAP-2 were increased. The percentage of NSE- and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group （P 〈 0. 05 ） . After induction, the number of LC3- positive dots and the expression of LC3-II in LiCl group were greater than those in control group （ P 〈 0. 05 ） . The expres-sion of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated-cells, and on the contra-ry, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA. CONCLUSION： Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy.