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

发布时间：2018-03-17 03:18

本文选题：神经干细胞　切入点：重组高迁移率组蛋白B　出处：《四川大学学报(医学版)》2017年03期 　论文类型：期刊论文

【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(P0.05),实验组RAGE、TLRs、MMP-9、NGF mRNA及蛋白表达均高于对照组(P0.05)。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。
[Abstract]:Objective to study the effect and mechanism of exogenous recombinant histone B1 HMGB1 on the proliferation and differentiation of neural stem cells. Methods SD rat cerebral cortex cells were cultured in serum-free neural stem cell culture medium. The neural stem cells were amplified and purified by subculture and purified. The nestin of neural stem cells was detected by immunofluorescence. The purity of neural stem cells was analyzed. CCK-8 was used to determine the effect of different concentrations of recombinant HMGB1 on the proliferation of neural stem cells. The optimal concentration of recombinant HMGB1 was selected for follow-up experiment. Effects of Recombinant HMGB1 on differentiation of Neural Stem cells by Cellular Immunofluorescence Assay the expression of advanced glycosylation end product receptor RAGEN mRNA-Toll-like receptor TLRsmRNAs, matrix metalloproteinase 9 mRNA-9 mRNAs, nerve growth factor nongfen mRNA expression and the expression of RAGEGE-TLsMMP-9NGF protein by real-time PCR. Results the purity of neural stem cells was 99% or more at the third passage of cultured rat cerebral cortex cells, and stimulated by recombinant HMGB1 10 ng / mL. The expression of 尾 -tubulin TUJ1 in the experimental group was higher than that in the control group (P 0.05), and the expression of NGF mRNA and protein in the experimental group was higher than that in the control group (P 0.05). Conclusion exogenous recombinant HMGB1 may be promoted by signal pathway such as RAGEG-TLRsMMP-9. Neural stem cells proliferate and differentiate into neurons.
【作者单位】：四川大学华西医院神经内科;
【基金】：国家自然科学基金(No.81371283和No.81671146)资助
【分类号】：R743.3

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