【摘要】： 中樞神經(jīng)系統(tǒng)(central nervous system,CNS)損傷后神經(jīng)功能的恢復(fù)非常困難,主要原因是因?yàn)槟X內(nèi)神經(jīng)元的再生能力極差。骨髓間充質(zhì)干細(xì)胞(bone marrowmesenchymal stem cells,BMSCs)可在自體獲得并能在體外穩(wěn)定擴(kuò)增,并可誘導(dǎo)分化成神經(jīng)細(xì)胞,有望用于神經(jīng)系統(tǒng)疾病的治療,故骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化相關(guān)的基礎(chǔ)醫(yī)學(xué)研究是十分重要的。 大量體外和體內(nèi)實(shí)驗(yàn)研究證明BMSCs可以分化為神經(jīng)細(xì)胞,對(duì)于神經(jīng)系統(tǒng)的損傷具有一定的修復(fù)作用。骨髓間充質(zhì)干細(xì)胞分化而來的神經(jīng)樣細(xì)胞,雖然表達(dá)了神經(jīng)元及其前體細(xì)胞的標(biāo)記物,但它能否發(fā)育為成熟的神經(jīng)元并表現(xiàn)其功能,仍然存在著很大的分歧,而且BMSCs向神經(jīng)樣細(xì)胞分化的分子機(jī)理不明,本課題研究將根據(jù)這些問題展開工作。 本工作先建立新生大鼠大腦皮層細(xì)胞原代培養(yǎng)體系,從該體系中獲得培養(yǎng)2 d后的無血清培養(yǎng)基作為條件培養(yǎng)基誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向神經(jīng)樣細(xì)胞分化。誘導(dǎo)培養(yǎng)24 h后,由BMSCs分化而來的神經(jīng)樣細(xì)胞具有典型的神經(jīng)元形態(tài)特征,用神經(jīng)細(xì)胞和BMSCs的標(biāo)記物鑒定此神經(jīng)樣細(xì)胞的表型,鑒定的結(jié)果是:Nestin(15.07%±1.86%)、β-Ⅲ-Tubulin(46.29%±2.58%)、GFAP(9.54%±1.42%)、NCAM(49.82%±3.46%)、CD106(96.86%±1.82%)、CD90(33.62%±2.48%)、CD44(32.44%±2.45%)、CD29(14.24%±1.84%);不表達(dá)NSE、CD45、CD71、CD34。Ⅲ-型β微管蛋白(β-Ⅲ-Tubulin)被認(rèn)為是早期神經(jīng)元標(biāo)記物,而神經(jīng)元特異性烯醇化酶(NSE)被廣泛認(rèn)為是成熟神經(jīng)元標(biāo)記物之一,結(jié)果顯示神經(jīng)樣細(xì)胞高比例表達(dá)β-Ⅲ-Tubulin,不表達(dá)NSE,提示由BMSCs分化而來的神經(jīng)樣細(xì)胞正處于BMSCs向成熟神經(jīng)元分化過程的過渡狀態(tài)。結(jié)果還顯示神經(jīng)樣細(xì)胞還陽(yáng)性表達(dá)CD106、CD90、CD44、CD29,不表達(dá)CD45、CD71、CD34,說明由BMSCs分化而來的神經(jīng)樣細(xì)胞仍具有BMSCs的表型特征,也提示其處于神經(jīng)分化的過渡狀態(tài)。 本工作還使用成纖維生長(zhǎng)因子(basic fibroblast growth factor,bFGF)、二甲基亞砜(dimethyl sulphoxide,DMSO)、丁羥基茴香醚(butyl hydroxy anisol,BHA)聯(lián)用的傳統(tǒng)方法誘導(dǎo)BMSCs向神經(jīng)樣細(xì)胞分化。BMSCs先經(jīng)預(yù)誘導(dǎo)液預(yù)誘導(dǎo)24 h,再經(jīng)化學(xué)誘導(dǎo)液誘導(dǎo)5h后,呈現(xiàn)出典型的神經(jīng)元形態(tài)特征。用神經(jīng)細(xì)胞和BMSCs的標(biāo)記物鑒定神經(jīng)樣細(xì)胞,結(jié)果是:Nestin(21.12%±2.28%)、β-Ⅲ-Tubulin(78.26%±3.52%)、NCAM(79.86%±2.88%)、NSE(70.82%±2.46%)、CD106(96.88%±2.75%)、CD29(64.94%±2.16%);不表達(dá)GFAP、CD90、CD44、CD45、CD71、CD34。結(jié)果說明由BMSCs分化而來的神經(jīng)樣細(xì)胞既具有神經(jīng)細(xì)胞的表型特征也具有BMSCs的部分表型特征,這些結(jié)果也提示傳統(tǒng)化學(xué)誘導(dǎo)方法誘導(dǎo)BMSCs分化而來的神經(jīng)樣細(xì)胞正處于BMSCs向成熟神經(jīng)元分化的過渡狀態(tài)。 本工作采用以上兩種方法誘導(dǎo)BMSCs向神經(jīng)樣細(xì)胞分化,再分別用含10%血清的正常培養(yǎng)基繼續(xù)培養(yǎng)神經(jīng)樣細(xì)胞24 h后,發(fā)現(xiàn)一個(gè)共同的現(xiàn)象:神經(jīng)樣細(xì)胞又返回BMSCs的形態(tài)。用神經(jīng)細(xì)胞和BMSCs的標(biāo)記物做免疫熒光鑒定,表型鑒定結(jié)果和BMSCs的表型特征類似。接著將鑒定由BMSCs分化而來的神經(jīng)樣細(xì)胞返回到具有BMSCs特征的細(xì)胞是否具有多向分化潛能,結(jié)果顯示其仍然具有多向分化潛能,表現(xiàn)為具有成神經(jīng)、成骨和成脂肪等分化能力。 真正的成熟神經(jīng)元是神經(jīng)終端分化細(xì)胞,很難逆轉(zhuǎn)為干細(xì)胞,說明本研究工作中所得到的神經(jīng)樣細(xì)胞有別于真正的神經(jīng)元,它們可能正處于BMSCs向成熟神經(jīng)元分化的過渡狀態(tài),仍然可以逆轉(zhuǎn)為BMSCs。本工作的結(jié)果證實(shí)了骨髓間充質(zhì)干細(xì)胞向神經(jīng)樣細(xì)胞分化的過程具有可塑性。本研究工作所得到的結(jié)果為日后利用骨髓間充質(zhì)干細(xì)胞治療神經(jīng)系統(tǒng)疾病提供一定的參考資料,為骨髓間充質(zhì)干細(xì)胞向神經(jīng)樣細(xì)胞分化的相關(guān)基礎(chǔ)研究提供了一定的實(shí)驗(yàn)依據(jù)。
[Abstract]:The recovery of nerve function after the central nervous system (CNS) injury is very difficult. The main reason is that the regeneration ability of neurons in the brain is very poor. The bone marrow mesenchymal stem cells (bone marrowmesenchymal stem cells, BMSCs) can be obtained in autologous and can be stabilized in vitro, and can be induced to differentiate into nerve cells. For the treatment of nervous system diseases, the basic medical research related to the differentiation of bone marrow mesenchymal stem cells into nerve cells is very important.
A large number of in vitro and in vivo experimental studies have shown that BMSCs can be differentiated into neural cells, which have a certain repair effect on the damage of the nervous system. Neural like cells differentiated from bone marrow mesenchymal stem cells express the markers of neurons and their precursor cells, but can they develop into mature neurons and demonstrate their function. There are great differences, and the molecular mechanism of BMSCs differentiating into neuron like cells is unknown.
In this work, the primary culture system of neonatal rat cerebral cortex cells was established. The serum-free medium after culture of 2 D was obtained as the conditioned medium to induce bone marrow mesenchymal stem cells to differentiate into neurolike cells. After induction and culture of 24 h, the neuron like cells differentiated from BMSCs had typical morphological characteristics of neurons, with the use of God. The phenotype of the nerve like cells was identified by cell and BMSCs markers, and the results were as follows: Nestin (15.07% + 1.86%), beta - -Tubulin (46.29% + 2.58%), GFAP (9.54% + 1.42%), NCAM (49.82% + 3.46%), CD106 (96.86% + 1.82%), CD90 (33.62% + 2.48%), CD44 (32.44% + 46.29%), CD29 (1.42%), and CD71, CD71, type III beta microtubule Protein (beta - III -Tubulin) is considered as an early marker of neuron, and neuron specific enolase (NSE) is widely regarded as one of the markers of mature neurons. The results show that the high proportion of neurolike cells express beta - III -Tubulin and does not express NSE. It suggests that neurolike cells derived from BMSCs are being differentiated from BMSCs into mature neurons. The results also showed that neurolike cells also expressed positive expression of CD106, CD90, CD44, CD29, and did not express CD45, CD71, CD34, indicating that the neuron like cells differentiated from BMSCs still had the phenotypic characteristics of BMSCs, and suggested that they were in the transition state of nerve differentiation.
This work also uses the basic fibroblast growth factor (bFGF), two methyl sulfoxide (dimethyl sulphoxide, DMSO), the traditional method of Ding Qiangji anisole (butyl hydroxy ANISOL, BHA) to induce the differentiation of neural like cells to the nerve like cells. Characteristics of typical neuronal morphology. Neural cells were identified by nerve cells and BMSCs markers. The results were: Nestin (21.12% + 2.28%), beta - -Tubulin (78.26% + 3.52%), NCAM (79.86% + 2.88%), NSE (70.82% + 2.46%), CD106 (96.88% + 2.75%), CD29 (64.94% + 2.16%), and CD90, CD44, CD45, CD71. The neurolike cells have both phenotypic characteristics of the nerve cells and some of the phenotypic characteristics of BMSCs. These results also suggest that the neurolike cells derived from the traditional chemical induction method are in the transition state of the differentiation of the mature neurons from the BMSCs to the mature neurons of the BMSCs.
This work uses these two methods to induce BMSCs to differentiate into neuron like cells, and then continue to cultivate nerve like cells with 24 h with the normal medium containing 10% serum, and find a common phenomenon: nerve like cells return to the morphology of BMSCs. Immunofluorescence identification with nerve cells and BMSCs markers, phenotypic identification results and BMSCs The phenotypic characteristics are similar. Then the identification of neuron like cells differentiated from BMSCs to whether cells with BMSCs characteristics have multiple differentiation potential, the results show that the cells still have multiple differentiation potential, showing the ability to differentiate into neurogenic, osteogenic and adipose tissue.
The real mature neurons are neural terminal differentiated cells, which are difficult to reverse to stem cells. It shows that the neuron like cells in this study are different from the real neurons. They may be in the transition state of BMSCs to mature neurons, and can still be reversed to BMSCs.. The process of cell differentiation to neurolike cells is plastic. The results obtained in this study provide some reference for the future use of bone marrow mesenchymal stem cells in the treatment of nervous system diseases, and provide some experimental basis for the basic research on differentiation of bone marrow mesenchymal stem cells to neurolike cells.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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