博爾納病病毒核蛋白調(diào)控神經(jīng)干細(xì)胞存活、增殖及分化的初步研究
發(fā)布時(shí)間：2019-04-01 23:09
【摘要】：背景與目的 神經(jīng)干細(xì)胞(Neural Stem Cells,NSCs)在體外培養(yǎng)體系中以神經(jīng)球的方式生長(zhǎng)增殖,而在干細(xì)胞研究中,許多實(shí)驗(yàn)方法都是針對(duì)單個(gè)細(xì)胞進(jìn)行的。將NSCs安全有效地單細(xì)胞化是實(shí)驗(yàn)中的必要步驟。現(xiàn)有多種離散神經(jīng)球的方法,但在安全性或有效性方面存在各種缺陷,對(duì)實(shí)驗(yàn)過程造成困擾。本實(shí)驗(yàn)探討一種離散神經(jīng)球的安全有效的方法,為后繼實(shí)驗(yàn)奠定基礎(chǔ)。 方法 1、體外分離、培養(yǎng)新生24h內(nèi)的Sprague—Dawley(SD)大鼠海馬源性NSCs并進(jìn)行NSCs的Nestin鑒定。 2、實(shí)驗(yàn)設(shè)立以下4組(1)胰酶消化組:未控制神經(jīng)球體積,離散時(shí)胰酶消化30min;(2)單純吹打組:未控制神經(jīng)球體積,離散時(shí)僅用巴斯德吸管吹打;(3)濾網(wǎng)研磨組:未控制神經(jīng)球體積,離散時(shí)采用不銹鋼濾網(wǎng)研磨;(4)控制神經(jīng)球體積結(jié)合胰酶短時(shí)消化組:對(duì)神經(jīng)球體積加以控制,離散時(shí)胰酶短時(shí)消化。 3、分別在離散后5min及離散后1d,觀察各組神經(jīng)球單細(xì)胞化效果及NSCs生長(zhǎng)情況。 4、分別在離散后5min及離散后1d,對(duì)各組細(xì)胞進(jìn)行臺(tái)盼藍(lán)染色細(xì)胞計(jì)數(shù),計(jì)算細(xì)胞存活率。 結(jié)果 胰酶消化較長(zhǎng)時(shí)間(30min)可以得到大量單細(xì)胞但是難以存活;單純巴斯德吸管吹打離散神經(jīng)球效果差;不銹鋼濾網(wǎng)研磨對(duì)細(xì)胞損傷大,導(dǎo)致細(xì)胞存活率低下;而采用控制神經(jīng)球體積結(jié)合胰酶短時(shí)消化可以獲得大量單細(xì)胞并且細(xì)胞存活率明顯較高(離散后5min 92.2%和離散后1d 82.0%,p0.05)。 結(jié)論 控制神經(jīng)球體積(直徑約50μm左右)結(jié)合胰酶短時(shí)(約5min左右)消化法是離散神經(jīng)球的一種安全有效的方法。 背景與目的 博爾納病病毒((Borna Disease Virus,BDV)是一種具有高度嗜神經(jīng)性的病毒。近年,有大量研究發(fā)現(xiàn)該病毒感染與人類神經(jīng)精神疾病包括抑郁癥的發(fā)生有關(guān)。但其確切機(jī)制仍未明了。核蛋白是BDV的主要蛋白之一,由p40基因片段編碼,在細(xì)胞中含量最豐富、變異程度最小。一些研究顯示ERK1/2信號(hào)通路在NSCs的存活、增殖和分化中發(fā)揮重要作用。本實(shí)驗(yàn)用含有博爾納病病毒核蛋白(BDV p40)基因的真核表達(dá)質(zhì)粒pEGFP-p40轉(zhuǎn)染新生24h內(nèi)SD大鼠海馬源性NSCs,觀察其增殖、存活及分化的改變,以及對(duì)NSCs ERK1/2信號(hào)通路的影響,試圖了解BDV p40對(duì)NSCs的作用,從而揭示BDV引起神經(jīng)精神疾病的部分發(fā)病機(jī)制。 方法 1、用陽(yáng)離子脂質(zhì)體法將pEGFP-N1-p40及pEGFP-N1質(zhì)粒分別轉(zhuǎn)染到NSCs中,在熒光顯微鏡下觀察轉(zhuǎn)染效率,用RT-PCR鑒定BDV p40在NSCs中的表達(dá), 2、實(shí)驗(yàn)設(shè)置3組:未轉(zhuǎn)染組、pEGFP-N1空轉(zhuǎn)組及pEGFP-N1-p40轉(zhuǎn)染組,用CCK-8試劑盒檢測(cè)細(xì)胞存活的改變,用BrdU攝入實(shí)驗(yàn)檢測(cè)細(xì)胞增殖的改變,并經(jīng)免疫組化檢測(cè)轉(zhuǎn)染后14d細(xì)胞貼壁分化為神經(jīng)元、星型膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞的比例變化。用Western Blot檢測(cè)ERK1/2磷酸化的改變。 結(jié)果 1、成功建立表達(dá)BDV p40的NSCs模型。在熒光顯微鏡下觀察到pEGFP-N1空轉(zhuǎn)組和pEGFP-N1-p40轉(zhuǎn)染組約10%NSCs在胞漿和/或胞核可見綠色熒光,未轉(zhuǎn)染組無(wú)綠色熒光表達(dá);PCR結(jié)果顯示只有pEGFP-N1-p40轉(zhuǎn)染組細(xì)胞有BDV p40基因表達(dá),而pEGFP-N1對(duì)照組及未轉(zhuǎn)染組細(xì)胞內(nèi)均未見表達(dá)。 2、(1)BDV p40抑制NSC s的存活。(2)BDV p40抑制NSCs的增殖。(3)在轉(zhuǎn)染后貼壁分化14d時(shí)3組細(xì)胞分化為神經(jīng)元、星型膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞的比例未見顯著差異。(4)Western Blot結(jié)果顯示BDV p40下調(diào)了磷酸化ERK1/2在蛋白水平的表達(dá)。 結(jié)論 BDV p40抑制NSCs的存活、增殖,但是對(duì)NSCs的分化方向沒有明顯的影響。BDV p40有可能通過下調(diào)磷酸化ERK1/2活性對(duì)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373
本文編號(hào)：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 鄧婧;博爾納病病毒對(duì)人少突膠質(zhì)細(xì)胞增殖與凋亡影響的初步研究[D];重慶醫(yī)科大學(xué);2012年
，本文編號(hào)：2451996
本文鏈接：http://sikaile.net/xiyixuelunwen/2451996.html
最近更新
教材專著
