本文選題：軀干神經(jīng)嵴干細(xì)胞　切入點：背根神經(jīng)節(jié)　出處：《蘇州大學(xué)》2011年博士論文

【摘要】：組織工程技術(shù)為周圍神經(jīng)損傷的治療帶來了新的希望,種子細(xì)胞的優(yōu)化精選是組織工程技術(shù)的關(guān)鍵。施旺氏細(xì)胞是周圍神經(jīng)系統(tǒng)的主要結(jié)構(gòu)和功能細(xì)胞,是公認(rèn)的種子細(xì)胞,然而其來源有限。軀干神經(jīng)嵴干細(xì)胞,可分化為組成周圍神經(jīng)系統(tǒng)的絕大部分神經(jīng)元和膠質(zhì)細(xì)胞,是周圍神經(jīng)系統(tǒng)的原基,因此將軀干神經(jīng)嵴干細(xì)胞應(yīng)用于神經(jīng)組織工程有著良好的應(yīng)用前景。 干細(xì)胞的分化受到體內(nèi)外多種因素的調(diào)節(jié),具有多向性和不確定性,應(yīng)用神經(jīng)干細(xì)胞移植來治療神經(jīng)系統(tǒng)損傷或疾病需要有目的地引導(dǎo)神經(jīng)干細(xì)胞向我們所需要的特定細(xì)胞類型分化。因此,誘導(dǎo)神經(jīng)干細(xì)胞的定向分化是一項具有重要意義的研究。bFGF具有多功能的特性,即它既能促進(jìn)神經(jīng)元的分化,也能促進(jìn)神經(jīng)膠質(zhì)細(xì)胞的分化并且它的這種功能變化是和體內(nèi)發(fā)育時相的變化一致的。即在神經(jīng)發(fā)育的早期促進(jìn)神經(jīng)元分化,而在發(fā)育晚期則促進(jìn)神經(jīng)膠質(zhì)細(xì)胞分化。周圍神經(jīng)系統(tǒng)中,bFGF是周圍神經(jīng)系統(tǒng)干細(xì)胞分化為施旺氏細(xì)胞祖細(xì)胞的正向決定因素,因此利用bFGF定向誘導(dǎo)背根節(jié)神經(jīng)干細(xì)胞沿施旺氏細(xì)胞方向分化將是一個很好的嘗試。本文首先體外分離培養(yǎng)背根節(jié)神經(jīng)干細(xì)胞,然后體外誘導(dǎo)其沿施旺氏細(xì)胞方向分化及對其功能鑒定;在此基礎(chǔ)上對bFGF體外誘導(dǎo)神經(jīng)干細(xì)胞沿施旺氏細(xì)胞分化的機(jī)制進(jìn)行研究。 第一部分背根節(jié)神經(jīng)干細(xì)胞的培養(yǎng),鑒定及定向誘導(dǎo)其沿施旺氏細(xì)胞方向分化 目的: 建立成體大鼠背根節(jié)神經(jīng)干細(xì)胞的培養(yǎng)方法及定向誘導(dǎo)其沿施旺氏細(xì)胞方向分化的方法,為周圍神經(jīng)系統(tǒng)再生提供真正的周圍神經(jīng)系統(tǒng)來源的種子細(xì)胞。 方法: 1.取成年SD大鼠的背根神經(jīng)節(jié),體外懸浮培養(yǎng)獲得原代神經(jīng)干細(xì)胞球后,利用無限稀釋法獲得單細(xì)胞克隆,傳代2次后對其干細(xì)胞特性進(jìn)行鑒定,主要包括特異性標(biāo)志物,增殖率及分化潛能; 2.將克隆化的神經(jīng)干細(xì)胞球分5組誘導(dǎo)分化,即NGF組;bFGF組;NRG組;FBS組;及對照組。誘導(dǎo)7 d后分別行S100免疫細(xì)胞化學(xué)和Westernblot。免疫細(xì)胞化學(xué)鑒定時隨機(jī)選取10個視野統(tǒng)計各組的S100陽性細(xì)胞比例;同時提取蛋白通過Western blot檢測各組S100的表達(dá)水平。 3. bFGF誘導(dǎo)背根神經(jīng)節(jié)干細(xì)胞分化的短時程和長時程效應(yīng)檢測,即一組加入分化培養(yǎng)液24 h后洗去bFGF繼續(xù)培養(yǎng);另一組則始終用含有bFGF的分化培養(yǎng)液。7 d后行S100免疫細(xì)胞化學(xué)和Westernblot。 4. BrdU標(biāo)記檢測bFGF作用24小時內(nèi)細(xì)胞的增殖:在加入bFGF誘導(dǎo)細(xì)胞分化的同時加入BrdU標(biāo)記液,24 h后免疫細(xì)胞化學(xué)檢測。 5.利用免疫細(xì)胞化學(xué),傳代培養(yǎng),體外成髓鞘實驗等方法對bFGF誘導(dǎo)所獲得的細(xì)胞進(jìn)行細(xì)胞學(xué)和功能學(xué)的鑒定;通過雙向電泳技術(shù)比較原代培養(yǎng)的施旺氏細(xì)胞和由背根節(jié)干細(xì)胞誘導(dǎo)分化的細(xì)胞蛋白表達(dá)譜差異。 結(jié)果: 1.通過體外分離培養(yǎng)的方法以及單克隆實驗獲得了穩(wěn)定傳代的背根節(jié)神經(jīng)干細(xì)胞。它表達(dá)神經(jīng)嵴干細(xì)胞特異性Marker,而且具有較高的增殖率和分化潛能。 2.誘導(dǎo)細(xì)胞分化的5組中的bFGF處理組, S100β陽性細(xì)胞的比例高于其他各組相比,Western Blot結(jié)果與此一致。 3.誘導(dǎo)背根神經(jīng)節(jié)干細(xì)胞分化的短時程和長時程效應(yīng)檢測結(jié)果顯示在bFGF在作用24 h時間內(nèi)影響細(xì)胞的分化,而對細(xì)胞的增殖沒有影響。 4.分化的細(xì)胞具有施旺氏細(xì)胞的形態(tài)學(xué)和功能學(xué)特征。 結(jié)論: 我們成功建立了培養(yǎng)背根節(jié)神經(jīng)干細(xì)胞的方法及定向誘導(dǎo)其向施旺氏細(xì)胞分化的方法,為周圍神經(jīng)再生提供了真正周圍神經(jīng)系統(tǒng)來源的種子細(xì)胞。 第二部分bFGF誘導(dǎo)神經(jīng)干細(xì)胞沿施旺氏細(xì)胞方向分化的機(jī)制研究 堿性成纖維細(xì)胞生長因子(bFGF)屬于有絲分裂原,通過與成纖維細(xì)胞生長因子受體(FGFRs)結(jié)合,激活MAPK,PI3K,PLCγ等信號通路,在影響細(xì)胞增殖、分化及生存方面起關(guān)鍵作用。成纖維生長因子受體有4種,配體與不同的受體結(jié)合會產(chǎn)生不同的效應(yīng)。神經(jīng)干細(xì)胞發(fā)育分化過程中的基因調(diào)控是一個復(fù)雜的過程,主要由快速反應(yīng)基因和轉(zhuǎn)錄因子的激活來完成。 目的: 探討bFGF對背根節(jié)神經(jīng)干細(xì)胞沿施旺氏細(xì)胞方向分化的過程中結(jié)合的受體及可能的信號轉(zhuǎn)導(dǎo)機(jī)制以及所涉及的快速反應(yīng)基因和轉(zhuǎn)錄因子調(diào)控作用,以揭示其誘導(dǎo)分化的機(jī)制。 方法: 1. RT-PCR和Western Blotting檢測成纖維生長因子受體在神經(jīng)球中的表達(dá),化學(xué)交聯(lián)和免疫共沉淀檢測與bFGF結(jié)合的受體。 2. Western Blotting檢測bFGF下游的主要的信號通路被激活情況及分化過程中參與的信號轉(zhuǎn)導(dǎo)通路。 3.檢測bFGF激活的信號轉(zhuǎn)導(dǎo)通路所起的生物學(xué)效應(yīng)。在加入bFGF的同時加入MEK抑制劑U0126, PI3K抑制劑LY294002及BrdU標(biāo)記液,收取細(xì)胞蛋白或固定細(xì)胞,Western Blotting或組化檢測細(xì)胞的增殖與分化情況。 4.利用TransAMTM Assay kits (Active Motif)報告實驗檢測bFGF作用的細(xì)胞的快速反應(yīng)基因c-jun, c-fos和Egr2(Krox20)的DNA的結(jié)合活性的變化;半定量RT-PCR檢測與雪旺氏細(xì)胞分化相關(guān)的轉(zhuǎn)錄因子Mash-1及Sox10的表達(dá)變化。 結(jié)果: 1. RT-PCR和Western Blot結(jié)果顯示背根節(jié)神經(jīng)干細(xì)胞高表達(dá)FGFR1-3,而低表達(dá)FGFR4。體外化學(xué)交聯(lián)和免疫共沉淀實驗結(jié)果以及細(xì)胞內(nèi)免疫共沉淀實驗結(jié)果證明bFGF與FGFR1結(jié)合激活下游信號通路。 2.與對照組相比,bFGF刺激可持續(xù)激活Erk1/2及Akt的磷酸化,表明Erk1/2和PI3K/AKT兩條信號通路在bFGF的生物功能中起作用。 3.背根節(jié)神經(jīng)干細(xì)胞體外分化24小時,bFGF在不影響分化細(xì)胞的增殖和存活的情況下通過ERK1/2信號通路促進(jìn)細(xì)胞向雪旺氏細(xì)胞方向分化。bFGF的刺激可在背根節(jié)神經(jīng)干細(xì)胞內(nèi)引起一系列的反應(yīng),包括ERK1/2和PI3K/AKT通路的激活,以及快速反應(yīng)基因c-Jun的活化和轉(zhuǎn)錄因子Mash-1的下調(diào)。如果在bFGF處理的同時用ERK1/2上游蛋白MEK1/2的抑制劑U0126阻斷ERK1/2的磷酸化,則bFGF介導(dǎo)的c-Jun的活化和轉(zhuǎn)錄因子Mash-1的下調(diào)及細(xì)胞沿施旺氏細(xì)胞分化被抑制。與之相對的,如果在bFGF處理的同時用PI3K的抑制劑LY294002阻斷AKT的磷酸化,則對則bFGF介導(dǎo)的c-Jun的活化和轉(zhuǎn)錄因子Mash-1的下調(diào)及細(xì)胞沿施旺氏細(xì)胞分化并無影響。 結(jié)論: bFGF綁定FGFR1受體通過Erk1/2信號通路引起快速反應(yīng)基因Jun的激活,下調(diào)轉(zhuǎn)錄因子Mash-1的基因表達(dá)水平,完成其在干細(xì)胞分化過程中的信號轉(zhuǎn)導(dǎo)和生物功能。
[Abstract]:Tissue engineering technology brings new hope for the treatment of peripheral nerve injury, the optimization selection of seed cells is the key technology of tissue engineering. Schwann cells are the main structure and function of peripheral nervous system cells, seed cells is recognized, but its source is limited. The trunk neural crest stem cells, can differentiate into around the nervous system of most neurons and glial cells, peripheral nerve system primordia, will therefore trunk neural crest stem cells in neural tissue engineering has a good application prospect.
The differentiation of stem cells in vivo is regulated by many factors, with diversity and uncertainty, neural stem cell transplantation for treatment of nervous system injury or disease is necessity to induce neural stem cells to specific cell type differentiation we need. Therefore, induction of differentiation of neural stem cells is an important characteristic the study of the meaning of.BFGF with multi functions, which can promote the differentiation of neurons, but also can promote the differentiation of glial cells and the function of its changes and development in vivo was consistent. In the early stage of neural development promotes neuronal differentiation, whereas in the late development promote the differentiation of glial cells. In the peripheral nervous system, bFGF is the peripheral nervous system stem cells differentiate into Schwann cells was positive determinants of progenitor cells, so the use of bFGF induced dorsal root ganglion Stem cells Schwann cell differentiation is a very good attempt. This paper firstly isolated and cultured in vitro DRG neural stem cells in vitro, then induce Schwann cell differentiation and its functional identification; on the basis of bFGF in vitro induction of neural stem cells and Schwann's cell differentiation mechanism along to conduct the research.
The first part of the culture of dorsal root ganglion neural stem cells, identification and directional induction of its direction differentiation along Schwann cells
Objective:
Methods to establish adult rat dorsal root ganglion neural stem cells and to orientate their differentiation along Schwann cells, so as to provide real peripheral nerve system seed cells for regeneration of peripheral nervous system.
Method:
1. the dorsal root ganglia of adult SD rats were cultured in vitro, and then the primary neural stem cell balls were obtained after suspension culture. The single cell clones were obtained by infinite dilution method, and their stem cell characteristics were identified after 2 passages, including specific markers, proliferation rate and differentiation potential.
2. cloned NSCs were divided into 5 groups: differentiation, NGF group; bFGF group; NRG group; FBS group; and the control group. S100 positive cells were induced after 7 d S100 immunocytochemistry and Westernblot. immunocytochemistry were performed at 10 randomly selected groups of proteins through statistical perspective; the expression level of Western blot was detected by S100 extraction at the same time.
3. bFGF induced short time and long term effects of DRG differentiation, namely, a group of 24 h after adding differentiated culture fluid, washed bFGF and continued to culture; the other group always used bFGF containing differentiation medium,.7 D, S100 immunocytochemistry and Westernblot..
4. BrdU markers were used to detect the proliferation of bFGF cells in 24 hours, while adding bFGF to induce cell differentiation and adding BrdU marker, and immunocytochemical detection after 24 h.
Passage 5. by immunocytochemistry, culture in vitro myelination experiment method was used to identify the cytological and functional studies on bFGF induced cells obtained by two-dimensional electrophoresis; expression comparison of primary cultured Schwann cells and DRG by differentiation of stem cells protein spectrum difference.
Result:
1., a stable passage of dorsal root ganglion neural stem cells was obtained through in vitro isolation and culture and monoclonal experiments. It expressed specific Marker of neural crest stem cells, and had high proliferation and differentiation potential.
2. the proportion of S100 beta positive cells in the bFGF treated group of 5 groups induced by cell differentiation was higher than that in the other groups, and the results of Western Blot were consistent with this.
3., the short-term and long-term effects of induction of DRG differentiation showed that bFGF could affect cell differentiation within 24 h time, but had no effect on cell proliferation.
4. the differentiated cells have morphological and functional characteristics of Schwann cells.
Conclusion:
We successfully established a method for culturing dorsal root ganglion neural stem cells and directed the induction of differentiation into Schwann cells, providing a real peripheral nerve system seed cell for peripheral nerve regeneration.
Study on the mechanism of bFGF induced neural stem cells differentiation along Schwann cells in the second part of the neural stem cells
紕辨，

本文編號：1657842