贴壁和悬浮培养的神经前体细胞生物学特性研究及酒精对神经前体细胞Cx43表达的影响

发布时间：2018-06-13 21:40

  本文选题：神经前体细胞 + 悬浮培养法　； 参考：《山东大学》2009年硕士论文

【摘要】： 研究背景: 神经前体细胞(neural precursor cells,NPCs)是中枢神经系统中处于不同发育阶段的不成熟神经细胞的总称,具有自我更新、增殖和分化为各类神经细胞的潜能,是公认的神经损伤修复的种子细胞。应用体外扩增的NPCs对神经系统创伤、变性及退行性疾病的治疗具有广阔的应用前景。1992年,Reynolds首先建立了NPCs的体外悬浮培养方法,开创了NPCs体外培养、扩增及其应用的新纪元。1995年Gage建立了NPCs的贴壁培养方法,大大推动了对NPCs的相关研究。但目前国际上对NPCs的研究仍然处于初级阶段,对两种不同培养方法获得的NPCs生物学特性研究资料还非常缺乏。我国开展NPCs的研究起步较晚,目前主要使用悬浮培养的方法开展工作。为了深入开展对NPCs相关研究,本课题在完善NPCs贴壁培养方法的基础上,对比研究了两种培养条件下的NPCs的生物学特性,为深入开展NPCs的基础研究和临床应用研究提供实验依据。 胎儿酒精综合征(fetal alcohol syndrome,FAS)是母亲在妊娠期间过量饮酒造成的子代出生缺陷,其症状包括一系列的中枢神经系统发育异常。体内、外实验发现,酒精可抑制NPCs的增殖,并导致部分NPCs死亡,这被认为是FAS发病的原因之一。但对于酒精损伤NPCs的机理,目前了解还比较少。文献报道,连接蛋白43(connexin 43,Cx43)在NPCs增殖过程中具有重要的作用。酒精对NPCs增殖的抑制作用是否与Cx43的表达有关,目前尚未见相关报道。 研究目的: 1、对比研究贴壁培养法和悬浮培养法在体外培养获得的NPCs的生物学特性,为深入开展NPCs的基础研究和临床应用研究提供实验依据。 2、建立体外贴壁培养NPCs的酒精损伤模型,研究酒精对体外培养的NPCs中Cx43表达的影响,探讨酒精抑制NPCs增殖的作用机理。 研究方法:以孕14d的胎鼠端脑为实验材料,分别采用贴壁培养和悬浮培养方法进行NPCs的原代、传代及冻存复苏后培养,进行下列实验: 1、两种培养方法获得的NPCs的生物学特性对比实验 (1)倒置相差显微镜下,每天对培养细胞进行形态学观察,并拍照; (2)用免疫荧光细胞化学方法及流式细胞术鉴定培养细胞中NPCs的纯度; (3)用BrdU掺入方法分析NPCs的增殖能力; (4)用细胞计数方法分析两种原代培养方法获得的NPCs的生长曲线; (5)采用去除生长因子的方法诱导NPCs分化,用免疫荧光方法鉴定各类分化细胞并分析其比例特征。 2、酒精对NPCs的Cx43的表达的影响 (1)建立贴壁培养NPCs酒精损伤实验模型,采用倒置相差显微镜观察细胞形态变化,并拍照; (2)采用MTT法检测NPCs琥珀酸脱氢酶的变化,分析酒精对NPCs活性的影响; (3)采用免疫荧光方法观察体外贴壁培养的NPCs中Cx43的表达情况; (4)采用Western-blot法检测NPCs中Cx43的表达量,分析酒精对NPCs中Cx43表达的影响; (5)通过刻痕标记/荧光扩散实验,观察荧光黄(Lucifer Yellow,LY)在NPCs之间的扩散距离,分析酒精对NPCs间隙连接功能的影响。 实验结果: 1、两种培养方法获得的NPCs的生物学特性对比实验 (1)形态学观察贴壁培养的NPCs表现出典型的神经上皮细胞的形态特征,细胞群体呈集落状生长,随着培养时间延长,细胞呈放射状向外延伸;悬浮培养的NPCs呈球状克隆生长,细胞多数呈圆形,细胞连接紧密。传代以及冻存复苏后培养的NPCs形态均无明显变化; (2) NPCs纯度鉴定原代贴壁培养细胞中nestin阳性细胞率由第3天的38.69%增加至第9天的93.76%,而原代悬浮培养细胞第9天的nestin阳性细胞率仅为59.75%;两者传代后第2天,贴壁培养细胞的nestin阳性细胞率可达98.36%,悬浮培养细胞为83.34%;冻存复苏后培养第4天,两种方法获得的NPCs纯度达均可达到99.9%; (3) NPCs的增殖能力检测原代贴壁培养的NPCs的增殖指数,由培养第3天的13.68%增加至最高的第7天的38.7%;悬浮培养第7天的NPCs的增殖指数亦达最高,但仅为33.47%;贴壁培养细胞在传代后第2天和复苏后培养第4天,增殖指数分别可达39.08%和39.25%,而悬浮培养的NPCs这两种条件下的增殖指数仅为36.95%和36.73%; (4)原代培养NPCs生长曲线分析两种培养方法获得的生长曲线均为“S”形曲线,但在细胞数量上有明显差异。贴壁培养第12天时,细胞数量最多,为初始接种数的68倍,继续培养细胞数量开始降低;悬浮培养第11天时,细胞数量即达最多,但仅为初始接种量的8.4倍; (5) NPCs的分化能力分析贴壁培养的NPCs诱导分化后培养7d,神经元、星形胶质细胞和少突胶质细胞的比例分别为14.13%,29.99%和7.21%;而悬浮培养的NPCs诱导分化7d后,上述三者的比例分别为17.72%,24.81%和8.15%。 2、酒精对NPCs的Cx43的表达的影响 (1)形态学观察贴壁培养的NPCs经不同浓度酒精(25、50和100mmol/L)损伤24h后,细胞会表现为不同程度的胞体变圆和突起回缩,高浓度酒精(100mmol/L)可使培养物中出现明显的细胞碎片; (2) MTT法检测细胞活性三种酒精浓度下的细胞存活率分别为79.25%、70.60%和67.93%; (3)免疫荧光化学法检测NPCs中Cx43表达特点Cx43广泛分布于NPCs的细胞表面,并且在相邻NPCs胞体接触位置分布较为集中; (4) Western-blot检测NPCs中Cx43表达量的变化三种浓度的酒精作用NPCs24h后,Cx43/Actin值分别为:0.82、0.67、0.46和0.19; (5)刻痕标记/荧光扩散实验检测NPCs间隙连接功能的变化LY的传播距离及荧光信号强度随酒精损伤浓度的增加而变短或减弱。 实验结论: 1、贴壁培养法获得的NPCs便于进行细胞计数和形态学观察。 2、原代和传代贴壁培养的细胞nestin阳性细胞率均高于悬浮培养细胞。 3、原代和传代贴壁培养获得的NPCs均具有较强的自我扩增能力,而悬浮培养获得的NPCs自我扩增能力相对较差;两种NPCs冻存复苏后均能保持较高的自我扩增能力。 4、贴壁培养获得的NPCs连续传代5代后扩增能力显著下降,而悬浮培养获得的NPCs在连续传代10次以上仍能较好地保持扩增能力。 5、贴壁和悬浮培养获得的NPCs,分化能力无显著差别。 6、贴壁培养的NPCs可以表达Cx43,酒精可以通过抑制NPCs中Cx43的表达来影响NPCs的间隙连接功能。
[Abstract]:Research background:
Neural precursor cells (NPCs) is the general name of immature nerve cells in the different developmental stages of the central nervous system. It has the potential of self renewal, proliferation and differentiation into all kinds of nerve cells. It is the recognized seed cell of neural injury repair. In vitro amplification of NPCs should be used for nerve system trauma, degeneration and regression. The treatment of progressive diseases has a broad prospect of application in.1992 years. Reynolds first established the method of NPCs in vitro suspension culture, created a new era of NPCs in vitro culture, amplification and application of.1995 Gage to establish a NPCs adherent culture method, which greatly promoted the related research on NPCs. However, the research on NPCs is still in the world at present. At the primary stage, the research data on the biological characteristics of NPCs obtained by two different culture methods are still very short. The research on NPCs in China started relatively late. At present, the main use of the method of suspension culture is carried out. In order to carry out the related research on NPCs, this subject has studied two kinds of methods on the basis of improving the method of NPCs wall cultivation. The biological characteristics of NPCs under the condition of culture provide experimental evidence for further research on basic research and clinical application of NPCs.
Fetal alcohol syndrome (FAS) is a mother's birth defect caused by excessive drinking during pregnancy. Its symptoms include a series of central nervous system dysplasia. In vivo, it is found that alcohol inhibits the proliferation of NPCs and causes partial NPCs death, which is considered to be one of the causes of the pathogenesis of FAS. The mechanism of alcohol damage to NPCs is still relatively small. It is reported that connexin 43 (connexin 43, Cx43) plays an important role in the process of NPCs proliferation. The inhibition of alcohol to NPCs proliferation is related to the expression of Cx43, and there is no related report yet.
The purpose of the study is:
1, compare the biological characteristics of NPCs obtained in vitro culture and suspension culture in vitro, and provide experimental basis for further research on basic and clinical application of NPCs.
2, the alcohol damage model of NPCs was established in vitro, and the effect of alcohol on the expression of Cx43 in NPCs in vitro was studied, and the mechanism of alcohol inhibition on the proliferation of NPCs was discussed.
Methods: the fetal rat end brain of pregnant 14d was used as the experimental material. The original NPCs, passages and cryopreservation and resuscitation were carried out by the method of adherent culture and suspension culture, and the following experiments were carried out.
1. Comparison of biological characteristics of NPCs obtained by two methods.
(1) under the inverted phase contrast microscope, the cultured cells were observed and photographed daily.
(2) the purity of NPCs in cultured cells was identified by immunofluorescence cytometry and flow cytometry.
(3) the BrdU incorporation method was used to analyze the proliferation ability of NPCs.
(4) the growth curve of NPCs obtained from two primary culture methods was analyzed by cell counting.
(5) NPCs differentiation was induced by the removal of growth factors, and all differentiated cells were identified by immunofluorescence and their proportional characteristics were analyzed.
2, the effect of alcohol on the expression of Cx43 in NPCs
(1) establishment of adherence culture NPCs alcohol injury experimental model, using inverted phase contrast microscope to observe cell morphological changes and take pictures.
(2) MTT method was used to detect the change of NPCs succinate dehydrogenase, and the effect of alcohol on NPCs activity was analyzed.
(3) immunofluorescence method was used to observe the expression of Cx43 in NPCs cultured in vitro.
(4) the expression of Cx43 in NPCs was detected by Western-blot, and the effect of alcohol on Cx43 expression in NPCs was analyzed.
(5) the diffusion distance between Lucifer Yellow (LY) and NPCs was observed through the mark / fluorescence diffusion test, and the effect of alcohol on the NPCs gap junction function was analyzed.
Experimental results:
1. Comparison of biological characteristics of NPCs obtained by two methods.
(1) the morphological observation and adherent culture of NPCs showed the morphological characteristics of the typical neuroepithelial cells. The cell population was colonially growing. With the prolongation of the culture time, the cells were radially extending outward, and the NPCs in suspension culture was globular clone growth. Most of the cells were round, the cells were connected tightly. The passages and the cultured NPCs after the cryopreservation were resuscitation. There was no obvious change in morphology.
(2) the percentage of nestin positive cells in primary cultured cells with NPCs purity was increased from 38.69% to 93.76% in ninth days, while the rate of nestin positive cells in the primary suspension culture cells was only 59.75% in ninth days. The nestin positive cell rate of the adherent culture cells was 98.36% and the suspension culture cells were 83.34% after the third days of passage. After fourth days of cultivation, the purity of NPCs obtained by two methods reached 99.9%.
(3) the proliferation index of NPCs's proliferation ability detected by primary adherent culture, the proliferation index of NPCs increased from 13.68% to the highest in third days to 38.7% in the highest seventh days; the proliferation index of NPCs in suspension culture for seventh days was also the highest, but only 33.47%; the adherent culture cells were second days after the passage and fourth days after the resuscitation, and the proliferation index could reach 39.08% and 39.2, respectively. 5%, the proliferation index of suspension culture NPCs under these two conditions is only 36.95% and 36.73%.
(4) the growth curve of the primary culture NPCs was analyzed by the two culture methods. The growth curves were all "S" curves, but there were significant differences in the number of cells. The number of cells was the most, 68 times the number of initial inoculation, and the number of cells continued to decrease at the twelfth day of adherent culture, and the number of cells was the most, but only at eleventh days in suspension culture, but only the number of cells was the most, but only the number of cells was the most. 8.4 times as much as the initial inoculation.
(5) the differentiation ability of NPCs was analyzed by the adherent culture of NPCs induced 7d, and the proportion of neurons, astrocytes and oligodendrocytes were 14.13%, 29.99% and 7.21%, respectively, while the proportion of the above three were 17.72%, 24.81% and 8.15%. respectively after the suspension culture was induced to differentiate 7d.
2, the effect of alcohol on the expression of Cx43 in NPCs
(1) morphological observation adhered to the wall culture of NPCs after different concentrations of alcohol (25,50 and 100mmol/L) damaged 24h, cells showed different degrees of cell body round and protuberance retraction, high concentration of alcohol (100mmol/L) can make obvious cell fragments in the culture.
(2) cell viability detected by MTT assay was 79.25%, 70.60% and 67.93%, respectively, under three ethanol concentrations.
(3) immunofluorescence assay was used to detect the expression of Cx43 in NPCs. Cx43 was widely distributed on the surface of NPCs cells, and the distribution of adjacent NPCs cells was more concentrated.
(4) Western-blot detected the change of Cx43 expression in NPCs. The Cx43/Actin values of three concentrations of alcohol after NPCs24h were 0.82,0.67,0.46 and 0.19 respectively.
(5) marks / fluorescence diffusion tests were used to detect the changes of NPCs gap junction function. The propagation distance of LY and the intensity of fluorescence signal were shortened or weakened with the increase of alcohol damage concentration.
Experimental conclusions:
1, NPCs obtained by adherent culture facilitates cell counting and morphological observation.
2, the percentage of nestin positive cells in primary and passage culture cells was higher than that in suspension culture cells.
3, NPCs obtained from primary and subculture adherent culture had strong self amplification ability, while the NPCs self amplification ability of suspension culture was relatively poor, and the two NPCs cryopreservation and resuscitation could maintain high self amplification ability.
4, the amplification ability of NPCs after 5 generations of continuous subculture decreased significantly, while the NPCs in suspension culture could still maintain the ability of amplification better than 10 times of continuous passage.
5, there was no significant difference in NPCs differentiation between adherent and suspension cultures.
6, adherent culture of NPCs can express Cx43, and alcohol can affect the gap junction function of NPCs by inhibiting the expression of Cx43 in NPCs.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.1

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3 李怡;极低频电磁场对中脑神经前体细胞分化的作用及其机制研究[D];第二军医大学;2003年

4 刘靖;Sertoli细胞促进大鼠胚胎中脑神经前体细胞分化的作用及其对Sertoli细胞DHH基因的相关研究[D];河北医科大学;2005年

5 张振兴;诱导成人成肌细胞转化为神经前体细胞及用于移植治疗局灶性脑缺血的实验研究[D];中国协和医科大学;2006年

6 林颖;体外共培养诱导嗅球神经前体细胞分化为功能性外周神经元[D];第四军医大学;2008年

7 吴旋;基因工程化胚胎干细胞用于AD治疗的基础实验研究[D];第三军医大学;2001年

8 杨彩侠;Desert Hedgehog诱导大鼠胚胎中脑神经前体细胞定向分化的研究[D];河北医科大学;2009年

9 李松;基质金属蛋白酶9在脑皮质发育障碍中的作用研究[D];第三军医大学;2012年

10 武明鑫;移植胚胎干细胞的衍生细胞治疗全横断脊髓损伤的实验研究[D];吉林大学;2006年

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1 孙涛;贴壁和悬浮培养的神经前体细胞生物学特性研究及酒精对神经前体细胞Cx43表达的影响[D];山东大学;2009年

2 吴秀中;低浓度毒死蜱对小鼠胚胎神经前体细胞周期性核运动及胚胎海马细胞的影响[D];浙江工业大学;2012年

3 谢珊珊;绞股蓝皂苷对酒精损伤神经前体细胞的保护作用及其对神经前体细胞增殖能力影响的研究[D];山东大学;2010年

4 王少帅;电针对高血压大鼠脑梗死后神经前体细胞移行的实验性研究[D];山西医科大学;2012年

5 刘宏志;大鼠骨髓间充质干细胞体外诱导分化为神经前体细胞的研究[D];中国医科大学;2005年

6 刘智全;不同接种密度培养胎鼠脊髓神经前体细胞的实验研究[D];大连医科大学;2008年

7 陈琰;大鼠中脑神经前体细胞的体外分离、培养和特性及Nurrl基因的克隆[D];中国医科大学;2002年

8 李志超;神经干/祖细胞移植治疗脊髓小脑共济失调大鼠的实验研究[D];第二军医大学;2005年

9 王辉;酒精对神经前体细胞损伤及其机制研究[D];山东大学;2007年

10 陶玉慧;神经前体细胞在成年小鼠脊髓的分布特征[D];南昌大学;2008年

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