外源性重组HMGB1对神经干细胞增殖分化的影响及其机制研究  

The Effect of Exogenous Recombinant HMGB1 on Neural Stem Cells and Related Mechanism

作　　者：张会敏[1] 吴波[1] 曹甜[1] 严玉颖 刘鸣[1] 

机构地区：[1]四川大学华西医院神经内科,成都610041

出　　处：《四川大学学报（医学版）》2017年第3期394-398,409,共6页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.81371283和No.81671146)资助

摘　　要：目的研究外源性重组高迁移率组蛋白B1(HMGB1)对神经干细胞增殖及分化的影响及其作用机制。方法在无血清的神经干细胞培养基中培养SD鼠大脑皮层细胞,传代扩增及纯化神经干细胞,免疫荧光检测神经干细胞标记物巢蛋白(nestin),分析神经干细胞纯度。CCK-8测定加入不同浓度的重组HMGB1对神经干细胞增殖活性的影响,选择重组HMGB1的最适浓度进行后续实验;细胞免疫荧光检测重组HMGB1对神经干细胞分化的影响,Real-time PCR检测晚期糖基化终末产物受体(RAGE)mRNA、Toll样受体(TLRs)mRNA、基质金属蛋白酶9(MMP-9)mRNA、神经生长因子(NGF)mRNA的表达,Western blot检测RAGE、TLRs、MMP-9、NGF蛋白的表达。结果大鼠大脑皮层细胞在培养至第3代时,nestin鉴定神经干细胞纯度可达99%及以上。在重组HMGB1 10ng/mL刺激下,神经干细胞增殖活性最高。实验组神经Ⅲ类β-微管蛋白(TUJ1)表达高于对照组(P<0.05),实验组RAGE、TLRs、MMP-9、NGF mRNA及蛋白表达均高于对照组(P<0.05)。结论外源性重组HMGB1或可通过RAGE、TLRs、MMP-9等信号通路促进神经干细胞增殖及其向神经元方向分化。Objective To explore the effect of exogenous recombinant high mobility group protein box1 （rHMGB1） on proliferation and differentiation of neural stem cells （NSCs） and the related mechanism. Methods SD rat cerebral cortex cells were cultured in serum-free medium, extending the culture and purification of neural stem cells. NSCs were identified by detecting nestin-label with immunofluorescence method. The NSCs proliferation activity after adding different concentrations of rHMGB1 was determined by CCK-8 assay and the optimal concentration of rHMGB1 for the follow-up experiments, was selected. The effect of rHMGB1 on NSCs differentiation was detected by immunofluorescence assay. The mRNA and protein expression of involved factors were studied by real-time PCR and Western blot separately. Results The neural cells isolated from the cortex of rat embryos showed the expression of nestin antigen and the neural stem cells purity could reach more than 99% when cultured to the third generation. Under the stimulation of 10 ng/mL rHMGB1, neural stem cells proliferation activity were the highest, therefore, 10 ng/mL rHMGB1 was selected to treat the experimental group. The expression of TUJ1 in the experimental group was higher than that in the control group （P〈0.05）. Real-time PCR and Western blot confirmed rHMGB1 could improve the expression of receptor for advanced glycation end products （RAGE）, Toll-like receptor 2 （TLR2）, Toll-like receptor 4 （TLR4）, matrix metalloproteinase 9 （MMP-9） and nerve growth factor （NGF） respectively at the level of mRNA and protein expression. Conclusion Exogenous rHMGB1 promoted rat NSCs proliferation and differentiation into neurons in vitro by activating RAGE, TLRs, MMP-9 signaling.

关 键 词：神经干细胞 重组高迁移率组蛋白B1 晚期糖基化终末产物受体 Toll样受体 基质金属蛋白酶9 神经生长因子 

分 类 号：R743.3[医药卫生—神经病学与精神病学]