人参皂苷Rg1对脑损伤后神经干细胞增殖、分化的作用及其表观遗传调控机制
发布时间：2018-08-12 18:37
【摘要】：神经干细胞(neural stem cells,NSCs)是可以自我增生和复制,并能分化为构成中枢神经系统的包括神经元、星形胶质细胞、少突胶质细胞的细胞类型。神经前体细胞(neural progenitor cells,NPCs)是神经干细胞在分化过程中初步分化形成的具备分化为单一成熟神经细胞的单能神经干细胞。在胚胎早期,脑室区NSCs是处于对称分裂状态中,从而达到增加数量的目的。接下来,神经干细胞再通过不对称分裂,分裂为神经干细胞和神经元来继续神经发生的过程。胚胎发展过程中,一部分神经干细胞是具备分裂成一个神经干细胞和一个NPCs的能力。神经发生后期,NSCs在分化成神经元的基础上,还可以分化产生为星型胶质细胞和少突胶质细胞。上述过程是受很多因素调节才得以发生的,其中表观遗传学是重要的调节因素。表观遗传学(epigenetics)是一种指不涉及DNA序列改变的基因或者蛋白表达的变化,并可以在发育和细胞增殖过程中稳定传递的遗传学,即可以从一个细胞被传递到其子代的基因调节类型。表观遗传性状的变化具有可继承性,又有一定程度的可逆性。表观遗传学在环境因素和转录因子等因素的调控下可以调节神经干细胞的增殖和分化。表观遗传学包括很多方式,对神经干细胞的增殖分化都存在着影响。因此揭示表观遗传学如何调节神经干细胞的增殖分化的机制,对中枢神经退行性病变的防治有着重要的意义。1、人参皂苷Rg1对离体缺血再灌注神经干细胞增殖分化的作用采用氧糖剥夺/再灌注(OGD/R)细胞模型模拟脑缺血再灌注造成的脑组织损伤,将体外分离培养的神经干细胞分为OGD/R后复氧2h、4h、6h共3个时间点,每个时间点分为对照组、缺血再灌注组和人参皂苷Rg1组,对照组细胞正常培养,缺血再灌注组和人参皂苷Rg1组缺氧培养4h后,再分别正常培养2h、4h、6h,各时间点结束后,各组神经干细胞增殖标记物(Brdu)、神经干细胞特异性标志物巢蛋白(Nestin)、神经元祖细胞特异性标记物(Tuj-1)及星形胶质细胞祖细胞标记物(Vimentin)的表达都应用免疫荧光双标染色方法进行检测,统计各组阳性细胞的个数、光密度、面密度。结果发现正常情况下,Nestin、Tuj-1和Vimentin均能在神经干细胞中有所表达。缺血再灌注组与对照组相比,免疫荧光观察发现,神经干细胞的阳性细胞数、光密度、面密度明显减少和降低,差异具有统计学意义(P0.05);人参皂苷Rg1组与缺血再灌注组相比,神经干细胞的阳性细胞数、光密度、面密度明显增加,差异具有统计学意义(P0.05)。而且随着复氧时间的延长,神经干细胞阳性细胞数、光密度、面密度逐渐增加。从形态学上说明人参皂苷Rg1可以在缺血缺氧的条件下通过促进神经干细胞的增殖分化,从而促进中枢神经损伤的修复。2、人参皂苷Rg1对缺血再灌注神经干细胞microRNA-21/210表达的影响实时荧光定量PCR的方法检测microRNA-21/210的表达量与神经干细胞增殖分化的关系,与对照组相比,缺血再灌注组miR-21在复氧4h和6h的表达呈现上调表达,miR-210在三个时间点均呈现上调表达,差异具有统计学意义(P0.05),这说明在缺氧可以促进miR-21和miR-210的表达,从而促进神经干细胞的增殖。人参皂苷Rg1干预后,与缺血再灌注组相比,人参皂苷Rg1组miR-21在在复氧4h和6h的表达呈现上调表达,miR-210在三个时间点均呈现上调表达差异具有统计学意义(P0.05),这说明人参皂苷Rg1可以促进缺氧状态下的神经干细胞中miR-21和miR-210的表达增加,促进神经干细胞在缺氧状态下的修复。这说明人参皂苷Rg1可能通过miR-21和miR-210参与脑损伤的神经干细胞修复。3、人参皂苷Rg1对缺血再灌注神经干细胞DNMT1/DNMT3a表达的影响实时荧光定量PCR发现,与对照组相比,三个时间点的缺血再灌注组中的DNMT1和DNMT3a的表达均有所增加,且差异有统计学意义(p0.05)。但人参皂苷Rg1药物干预后,与缺血再灌注组相比,三个时间点下人参皂苷Rg1组中的DNMT1表达是没有显著变化的,而DNMT3a的表达呈现显著上调,且差异有统计学意义(p0.05)。Western blot实验发现,神经干细胞中的DNMT3a在缺血再灌注组中,即缺血缺氧的条件下,与对照组相比,三个时间点的表达均是增加的,差异具有统计学意义(P0.05)。这说明DNMT3a可能促进神经干细胞在缺血缺氧条件下的自我修复和增殖。在人参皂苷Rg1的干预下,与缺血再灌注组相比,三个时间点的DNMT3a的表达也是增加的。说明人参皂苷Rg1可能通过DNMT3a促进神经干细胞的自我更新和增殖。综上所述,人参皂苷Rg1可以在缺血缺氧的条件下促进神经干细胞的增殖分化,并可以促进缺氧状态下的神经干细胞中miR-21和miR-210以及DNMT3a的表达增加。这证明人参皂苷Rg1可能通过增强miR-21/miR-210以及DNMT3a表达,从而能促进了缺血再灌注神经干细胞的存活、自我复制和分化,参与脑组织缺血、损伤的神经修复。
[Abstract]:Neural stem cells (NSCs) can self-proliferate and replicate and differentiate into cell types that comprise neurons, astrocytes and oligodendrocytes in the central nervous system. In the early embryonic stage, NSCs in the ventricular region are in a state of symmetrical division, thereby increasing the number of neural stem cells. Next, neural stem cells are divided asymmetrically into neural stem cells and neurons to continue the process of neurogenesis. During embryonic development, a part of the nerve is involved. Stem cells have the ability to divide into a neural stem cell and a NPCs. At the late stage of neurogenesis, NSCs can also differentiate into astrocytes and oligodendrocytes on the basis of differentiation into neurons. Epigenetics is a type of genetics that does not involve changes in DNA sequence or protein expression and can be transmitted steadily during development and cell proliferation. Reversibility. Epigenetics regulates the proliferation and differentiation of neural stem cells by environmental factors and transcription factors. Epigenetics involves many ways that affect the proliferation and differentiation of neural stem cells. Therefore, it is necessary to reveal how epigenetics regulates the proliferation and differentiation of neural stem cells and the central nervous system. The effect of ginsenoside Rg1 on the proliferation and differentiation of neural stem cells after ischemia-reperfusion in vitro was studied. The brain tissue injury induced by cerebral ischemia-reperfusion was simulated by oxygen-glucose deprivation/reperfusion (OGD/R) cell model. The neural stem cells isolated and cultured in vitro were divided into OGD/R 2 h, 4 h and 6 h after reoxygenation. At each time point, the cells were divided into control group, ischemia-reperfusion group and ginsenoside Rg1 group, control group were cultured normally, ischemia-reperfusion group and ginsenoside Rg1 group were cultured under hypoxia for 4 hours, then normal culture for 2 hours, 4 hours, 6 hours, after the end of each time point, neural stem cell proliferation markers (Brdu), neural stem cell specific markers nested eggs. The expression of Nestin, Tuj-1 and Vimentin were detected by immunofluorescence double labeling. The number, optical density and surface density of positive cells were counted. The results showed that Nestin, Tuj-1 and Vimentin could be found in neural stem cells under normal conditions. Compared with the control group, the number of positive cells, optical density and area density of neural stem cells in the ischemia-reperfusion group were significantly decreased (P 0.05), and the number of positive cells, optical density and area density of neural stem cells in the ginsenoside Rg1 group were significantly higher than those in the ischemia-reperfusion group. Morphologically, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, thereby promoting the repair of central nervous system injury. 2, ginsenoside R Effect of G1 on the expression of microRNA-21/210 in ischemia-reperfusion neural stem cells The difference was statistically significant (P 0.05), indicating that hypoxia can promote the expression of microRNAs-21 and microRNAs-210, thus promoting the proliferation of neural stem cells. The difference was statistically significant (P 0.05), indicating that ginsenoside Rg1 can promote the expression of microRNAs-21 and microRNAs-210 in neural stem cells under hypoxia, and promote the repair of neural stem cells under hypoxia. This suggests that ginsenoside Rg1 may participate in the repair of neural stem cells after brain injury through microRNAs-21 and microRNAs-210. The expression of DNMT1 and DNMT3a in the ischemia-reperfusion group at three time points was higher than that in the control group, and the difference was statistically significant (p0.05). The expression of DNMT1 in the Rg1 group was not significantly changed, but the expression of DNMT3a was significantly up-regulated (p0.05). Western blot showed that the expression of DNMT3a in neural stem cells was increased in the ischemia-reperfusion group, i.e. in the condition of ischemia-hypoxia, compared with the control group at three time points. The difference was statistically significant (P 0.05). This suggests that DNMT3a may promote the self-repair and proliferation of neural stem cells under hypoxic and ischemic conditions. In conclusion, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, and increase the expression of microRNAs-21, microRNAs-210 and DNMT3a in neural stem cells under hypoxic and ischemic conditions. It can promote the survival, self-replication and differentiation of neural stem cells after ischemia-reperfusion, and participate in the nerve repair of cerebral ischemia and injury.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285
本文编号：2179942
[Abstract]:Neural stem cells (NSCs) can self-proliferate and replicate and differentiate into cell types that comprise neurons, astrocytes and oligodendrocytes in the central nervous system. In the early embryonic stage, NSCs in the ventricular region are in a state of symmetrical division, thereby increasing the number of neural stem cells. Next, neural stem cells are divided asymmetrically into neural stem cells and neurons to continue the process of neurogenesis. During embryonic development, a part of the nerve is involved. Stem cells have the ability to divide into a neural stem cell and a NPCs. At the late stage of neurogenesis, NSCs can also differentiate into astrocytes and oligodendrocytes on the basis of differentiation into neurons. Epigenetics is a type of genetics that does not involve changes in DNA sequence or protein expression and can be transmitted steadily during development and cell proliferation. Reversibility. Epigenetics regulates the proliferation and differentiation of neural stem cells by environmental factors and transcription factors. Epigenetics involves many ways that affect the proliferation and differentiation of neural stem cells. Therefore, it is necessary to reveal how epigenetics regulates the proliferation and differentiation of neural stem cells and the central nervous system. The effect of ginsenoside Rg1 on the proliferation and differentiation of neural stem cells after ischemia-reperfusion in vitro was studied. The brain tissue injury induced by cerebral ischemia-reperfusion was simulated by oxygen-glucose deprivation/reperfusion (OGD/R) cell model. The neural stem cells isolated and cultured in vitro were divided into OGD/R 2 h, 4 h and 6 h after reoxygenation. At each time point, the cells were divided into control group, ischemia-reperfusion group and ginsenoside Rg1 group, control group were cultured normally, ischemia-reperfusion group and ginsenoside Rg1 group were cultured under hypoxia for 4 hours, then normal culture for 2 hours, 4 hours, 6 hours, after the end of each time point, neural stem cell proliferation markers (Brdu), neural stem cell specific markers nested eggs. The expression of Nestin, Tuj-1 and Vimentin were detected by immunofluorescence double labeling. The number, optical density and surface density of positive cells were counted. The results showed that Nestin, Tuj-1 and Vimentin could be found in neural stem cells under normal conditions. Compared with the control group, the number of positive cells, optical density and area density of neural stem cells in the ischemia-reperfusion group were significantly decreased (P 0.05), and the number of positive cells, optical density and area density of neural stem cells in the ginsenoside Rg1 group were significantly higher than those in the ischemia-reperfusion group. Morphologically, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, thereby promoting the repair of central nervous system injury. 2, ginsenoside R Effect of G1 on the expression of microRNA-21/210 in ischemia-reperfusion neural stem cells The difference was statistically significant (P 0.05), indicating that hypoxia can promote the expression of microRNAs-21 and microRNAs-210, thus promoting the proliferation of neural stem cells. The difference was statistically significant (P 0.05), indicating that ginsenoside Rg1 can promote the expression of microRNAs-21 and microRNAs-210 in neural stem cells under hypoxia, and promote the repair of neural stem cells under hypoxia. This suggests that ginsenoside Rg1 may participate in the repair of neural stem cells after brain injury through microRNAs-21 and microRNAs-210. The expression of DNMT1 and DNMT3a in the ischemia-reperfusion group at three time points was higher than that in the control group, and the difference was statistically significant (p0.05). The expression of DNMT1 in the Rg1 group was not significantly changed, but the expression of DNMT3a was significantly up-regulated (p0.05). Western blot showed that the expression of DNMT3a in neural stem cells was increased in the ischemia-reperfusion group, i.e. in the condition of ischemia-hypoxia, compared with the control group at three time points. The difference was statistically significant (P 0.05). This suggests that DNMT3a may promote the self-repair and proliferation of neural stem cells under hypoxic and ischemic conditions. In conclusion, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, and increase the expression of microRNAs-21, microRNAs-210 and DNMT3a in neural stem cells under hypoxic and ischemic conditions. It can promote the survival, self-replication and differentiation of neural stem cells after ischemia-reperfusion, and participate in the nerve repair of cerebral ischemia and injury.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285
【参考文献】
相关期刊论文 前10条
1 叶玉勤;苏鑫洪;贺晓生;;microRNA-132调控突触可塑性的作用研究[J];中华神经外科疾病研究杂志;2016年05期
2 姚志刚;吕蓓蓓;李加美;;组蛋白去乙酰化酶2在神经系统领域的研究进展[J];医学综述;2016年19期
3 丁玲芝;姜正林;高艳琴;王国华;;组蛋白去乙酰化酶抑制剂对缺血缺氧性脑损伤后神经保护的研究进展[J];复旦学报(医学版);2016年05期
4 曹治云;张志灯;陈旭征;黄争荣;王雷;陈立武;陈瑞琦;廖联明;;低氧条件下低氧诱导因子1α/miR-210调节回路对肿瘤能量代谢及血管生成的调控[J];中华细胞与干细胞杂志(电子版);2016年03期
5 任衍钢;白冠军;宋玉奇;卫红萍;;表观遗传学的起源与发展[J];生物学通报;2016年03期
6 崔晓萍;陈建梅;穆军山;叶建新;林敏;赵文龙;翁婧;林航;;PI3K-AKT通路对脑缺血损伤神经干细胞的增殖作用[J];中国康复医学杂志;2016年02期
7 李鹏;陈凡帆;谢伟;张昊;钟文军;涂兰波;曹志恺;全伟;;海马可溶性因子体外诱导分化大鼠内源性神经干细胞为胶质样细胞[J];广州医药;2016年01期
8 梁红霞;李依們;时书勤;邓锦波;;血管介导下的神经细胞迁移[J];河南大学学报(医学版);2015年04期
9 张姗姗;全日城;杨晓红;许勇钢;胡晓梅;刘锋;麻柔;;青黄散治疗骨髓增生异常综合征DNA磷硫酰化探析[J];中国中医基础医学杂志;2015年10期
10 许伟;谢兴文;赵永利;李宁;徐世红;宋敏;;Brdu标记的rBMSCs在大鼠体内的迁移[J];中国骨质疏松杂志;2015年04期
，本文编号：2179942
本文链接：https://www.wllwen.com/shoufeilunwen/mpalunwen/2179942.html
最近更新
教材专著
