本文選題：神經(jīng)前體細(xì)胞 + 懸浮培養(yǎng)法��； 參考：《山東大學(xué)》2009年碩士論文

【摘要】： 研究背景: 神經(jīng)前體細(xì)胞(neural precursor cells,NPCs)是中樞神經(jīng)系統(tǒng)中處于不同發(fā)育階段的不成熟神經(jīng)細(xì)胞的總稱,具有自我更新、增殖和分化為各類神經(jīng)細(xì)胞的潛能,是公認(rèn)的神經(jīng)損傷修復(fù)的種子細(xì)胞。應(yīng)用體外擴(kuò)增的NPCs對神經(jīng)系統(tǒng)創(chuàng)傷、變性及退行性疾病的治療具有廣闊的應(yīng)用前景。1992年,Reynolds首先建立了NPCs的體外懸浮培養(yǎng)方法,開創(chuàng)了NPCs體外培養(yǎng)、擴(kuò)增及其應(yīng)用的新紀(jì)元。1995年Gage建立了NPCs的貼壁培養(yǎng)方法,大大推動了對NPCs的相關(guān)研究。但目前國際上對NPCs的研究仍然處于初級階段,對兩種不同培養(yǎng)方法獲得的NPCs生物學(xué)特性研究資料還非常缺乏。我國開展NPCs的研究起步較晚,目前主要使用懸浮培養(yǎng)的方法開展工作。為了深入開展對NPCs相關(guān)研究,本課題在完善NPCs貼壁培養(yǎng)方法的基礎(chǔ)上,對比研究了兩種培養(yǎng)條件下的NPCs的生物學(xué)特性,為深入開展NPCs的基礎(chǔ)研究和臨床應(yīng)用研究提供實(shí)驗(yàn)依據(jù)。 胎兒酒精綜合征(fetal alcohol syndrome,FAS)是母親在妊娠期間過量飲酒造成的子代出生缺陷,其癥狀包括一系列的中樞神經(jīng)系統(tǒng)發(fā)育異常。體內(nèi)、外實(shí)驗(yàn)發(fā)現(xiàn),酒精可抑制NPCs的增殖,并導(dǎo)致部分NPCs死亡,這被認(rèn)為是FAS發(fā)病的原因之一。但對于酒精損傷NPCs的機(jī)理,目前了解還比較少。文獻(xiàn)報(bào)道,連接蛋白43(connexin 43,Cx43)在NPCs增殖過程中具有重要的作用。酒精對NPCs增殖的抑制作用是否與Cx43的表達(dá)有關(guān),目前尚未見相關(guān)報(bào)道。 研究目的: 1、對比研究貼壁培養(yǎng)法和懸浮培養(yǎng)法在體外培養(yǎng)獲得的NPCs的生物學(xué)特性,為深入開展NPCs的基礎(chǔ)研究和臨床應(yīng)用研究提供實(shí)驗(yàn)依據(jù)。 2、建立體外貼壁培養(yǎng)NPCs的酒精損傷模型,研究酒精對體外培養(yǎng)的NPCs中Cx43表達(dá)的影響,探討酒精抑制NPCs增殖的作用機(jī)理。 研究方法:以孕14d的胎鼠端腦為實(shí)驗(yàn)材料,分別采用貼壁培養(yǎng)和懸浮培養(yǎng)方法進(jìn)行NPCs的原代、傳代及凍存復(fù)蘇后培養(yǎng),進(jìn)行下列實(shí)驗(yàn): 1、兩種培養(yǎng)方法獲得的NPCs的生物學(xué)特性對比實(shí)驗(yàn) (1)倒置相差顯微鏡下,每天對培養(yǎng)細(xì)胞進(jìn)行形態(tài)學(xué)觀察,并拍照; (2)用免疫熒光細(xì)胞化學(xué)方法及流式細(xì)胞術(shù)鑒定培養(yǎng)細(xì)胞中NPCs的純度; (3)用BrdU摻入方法分析NPCs的增殖能力; (4)用細(xì)胞計(jì)數(shù)方法分析兩種原代培養(yǎng)方法獲得的NPCs的生長曲線; (5)采用去除生長因子的方法誘導(dǎo)NPCs分化,用免疫熒光方法鑒定各類分化細(xì)胞并分析其比例特征。 2、酒精對NPCs的Cx43的表達(dá)的影響 (1)建立貼壁培養(yǎng)NPCs酒精損傷實(shí)驗(yàn)?zāi)Ｐ?采用倒置相差顯微鏡觀察細(xì)胞形態(tài)變化,并拍照; (2)采用MTT法檢測NPCs琥珀酸脫氫酶的變化,分析酒精對NPCs活性的影響; (3)采用免疫熒光方法觀察體外貼壁培養(yǎng)的NPCs中Cx43的表達(dá)情況; (4)采用Western-blot法檢測NPCs中Cx43的表達(dá)量,分析酒精對NPCs中Cx43表達(dá)的影響; (5)通過刻痕標(biāo)記/熒光擴(kuò)散實(shí)驗(yàn),觀察熒光黃(Lucifer Yellow,LY)在NPCs之間的擴(kuò)散距離,分析酒精對NPCs間隙連接功能的影響。 實(shí)驗(yàn)結(jié)果: 1、兩種培養(yǎng)方法獲得的NPCs的生物學(xué)特性對比實(shí)驗(yàn) (1)形態(tài)學(xué)觀察貼壁培養(yǎng)的NPCs表現(xiàn)出典型的神經(jīng)上皮細(xì)胞的形態(tài)特征,細(xì)胞群體呈集落狀生長,隨著培養(yǎng)時(shí)間延長,細(xì)胞呈放射狀向外延伸;懸浮培養(yǎng)的NPCs呈球狀克隆生長,細(xì)胞多數(shù)呈圓形,細(xì)胞連接緊密。傳代以及凍存復(fù)蘇后培養(yǎng)的NPCs形態(tài)均無明顯變化; (2) NPCs純度鑒定原代貼壁培養(yǎng)細(xì)胞中nestin陽性細(xì)胞率由第3天的38.69%增加至第9天的93.76%,而原代懸浮培養(yǎng)細(xì)胞第9天的nestin陽性細(xì)胞率僅為59.75%;兩者傳代后第2天,貼壁培養(yǎng)細(xì)胞的nestin陽性細(xì)胞率可達(dá)98.36%,懸浮培養(yǎng)細(xì)胞為83.34%;凍存復(fù)蘇后培養(yǎng)第4天,兩種方法獲得的NPCs純度達(dá)均可達(dá)到99.9%; (3) NPCs的增殖能力檢測原代貼壁培養(yǎng)的NPCs的增殖指數(shù),由培養(yǎng)第3天的13.68%增加至最高的第7天的38.7%;懸浮培養(yǎng)第7天的NPCs的增殖指數(shù)亦達(dá)最高,但僅為33.47%;貼壁培養(yǎng)細(xì)胞在傳代后第2天和復(fù)蘇后培養(yǎng)第4天,增殖指數(shù)分別可達(dá)39.08%和39.25%,而懸浮培養(yǎng)的NPCs這兩種條件下的增殖指數(shù)僅為36.95%和36.73%; (4)原代培養(yǎng)NPCs生長曲線分析兩種培養(yǎng)方法獲得的生長曲線均為“S”形曲線,但在細(xì)胞數(shù)量上有明顯差異。貼壁培養(yǎng)第12天時(shí),細(xì)胞數(shù)量最多,為初始接種數(shù)的68倍,繼續(xù)培養(yǎng)細(xì)胞數(shù)量開始降低;懸浮培養(yǎng)第11天時(shí),細(xì)胞數(shù)量即達(dá)最多,但僅為初始接種量的8.4倍; (5) NPCs的分化能力分析貼壁培養(yǎng)的NPCs誘導(dǎo)分化后培養(yǎng)7d,神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞的比例分別為14.13%,29.99%和7.21%;而懸浮培養(yǎng)的NPCs誘導(dǎo)分化7d后,上述三者的比例分別為17.72%,24.81%和8.15%。 2、酒精對NPCs的Cx43的表達(dá)的影響 (1)形態(tài)學(xué)觀察貼壁培養(yǎng)的NPCs經(jīng)不同濃度酒精(25、50和100mmol/L)損傷24h后,細(xì)胞會表現(xiàn)為不同程度的胞體變圓和突起回縮,高濃度酒精(100mmol/L)可使培養(yǎng)物中出現(xiàn)明顯的細(xì)胞碎片; (2) MTT法檢測細(xì)胞活性三種酒精濃度下的細(xì)胞存活率分別為79.25%、70.60%和67.93%; (3)免疫熒光化學(xué)法檢測NPCs中Cx43表達(dá)特點(diǎn)Cx43廣泛分布于NPCs的細(xì)胞表面,并且在相鄰NPCs胞體接觸位置分布較為集中; (4) Western-blot檢測NPCs中Cx43表達(dá)量的變化三種濃度的酒精作用NPCs24h后,Cx43/Actin值分別為:0.82、0.67、0.46和0.19; (5)刻痕標(biāo)記/熒光擴(kuò)散實(shí)驗(yàn)檢測NPCs間隙連接功能的變化LY的傳播距離及熒光信號強(qiáng)度隨酒精損傷濃度的增加而變短或減弱。 實(shí)驗(yàn)結(jié)論: 1、貼壁培養(yǎng)法獲得的NPCs便于進(jìn)行細(xì)胞計(jì)數(shù)和形態(tài)學(xué)觀察。 2、原代和傳代貼壁培養(yǎng)的細(xì)胞nestin陽性細(xì)胞率均高于懸浮培養(yǎng)細(xì)胞。 3、原代和傳代貼壁培養(yǎng)獲得的NPCs均具有較強(qiáng)的自我擴(kuò)增能力,而懸浮培養(yǎng)獲得的NPCs自我擴(kuò)增能力相對較差;兩種NPCs凍存復(fù)蘇后均能保持較高的自我擴(kuò)增能力。 4、貼壁培養(yǎng)獲得的NPCs連續(xù)傳代5代后擴(kuò)增能力顯著下降,而懸浮培養(yǎng)獲得的NPCs在連續(xù)傳代10次以上仍能較好地保持?jǐn)U增能力。 5、貼壁和懸浮培養(yǎng)獲得的NPCs,分化能力無顯著差別。 6、貼壁培養(yǎng)的NPCs可以表達(dá)Cx43,酒精可以通過抑制NPCs中Cx43的表達(dá)來影響NPCs的間隙連接功能。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329.1

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10 笱玉蘭;朱遂強(qiáng);陳國華;張繼龍;羅利俊;阮旭中;;能合成和分泌GABA的永生化神經(jīng)前體細(xì)胞的構(gòu)建[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2007年06期

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