博尔纳病病毒核蛋白调控神经干细胞存活、增殖及分化的初步研究
发布时间：2019-04-01 23:09
【摘要】：背景与目的 神经干细胞(Neural Stem Cells,NSCs)在体外培养体系中以神经球的方式生长增殖,而在干细胞研究中,许多实验方法都是针对单个细胞进行的。将NSCs安全有效地单细胞化是实验中的必要步骤。现有多种离散神经球的方法,但在安全性或有效性方面存在各种缺陷,对实验过程造成困扰。本实验探讨一种离散神经球的安全有效的方法,为后继实验奠定基础。 方法 1、体外分离、培养新生24h内的Sprague—Dawley(SD)大鼠海马源性NSCs并进行NSCs的Nestin鉴定。 2、实验设立以下4组(1)胰酶消化组:未控制神经球体积,离散时胰酶消化30min;(2)单纯吹打组:未控制神经球体积,离散时仅用巴斯德吸管吹打;(3)滤网研磨组:未控制神经球体积,离散时采用不锈钢滤网研磨;(4)控制神经球体积结合胰酶短时消化组:对神经球体积加以控制,离散时胰酶短时消化。 3、分别在离散后5min及离散后1d,观察各组神经球单细胞化效果及NSCs生长情况。 4、分别在离散后5min及离散后1d,对各组细胞进行台盼蓝染色细胞计数,计算细胞存活率。 结果 胰酶消化较长时间(30min)可以得到大量单细胞但是难以存活;单纯巴斯德吸管吹打离散神经球效果差;不锈钢滤网研磨对细胞损伤大,导致细胞存活率低下;而采用控制神经球体积结合胰酶短时消化可以获得大量单细胞并且细胞存活率明显较高(离散后5min 92.2%和离散后1d 82.0%,p0.05)。 结论 控制神经球体积(直径约50μm左右)结合胰酶短时(约5min左右)消化法是离散神经球的一种安全有效的方法。 背景与目的 博尔纳病病毒((Borna Disease Virus,BDV)是一种具有高度嗜神经性的病毒。近年,有大量研究发现该病毒感染与人类神经精神疾病包括抑郁症的发生有关。但其确切机制仍未明了。核蛋白是BDV的主要蛋白之一,由p40基因片段编码,在细胞中含量最丰富、变异程度最小。一些研究显示ERK1/2信号通路在NSCs的存活、增殖和分化中发挥重要作用。本实验用含有博尔纳病病毒核蛋白(BDV p40)基因的真核表达质粒pEGFP-p40转染新生24h内SD大鼠海马源性NSCs,观察其增殖、存活及分化的改变,以及对NSCs ERK1/2信号通路的影响,试图了解BDV p40对NSCs的作用,从而揭示BDV引起神经精神疾病的部分发病机制。 方法 1、用阳离子脂质体法将pEGFP-N1-p40及pEGFP-N1质粒分别转染到NSCs中,在荧光显微镜下观察转染效率,用RT-PCR鉴定BDV p40在NSCs中的表达, 2、实验设置3组:未转染组、pEGFP-N1空转组及pEGFP-N1-p40转染组,用CCK-8试剂盒检测细胞存活的改变,用BrdU摄入实验检测细胞增殖的改变,并经免疫组化检测转染后14d细胞贴壁分化为神经元、星型胶质细胞、少突胶质细胞的比例变化。用Western Blot检测ERK1/2磷酸化的改变。 结果 1、成功建立表达BDV p40的NSCs模型。在荧光显微镜下观察到pEGFP-N1空转组和pEGFP-N1-p40转染组约10%NSCs在胞浆和/或胞核可见绿色荧光,未转染组无绿色荧光表达;PCR结果显示只有pEGFP-N1-p40转染组细胞有BDV p40基因表达,而pEGFP-N1对照组及未转染组细胞内均未见表达。 2、(1)BDV p40抑制NSC s的存活。(2)BDV p40抑制NSCs的增殖。(3)在转染后贴壁分化14d时3组细胞分化为神经元、星型胶质细胞、少突胶质细胞的比例未见显著差异。(4)Western Blot结果显示BDV p40下调了磷酸化ERK1/2在蛋白水平的表达。 结论 BDV p40抑制NSCs的存活、增殖,但是对NSCs的分化方向没有明显的影响。BDV p40有可能通过下调磷酸化ERK1/2活性对NSCs的存活、增殖起抑制作用。
[Abstract]:Background and Purpose Neural Stem Cells (NSCs) grow in a neurosphere in an in vitro culture system, whereas in stem cell studies many of the experimental methods are for individual cells It is necessary for NSCs to be safely and effectively single-cell. The present invention relates to a method for producing a plurality of discrete neurospheres, but there are various defects in the aspects of safety or effectiveness, The safety and effective method of a kind of discrete nerve ball is discussed in this experiment, which lays the foundation for subsequent experiments. No, no, no. Method 1. In vitro separation, Sprague-Dawley (SD) rat hippocampal-derived NSCs and Nes of NSCs were cultured in 24 h. tin identification.2. The following four groups (1) of the pancreatic enzyme digestion group were established: the volume of the neural ball was not controlled, the pancreatin was digested for 30 minutes at the time of dispersion; (2) the simple blow-up group: the volume of the nerve ball was not controlled, and it was only blown by the pasteur pipette at the time of dispersion; (3) the filter screen grinding group: not The volume of the nerve ball is controlled, the stainless steel filter screen is used for grinding when the volume of the nerve ball is discrete, (4) the volume of the nerve ball is controlled to be combined with the short-time digestion group of the pancreatin, the volume of the nerve ball is controlled, the dispersion is discrete, In that case of short-time digestion of the pancreatin, the single-cell effect of each group of neurospheres was observe after 5 min and 1 d after the dispersion, respectively. And the growth of NSCs.4. The cells were stained with trypan blue for 5 min and 1 d after the dispersion, respectively. cytometer The results showed that the pancreatic enzyme can be digested for a long time (30 min) to obtain a large number of single cells, but it is difficult to survive; the simple Pasteur pipet has poor effect on the discrete nerve ball, and the grinding of the stainless steel filter screen The large number of single cells can be obtained with the control of the volume of the neural ball and the short-time digestion of the pancreatin, and the cell survival rate is obviously higher (92.2% and 92.2% after the dispersion). Rear 1 D 82.0%, p0.05). It was concluded that the volume of the nerve-ball (about 50. mu.m in diameter) was combined with the pancreatin for a short time (about 5 min). digestion method is away from A safe and effective method for the treatment of scattered neurospheres. Background and Objective Borna Disease V The virus (BDV) is a highly neurogenic virus. In recent years, a large number of studies have been found viral infection and human neuropsychiatric The disease is related to the occurrence of depression. But the exact mechanism remains unknown. The nucleoprotein is one of the major proteins of the BDV and is composed of p40 Gene fragment encoding, the most abundant in the cell, the smallest variation. Some studies show the ERK1/2 signal The survival, proliferation and differentiation of NSCs play an important role in the survival, proliferation and differentiation of NSCs. In this experiment, a eukaryotic expression plasmid pEGFP-p40 containing the BDV p40 gene was used to transfect the rat hippocampal-derived NSCs in the newborn for 24 h, and the proliferation, survival and differentiation of the NSCs were observed, and the NSCs ERK1/2 signaling was also observed. The effect of BDV p40 on NSCs is an attempt to understand the effect of BDV p40 on NSCs , from Methods 1. pEGFP-N1-p40 and pEGFP-N1 plasmids were transfected into NSCs by cationic liposome. Efficiency and RT-PCR to identify the expression of BDV p40 in NSCs. Group, pEGFP-N1 lost group and pEGFP-N1-p40 transfection group, the change of cell survival was detected by means of CCK-8, and the changes of cell proliferation were detected by BrdU uptake. The proportion of the cells to the neuron, the astrocytes, the oligodendrocytes, change . Western Blot was used to detect ERK1/2 phosphorus. Results 1. The NSCs model for expressing BDV p40 was successfully established. The green fluorescence of pEGFP-N1 and pEGFP-N1-p40 was observed under the fluorescence microscope, and no green fluorescence expression was observed in the untransfected group. The results showed that only the cells transfected with pEGFP-N1-p40 were BDV. p40 gene expression, and pEGFP-N1 control group and no expression was found in the transfected group cells. (1 ) BDV p40 inhibits the survival of NSCs. (2) BDV p40 inhibits the proliferation of NSCs. (3) After transfection, the adherent differentiation 14 There was no significant difference in the ratio of 3 groups of cells to the neurons, astrocytes and oligodendrocytes. (4) Weaster n B The results showed that BDV p40 down-regulated the expression of phosphorylated ERK1/2 at the protein level. BDV p40 inhibited the survival and proliferation of NSCs, but did not have a significant effect on the differentiation direction of NSCs.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
本文编号：2451996
[Abstract]:Background and Purpose Neural Stem Cells (NSCs) grow in a neurosphere in an in vitro culture system, whereas in stem cell studies many of the experimental methods are for individual cells It is necessary for NSCs to be safely and effectively single-cell. The present invention relates to a method for producing a plurality of discrete neurospheres, but there are various defects in the aspects of safety or effectiveness, The safety and effective method of a kind of discrete nerve ball is discussed in this experiment, which lays the foundation for subsequent experiments. No, no, no. Method 1. In vitro separation, Sprague-Dawley (SD) rat hippocampal-derived NSCs and Nes of NSCs were cultured in 24 h. tin identification.2. The following four groups (1) of the pancreatic enzyme digestion group were established: the volume of the neural ball was not controlled, the pancreatin was digested for 30 minutes at the time of dispersion; (2) the simple blow-up group: the volume of the nerve ball was not controlled, and it was only blown by the pasteur pipette at the time of dispersion; (3) the filter screen grinding group: not The volume of the nerve ball is controlled, the stainless steel filter screen is used for grinding when the volume of the nerve ball is discrete, (4) the volume of the nerve ball is controlled to be combined with the short-time digestion group of the pancreatin, the volume of the nerve ball is controlled, the dispersion is discrete, In that case of short-time digestion of the pancreatin, the single-cell effect of each group of neurospheres was observe after 5 min and 1 d after the dispersion, respectively. And the growth of NSCs.4. The cells were stained with trypan blue for 5 min and 1 d after the dispersion, respectively. cytometer The results showed that the pancreatic enzyme can be digested for a long time (30 min) to obtain a large number of single cells, but it is difficult to survive; the simple Pasteur pipet has poor effect on the discrete nerve ball, and the grinding of the stainless steel filter screen The large number of single cells can be obtained with the control of the volume of the neural ball and the short-time digestion of the pancreatin, and the cell survival rate is obviously higher (92.2% and 92.2% after the dispersion). Rear 1 D 82.0%, p0.05). It was concluded that the volume of the nerve-ball (about 50. mu.m in diameter) was combined with the pancreatin for a short time (about 5 min). digestion method is away from A safe and effective method for the treatment of scattered neurospheres. Background and Objective Borna Disease V The virus (BDV) is a highly neurogenic virus. In recent years, a large number of studies have been found viral infection and human neuropsychiatric The disease is related to the occurrence of depression. But the exact mechanism remains unknown. The nucleoprotein is one of the major proteins of the BDV and is composed of p40 Gene fragment encoding, the most abundant in the cell, the smallest variation. Some studies show the ERK1/2 signal The survival, proliferation and differentiation of NSCs play an important role in the survival, proliferation and differentiation of NSCs. In this experiment, a eukaryotic expression plasmid pEGFP-p40 containing the BDV p40 gene was used to transfect the rat hippocampal-derived NSCs in the newborn for 24 h, and the proliferation, survival and differentiation of the NSCs were observed, and the NSCs ERK1/2 signaling was also observed. The effect of BDV p40 on NSCs is an attempt to understand the effect of BDV p40 on NSCs , from Methods 1. pEGFP-N1-p40 and pEGFP-N1 plasmids were transfected into NSCs by cationic liposome. Efficiency and RT-PCR to identify the expression of BDV p40 in NSCs. Group, pEGFP-N1 lost group and pEGFP-N1-p40 transfection group, the change of cell survival was detected by means of CCK-8, and the changes of cell proliferation were detected by BrdU uptake. The proportion of the cells to the neuron, the astrocytes, the oligodendrocytes, change . Western Blot was used to detect ERK1/2 phosphorus. Results 1. The NSCs model for expressing BDV p40 was successfully established. The green fluorescence of pEGFP-N1 and pEGFP-N1-p40 was observed under the fluorescence microscope, and no green fluorescence expression was observed in the untransfected group. The results showed that only the cells transfected with pEGFP-N1-p40 were BDV. p40 gene expression, and pEGFP-N1 control group and no expression was found in the transfected group cells. (1 ) BDV p40 inhibits the survival of NSCs. (2) BDV p40 inhibits the proliferation of NSCs. (3) After transfection, the adherent differentiation 14 There was no significant difference in the ratio of 3 groups of cells to the neurons, astrocytes and oligodendrocytes. (4) Weaster n B The results showed that BDV p40 down-regulated the expression of phosphorylated ERK1/2 at the protein level. BDV p40 inhibited the survival and proliferation of NSCs, but did not have a significant effect on the differentiation direction of NSCs.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
【引证文献】
相关硕士学位论文 前1条
1 邓婧;博尔纳病病毒对人少突胶质细胞增殖与凋亡影响的初步研究[D];重庆医科大学;2012年
，本文编号：2451996
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