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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 investigate the effect of exogenous recombinant high-mobility group histone protein B1 (HMGB1) on the proliferation and differentiation of neural stem cells and its mechanism. Methods SD rat cerebral cortex cells were cultured in serum-free neural stem cell culture medium, and the neural stem cells were passaged and purified. The neural stem cell marker nestin was detected by immunofluorescence and the purity of neural stem cells was analyzed. CCK-8 assayed the effect of different concentrations of recombinant HMGB1 on the proliferation of neural stem cells, select the optimal concentration of recombinant HMGB1 for follow-up experiments; immunofluorescence detection of recombinant HMGB1 on neural stem cells differentiation, Real-time PCR detection of advanced glycoprotein The expression of RAGE, TLRs and MMP-9 mRNA, Toll-like receptor (TLRs) mRNA, MMP-9 mRNA and NGF mRNA were detected by Western blot. 9, NGF protein expression. Results When cultured in the third generation of rat cerebral cortex cells, the purity of neural stem cells was up to 99% by nestin. Under the stimulation of 10ng / mL HMGB1, the proliferation activity of neural stem cells was the highest. The expression of TUJ1 in experimental group was higher than that in control group (P <0.05). The mRNA and protein expressions of RAGE, TLRs, MMP-9 and NGF in experimental group were higher than those in control group (P <0.05). Conclusion Exogenous recombinant HMGB1 can promote neural stem cell proliferation and its differentiation to neurons through RAGE, TLRs, MMP-9 and other signaling pathways.