Zhang Hui-Min, Wu Bo, Cao Tian, Yan Yu-Ying, Liu Ming
Department of Neurology, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2017 May;48(3):394-398.
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.
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.
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 (<0.05). Real-time PCR and Western blot confirmed rHMGB1 could improve the expression of receptor for advanced glycation end products (), Toll-like receptor 2 (), Toll-like receptor 4 (), matrix metalloproteinase 9 () and nerve growth factor(NGF) respectively at the level of mRNA and protein expression.
Exogenous rHMGB1 promoted rat NSCs proliferation and differentiation into neurons by activating RAGE, TLRs, MMP-9 signaling.
探讨外源性重组高迁移率族蛋白盒1（rHMGB1）对神经干细胞（NSCs）增殖和分化的影响及其相关机制。
采用无血清培养基培养SD大鼠大脑皮质细胞，进行神经干细胞的扩增培养与纯化。通过免疫荧光法检测巢蛋白标记来鉴定神经干细胞。采用CCK-8法测定添加不同浓度rHMGB1后神经干细胞的增殖活性，选择后续实验的最佳rHMGB1浓度。通过免疫荧光法检测rHMGB1对神经干细胞分化的影响。分别采用实时荧光定量PCR和蛋白质免疫印迹法研究相关因子的mRNA和蛋白表达。
从大鼠胚胎皮质分离的神经细胞呈巢蛋白抗原表达，培养至第三代时神经干细胞纯度可达99%以上。在10 ng/mL rHMGB1刺激下，神经干细胞增殖活性最高，因此选择10 ng/mL rHMGB1处理实验组。实验组中TUJ1的表达高于对照组（<0.05）。实时荧光定量PCR和蛋白质免疫印迹法证实rHMGB1可分别在mRNA和蛋白表达水平上提高晚期糖基化终末产物受体（RAGE）、Toll样受体2（TLR2）、Toll样受体4（TLR4）、基质金属蛋白酶9（MMP-9）和神经生长因子（NGF）的表达。
外源性rHMGB1通过激活RAGE、TLRs、MMP-9信号通路促进大鼠神经干细胞增殖并分化为神经元。
