本文選題：神經(jīng)干細(xì)胞　切入點：骨髓基質(zhì)細(xì)胞　出處：《河北醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:神經(jīng)干細(xì)胞(neural stem cells,NSCs)是具有分化為神經(jīng)元細(xì)胞、星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞的能力,可自我更新并提供腦組織細(xì)胞的干細(xì)胞。NSCs的出現(xiàn)為臨床治療神經(jīng)退行性疾病以及神經(jīng)系統(tǒng)損傷帶來了新的希望。但無論是體外還是體內(nèi)研究的結(jié)果,NSCs分化為神經(jīng)元的比例都明顯低于膠質(zhì)細(xì)胞,致使難以達(dá)到理想的替代因損傷和疾病等原因造成的神經(jīng)元缺失的目的。 骨髓基質(zhì)細(xì)胞(bone marrow stromal cells,BMSCs)是來源于骨髓的多潛能干細(xì)胞,其主要功能是支持和營養(yǎng)造血細(xì)胞。近年來研究發(fā)現(xiàn)BMSCs能分泌許多細(xì)胞因子如腦源性神經(jīng)營養(yǎng)因子(BDNF)、堿性成纖維生長因子(bFGF)、血管內(nèi)皮生長因子(VEGF)、白細(xì)胞介素(IL)等,而且這些細(xì)胞因子對NSCs的分化、存活及增殖也有一定影響。本實驗室研究已證實BMSCs能夠誘導(dǎo)中腦NSCs分化為高比例神經(jīng)元,并且是BMSCs分泌至培養(yǎng)液中的可溶性分子在這一過程中發(fā)揮了重要作用,但究竟是這些可溶性分子通過什么途徑在起作用目前尚不清楚。 絲裂酶原活化蛋白激酶(mitogen-activated protein kinase, MAPK)普遍存在于多種生物細(xì)胞內(nèi),MAPK將細(xì)胞外信號轉(zhuǎn)導(dǎo)至胞內(nèi),從而參與細(xì)胞的生長、分化和細(xì)胞凋亡等過程。本實驗旨在利用Neurobasal培養(yǎng)液制成的BMSCs條件培養(yǎng)液(Neurobasal-conditioned medium,N-CM)培養(yǎng)NSCs,通過在培養(yǎng)體系中加入MAPKs信號轉(zhuǎn)導(dǎo)通路的抑制劑,觀察其對NSCs分化為神經(jīng)元和星型膠質(zhì)細(xì)胞的影響,明確MAPKs信號轉(zhuǎn)導(dǎo)通路在這一過程中的作用,初步探討了BMSCs調(diào)節(jié)NSCs分化的信號轉(zhuǎn)導(dǎo)機(jī)制。 方法:分離SD大鼠股骨和脛骨,沖洗骨髓腔,將細(xì)胞懸液離心后種入75ml培養(yǎng)瓶內(nèi)。取3~ 6代的BMSCs,待細(xì)胞鋪滿瓶底85%后,棄去培養(yǎng)液,更換為Neurobasal培養(yǎng)液6ml,培養(yǎng)24 h后,離心收集上清即為N-CM。取新生大鼠中腦,機(jī)械分散成單細(xì)胞后,離心棄上清,加入DMEM/F12(1∶1)添加2% B27的無血清培養(yǎng)基,同時加入20ng/ml bFGF,將細(xì)胞種入培養(yǎng)瓶中。在分離培養(yǎng)7d左右單個的NSCs便可增殖形成球體(NSCs球)。此后,每5～7d傳代1次。將二代或三代NSCs球均勻種植于預(yù)先包被多聚賴氨酸的35 mm培養(yǎng)皿中,待NSCs球貼壁后更換培養(yǎng)液。實驗分為三組:(1)自然分化組:用單純Neurobasal培養(yǎng)液培養(yǎng)NSCs;(2)對照組:用N-CM培養(yǎng)NSCs;(3)抑制劑組:在N-CM培養(yǎng)的NSCs中分別加入三種MAPKs信號轉(zhuǎn)導(dǎo)通路抑制劑,濃度為:PD98059 5μM,SB203580 4μM,Genistin 4μM。培養(yǎng)3d后,進(jìn)行免疫細(xì)胞化學(xué)染色。在倒置熒光顯微鏡下每個培養(yǎng)皿隨機(jī)選擇10個視野,分別計數(shù)每個視野內(nèi)MAP-2及GFAP陽性細(xì)胞數(shù)和同一個視野中的細(xì)胞總數(shù),得到該視野陽性細(xì)胞百分比。各組實驗獨立重復(fù)3次,進(jìn)行相同的計數(shù)過程,最后取均值。結(jié)果采用SPSS13.0統(tǒng)計軟件處理,所有計量資料均采用均數(shù)±標(biāo)準(zhǔn)差表示,各組之間采用t檢驗進(jìn)行顯著性分析,以p0.05為差異有顯著性。 結(jié)果: 1.N-CM對NSCs分化的影響 自然分化組中細(xì)胞總體生長狀態(tài)很差,細(xì)胞核多有固縮、破碎。神經(jīng)元稀少,體積小、突起不明顯。遷移距離較近,多聚集在一起。星形膠質(zhì)細(xì)胞數(shù)量多,突起粗,多聚集在神經(jīng)球的中央,呈放射狀排列。這兩種細(xì)胞占神經(jīng)干細(xì)胞球中分化出的細(xì)胞的大多數(shù)。而用N-CM培養(yǎng)NSCs,細(xì)胞生長狀況良好,細(xì)胞核大小均一,很少有破碎。神經(jīng)元較自然分化組多,突起較長,遷移距離遠(yuǎn),分布較均勻。星形膠質(zhì)細(xì)胞較自然分化組數(shù)量少,胞體細(xì)長,分布較分散。N-CM組NSCs分化為神經(jīng)元的比例為(34.26±9.31%),高于自然分化組(18.02±7.69%)(p0.01);星形膠質(zhì)細(xì)胞的比例為(36.92±10.14%),低于自然分化組(60.11±8.99%)(p0.01),說明N-CM對NSCs分化為神經(jīng)元具有上調(diào)作用,而對星形膠質(zhì)細(xì)胞有抑制作用。 2.含三種抑制劑的N-CM誘導(dǎo)NSCs分化 PD98059組和SB203580組細(xì)胞生長狀況較好,胞核較均一,有少量破碎。神經(jīng)元較對照組數(shù)量少,突起不明顯,遷移距離不遠(yuǎn),分布尚均勻,這兩組NSCs分化為神經(jīng)元的比例為(20.37±9.25%)和(22.14±8.47%)均明顯低于對照組(34.26±9.31%)(p0.01)。星形膠質(zhì)細(xì)胞粗大、扁平,突起多,形態(tài)不規(guī)則,較對照組數(shù)量多,遷移距離遠(yuǎn),分布分散。這兩組NSCs分化為星形膠質(zhì)細(xì)胞的比例為(47.43±11.45%)和(48.72±10.58%)均明顯高于對照組(36.92±10.14%)(p0.05)。而Genistin組與對照組相比,神經(jīng)元和星形膠質(zhì)細(xì)胞的形態(tài)、遷移距離及數(shù)量統(tǒng)計均無明顯差別(p 0.05)。 結(jié)論:本研究證實:1.BMSCs分泌的可溶性分子能夠誘導(dǎo)NSCs分化為高比例的神經(jīng)元。2. BMSCs分泌的可溶性分子不僅能夠調(diào)節(jié)NSCs的分化,還能夠影響神經(jīng)元和膠質(zhì)細(xì)胞形態(tài)、突起長短和遷移等細(xì)胞形態(tài)學(xué)和行為學(xué)。3. BMSCs分泌的可溶性分子誘導(dǎo)NSCs分化為高比例的神經(jīng)元與ERK1/2信號轉(zhuǎn)導(dǎo)通路和p38信號轉(zhuǎn)導(dǎo)通路有關(guān),而與SAPK/JNK信號轉(zhuǎn)導(dǎo)通路無關(guān),初步揭示了其信號轉(zhuǎn)導(dǎo)機(jī)制。
[Abstract]:Objective: neural stem cells (neural stem cells, NSCs) can differentiate into neurons, astrocytes, oligodendrocytes, self renewing stem cells and provide.NSCs brain cells for the treatment of neurodegenerative diseases and nerve system injury brought new hope. But whether it is in vitro and in vivo results, NSCs the percentage of neurons were significantly lower than those of glial cells, resulting in the loss of neurons is difficult to achieve the ideal alternative due to injury and disease and other purposes.
Bone marrow stromal cells (bone marrow stromal cells, BMSCs) is derived from bone marrow pluripotent stem cells, its main function is to support and nutrition of hematopoietic cells. Recent studies have found that BMSCs can secrete many cytokines such as brain-derived neurotrophic factor (BDNF), basic fiber growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin (IL), and these cytokines on NSCs differentiation, proliferation and survival to a certain extent. The laboratory studies have confirmed that BMSCs can induce NSCs to differentiate into a high proportion of midbrain neurons, and BMSCs is secreted into the culture medium soluble molecules in this process play an important role, but whether these soluble molecules through what way in action is unclear.
Mitogen activated protein kinase (mitogen-activated protein, kinase, MAPK) exists in a variety of biological cells, MAPK extracellular signals to intracellular, which involved in cell growth, differentiation and apoptosis. This study aims to develop the solution made of BMSCs medium with Neurobasal (Neurobasal-conditioned medium N-CM) training NSCs, produced by the inhibitor of MAPKs signal transduction pathway in the system, to observe the differentiation of NSCs into neurons and astrocytes, define the function of MAPKs signaling pathway in this process, discusses the signal transduction mechanism for regulating NSCs differentiation of BMSCs.
鏂規(guī)硶:鍒嗙SD澶ч紶鑲￠鍜岃儷楠，

本文編號：1598863