【摘要】： 研究背景和目的: 神經(jīng)干細(xì)胞具有不斷增殖和自我更新能力,并可以分化為神經(jīng)元、星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞,能為中樞神經(jīng)系統(tǒng)功能重建和神經(jīng)再生提供新的供體源,而且能較好地解決神經(jīng)移植治療的倫理道德及供體不足等問題,為神經(jīng)系統(tǒng)的許多疾病如帕金森氏病的治療帶來了新希望。 室管膜前下區(qū)(Anterior subventriculor zone, SVZa)是公認(rèn)的神經(jīng)干細(xì)胞富集區(qū),該區(qū)的神經(jīng)干細(xì)胞在體內(nèi)背-腹信號控制下,不斷沿著一條局限化的遷移通道-吻側(cè)遷移流(Rostral migratory stream,RMS)向嗅球(Olfactory bulb,OB)遷移,最后在嗅球分化為具有OB表型特征的中間神經(jīng)元,包括:多巴胺能神經(jīng)元和γ-氨基丁酸能神經(jīng)元。并且SVZa神經(jīng)干細(xì)胞除具有一般干細(xì)胞的自我更新能力和多分化潛能外,還具有以下特點:(1)自產(chǎn)生起即具備發(fā)育為神經(jīng)元的潛能;(2)能長距離定向遷移;(3)在遷移過程中始終維持增殖狀態(tài)和神經(jīng)元分化潛能而不進(jìn)一步分化。故SVZa神經(jīng)干細(xì)胞成為研究神經(jīng)干細(xì)胞增殖、遷移和分化的最佳模型。 神經(jīng)干細(xì)胞的分化主要取決于兩個方面:一是干細(xì)胞的內(nèi)在遺傳基因,二是干細(xì)胞所處的外部微環(huán)境。相同來源的干細(xì)胞處于不同的微環(huán)境中可以分化為不同類型不同譜系的細(xì)胞,因此目前對于干細(xì)胞分化的研究除了其自身基因的調(diào)控外,多側(cè)重于外來信號的調(diào)控。BMP2作為轉(zhuǎn)化生長因子-β(TGF-β)超家族成員,是干細(xì)胞所處局部微環(huán)境中非常重要的一種細(xì)胞因子。其在體外條件下對室管膜前下區(qū)神經(jīng)干細(xì)胞分化為多巴胺能神經(jīng)元作用如何還需要進(jìn)一步的實驗研究。 另外,既往研究表明,單一因子誘導(dǎo)神經(jīng)干細(xì)胞分化為多巴胺能神經(jīng)元作用非常有限,這就要求探求多種因子聯(lián)合作用從而使分化為多巴胺能神經(jīng)元比例增加。ACM作為星形膠質(zhì)細(xì)胞體外培養(yǎng)時的細(xì)胞外液,其內(nèi)含有膠質(zhì)細(xì)胞分泌的豐富的可溶性生物活性物質(zhì),如:神經(jīng)生長因子(NGF)、膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子(GDNF)、腦源性神經(jīng)營養(yǎng)因子(BDNF)等,且與神經(jīng)干細(xì)胞所處的體內(nèi)環(huán)境較為接近,其促分化作用是單一神經(jīng)營養(yǎng)因子所無法比擬的。因此,本研究首先分離培養(yǎng)出SVZa神經(jīng)干細(xì)胞作為研究對象,然后再以不同濃度的BMP2誘導(dǎo)其分化,并分別檢測其分化為多巴胺能神經(jīng)元的比例。最后再以BMP2和ACM誘導(dǎo)SVZa神經(jīng)干細(xì)胞分化,研究它們在SVZa神經(jīng)干細(xì)胞分化為多巴胺能神經(jīng)元中的作用,希望為SVZa神經(jīng)干細(xì)胞的定向分化研究提供一些實驗資料。 方法: 1、運(yùn)用細(xì)胞培養(yǎng)技術(shù)對SVZa神經(jīng)干細(xì)胞進(jìn)行分離培養(yǎng),并將收獲的細(xì)胞克隆球行免疫細(xì)胞學(xué)的Nestin鑒定,然后將細(xì)胞克隆球分化后行NF、GFAP、CNP的免疫細(xì)胞學(xué)鑒定,以明確其具有干細(xì)胞特性和多分化潛能。 2、將體外培養(yǎng)所得的SVZa神經(jīng)干細(xì)胞,分別以0、0.1、1、10、20、100(ng/ml)濃度的BMP2誘導(dǎo)其分化,通過免疫組化和流式細(xì)胞儀技術(shù)檢測不同濃度情況下SVZa神經(jīng)干細(xì)胞分化為TH+(酪氨酸羥化酶)神經(jīng)元的比例。 3、將體外培養(yǎng)所得的SVZa神經(jīng)干細(xì)胞,分為對照組、BMP2組、ACM組、BMP2+ACM組分別予以誘導(dǎo)分化,通過免疫組化和流式細(xì)胞儀技術(shù)檢測各組中SVZa神經(jīng)干細(xì)胞分化為TH+細(xì)胞的比例。 結(jié)果: 1、在體外無血清培養(yǎng)條件下,可以收獲大量懸浮生長的細(xì)胞克隆球,Nestin免疫細(xì)胞學(xué)反應(yīng)陽性,分化后的NF、GFAP、CNP免疫細(xì)胞學(xué)反應(yīng)也呈陽性反應(yīng)。 2、SVZa神經(jīng)干細(xì)胞分別以0、0.1、1、10、20、100(ng/ml)濃度的BMP2誘導(dǎo)分化后,經(jīng)檢測0.1～100(ng/ml)組的TH+細(xì)胞比例均明顯高于0ng/ml組,10ng/ml組達(dá)最高峰,各組間差異均有統(tǒng)計學(xué)意義(P㩳0.05)。 3、SVZa神經(jīng)干細(xì)胞分為對照組、BMP2組、ACM組、BMP2+ACM組誘導(dǎo)分化后,免疫組化和流式細(xì)胞儀檢測結(jié)果提示:各實驗組的TH+細(xì)胞比例均明顯高于對照組,其中BMP2+ACM組分化比例最高,差異有統(tǒng)計學(xué)意義(P㩳0.05) 結(jié)論: 1、從孕16天Wistar大鼠SVZa腦組織中成功分離得到具有不斷增殖和自我更新能力,并可以分化為神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞的神經(jīng)干細(xì)胞。 2、BMP2可以促進(jìn)SVZa神經(jīng)干細(xì)胞分化為多巴胺能神經(jīng)元,但不完全是劑量依賴性的,而是有一個最佳濃度,即10ng/ml的BMP2作用最明顯。 3、ACM可以促進(jìn)SVZa神經(jīng)干細(xì)胞向多巴胺能神經(jīng)元分化,而且BMP2與ACM具有正協(xié)同作用。
[Abstract]:Background and purpose of the study: Neural stem cells have the ability to proliferate and self-update and can be differentiated into neurons, astrocytes, oligodendrocytes, which can provide a new way for the functional reconstruction and nerve regeneration of the central nervous system The donor source can better solve the problems of the ethics and the donor deficiency of the nerve graft treatment, and brings the treatment of a plurality of diseases of the nervous system such as Parkinson's disease The new hope is that the anterior inferior zone (SVZa) of the ependymal membrane is a well-established neural stem cell-rich region, and the neural stem cells in the region are under the control of the in vivo back-and-abdominal signals, and the olfactory bulb (Olfactory bul) is continuously transported along a localized migration channel-side migration stream (RMS) under the control of the in vivo back-abdominal signal. b, OB) migration, and finally, differentiation of the olfactory bulb into an intermediate neuron having an OB phenotypic characteristic, including: a dopaminergic neuron and a HCO3-ammonia; The SVZa neural stem cell has the following characteristics in addition to the self-renewal capacity and the multi-differentiation potential of the general stem cells: (1) the self-generation of the neural stem cells has the following characteristics: (1) the self-generation of the neural stem cells has the potential to develop into the neuron; (2) the SVZa neural stem cell can Long-distance directional migration; (3) always maintain the proliferative state and the potential of neuronal differentiation during the migration Therefore, the SVZa neural stem cells become the study of the proliferation and migration of neural stem cells. The differentiation is the best model. The differentiation of neural stem cells depends on two aspects: one is the inner genetic gene of the stem cells, and the other is the second. The stem cells of the same origin are in different microenvironment and can be differentiated into different types of cells, so the present study on stem cell differentiation, in addition to its own gene regulation, Multi-focus on the regulation of external signals. BMP2 is a member of the superfamily of the transformation growth factor-1 (TGF-1), which is the local microenvironment of the stem cells. A cytokine that is very important. How can the differentiation of neural stem cells in the anterior inferior region of the ependytomembrane into dopaminergic neurons under in vitro conditions Further experimental studies are also required. In addition, previous studies have shown that the differentiation of a single factor-induced neural stem cell into dopaminergic neurons is very limited, which requires a combination of multiple factors. AACM, as the extracellular fluid in the in vitro culture of astrocytes, contains abundant soluble bioactive substances, such as nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), brain, The derived neurotrophic factor (BDNF) and the like are close to the in-vivo environment in which the neural stem cells are located, and the differentiation effect In this study, the SVZa neural stem cells were isolated and cultured as the research object, then the differentiation was induced by BMP2 at different concentrations. In the end, the differentiation of SVZa neural stem cells was induced by BMP2 and ACM, and the role of them in the differentiation of the SVZa neural stem cells into dopaminergic neurons was studied, and it was desired to be SVZa neural stem cells orientation Methods:1. The SVZa neural stem cells were isolated and cultured by a cell culture technique, and the harvested cells were identified by the Nestin identification of the immunocytological cell line, and then the cells were cloned into the cells. The immunity of NF, GFAP and CNP after differentiation The SVZa neural stem cells obtained from in-vitro culture were induced by BMP2 at 0, 0.1,1,10,20,100 (ng/ ml), respectively, and SV was detected by immunohistochemistry and flow cytometry. Za neural stem cells were differentiated into TH + (tyrosine hydroxylase) neurons.3. The SVZa neural stem cells obtained in vitro were divided into control group, BMP2 group, ACM group and BMP2 + ACM group, respectively. cell The ratio of SVZa neural stem cells to TH + cells in each group was detected by the instrument technique. Results:1. In the absence of serum culture in vitro, a large number of cell clone balls of suspension growth could be harvested, Nesti The positive and differentiated NF, GFAP and CNP were positive. The ratio of TH + cells in 0.1 to 100 (ng/ ml) group was significantly higher after the differentiation of the SVZa neural stem cells at 0, 0.1,1,10,20,100 (ng/ ml). Compared with the control group, the control group, the BMP2 group, the ACM group and the BMP2 + ACM group were divided into the control group, the BMP2 group, the ACM group and the BMP2 + ACM group to induce the differentiation. Tip of the cytometric test: TH of each experimental group + cells The ratio of BMP2 + ACM group was higher than that of control group, and the difference was significant (P? 0.05). the successful isolation of Za brain tissue has the ability to continuously proliferate and self-update, and can be differentiated into neural stem cells of neurons, astrocytes and oligodendrocytes. BMP2 can promote S VZa neural stem cells are differentiated into dopaminergic neurons, but are not completely dose-dependent, but have an optimal concentration, i.e.,10 ng
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 高國一,盧亦成,江基堯,李莉,張玲珍,朱誠;免疫磁珠法分選胚胎大鼠腦神經(jīng)干細(xì)胞的初步研究[J];第二軍醫(yī)大學(xué)學(xué)報;2000年11期

2 嚴(yán)稽文;黃其林;;星形膠質(zhì)細(xì)胞條件培養(yǎng)液對缺氧損傷神經(jīng)元的保護(hù)作用[J];神經(jīng)解剖學(xué)雜志;2007年03期

3 高方友;楊輝;張治元;何家全;劉仕勇;邱克軍;;Dlx5 mRNA在室管膜前下區(qū)神經(jīng)干細(xì)胞中的表達(dá)規(guī)律及其對神經(jīng)元分化的影響[J];中華創(chuàng)傷雜志;2006年05期

，

本文編號：2444380