本文關(guān)鍵詞：軀干神經(jīng)嵴干細(xì)胞的分離、純化及其向雪旺細(xì)胞分化的實驗研究　出處：《山東大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 神經(jīng)嵴 干細(xì)胞 分化 雪旺細(xì)胞 髓鞘堿性蛋白

【摘要】： 周圍神經(jīng)損傷是臨床上的常見病、多發(fā)病。神經(jīng)損傷后致使支配區(qū)的感覺及運動障礙,常常導(dǎo)致肌肉癱瘓、萎縮,甚至留下終生殘疾。尋找比較理想的治療方法一直是當(dāng)今創(chuàng)傷外科和康復(fù)醫(yī)學(xué)等學(xué)科的一個研究熱點。近年來,組織工程技術(shù)的進展為周圍神經(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。 雪旺細(xì)胞(Schwann cell,SCs)是周圍神經(jīng)系統(tǒng)的主要結(jié)構(gòu)和功能細(xì)胞,起源于神經(jīng)嵴干細(xì)胞。周圍神經(jīng)損傷后,它既可引導(dǎo)神經(jīng)軸索的定向生長,又能分泌多種神經(jīng)營養(yǎng)因子,改善神經(jīng)再生的微環(huán)境,促進周圍神經(jīng)再生及其功能恢復(fù),是公認(rèn)的種子細(xì)胞。然而自體來源的SCs在適當(dāng)條件下難以獲得大量子代雪旺細(xì)胞;異體來源的SCs則存在免疫排斥反應(yīng)等,因此有必要繼續(xù)尋找更為有效的種子細(xì)胞。 神經(jīng)嵴是脊椎動物胚胎早期從神經(jīng)管背外側(cè)遷移出來的位于神經(jīng)管和外胚層之間的兩條縱向細(xì)胞帶。神經(jīng)嵴細(xì)胞能夠進行自我更新,并可分化為神經(jīng)元、神經(jīng)膠質(zhì)細(xì)胞、平滑肌樣細(xì)胞等多種類型的細(xì)胞,也稱為神經(jīng)嵴干細(xì)胞(neuralcrest stem cell,NCSC)。自軀干部神經(jīng)管區(qū)域遷移出來的神經(jīng)嵴干細(xì)胞,即軀干神經(jīng)嵴干細(xì)胞(trunk neural crest stem cell,TNCSC),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,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。研究表明,神經(jīng)嵴干細(xì)胞可以向神經(jīng)譜系、成骨細(xì)胞等多細(xì)胞系方向進行分化,但是由于神經(jīng)嵴干細(xì)胞對體外培養(yǎng)的要求比較苛刻,且取得純度較高的細(xì)胞存在一定的困難,所以對促使其分化為成熟雪旺細(xì)胞的影響因素和機制及雪旺細(xì)胞與NCSC之間相互的作用鮮有報道。 探索如何獲取軀干神經(jīng)嵴干細(xì)胞,并增加其向雪旺細(xì)胞分化的數(shù)量,為組織工程技術(shù)提供合適的種子細(xì)胞,從而促進周圍神經(jīng)再生和功能恢復(fù),是目前亟待解決的問題。 基于以上理念,我們設(shè)計了本項實驗:取孕10.5d Wistar大鼠的胚胎神經(jīng)管分離,利用軀干神經(jīng)管植塊法進行培養(yǎng)、純化神經(jīng)嵴干細(xì)胞,行p75和nestin免疫細(xì)胞化學(xué)雙重染色鑒定培養(yǎng)的軀干神經(jīng)嵴干細(xì)胞,并對其克隆形成能力、增殖、自然分化等生物學(xué)特性進行了初步觀察;將傳代的軀干神經(jīng)嵴干細(xì)胞置于雪旺細(xì)胞條件培養(yǎng)液中培養(yǎng)觀察,利用MTT分析、細(xì)胞計數(shù)、免疫細(xì)胞化學(xué)染色等方法觀察軀干神經(jīng)嵴干細(xì)胞存活、增殖、定向分化為功能性雪旺細(xì)胞的情況,旨在通過體外培養(yǎng)獲得足夠量的雪旺細(xì)胞,為細(xì)胞移植修復(fù)周圍神經(jīng)損傷提供實驗依據(jù)。 本實驗結(jié)果顯示,軀干神經(jīng)管植塊培養(yǎng)貼壁24h后可見細(xì)胞自組織塊邊緣遷出,48h后細(xì)胞以神經(jīng)管組織為中心呈輻射狀分布,形態(tài)多為梭形,少量呈多角形,胞體豐滿,胞核較大,免疫細(xì)胞化學(xué)染色結(jié)果顯示,神經(jīng)嵴干細(xì)胞的特異性抗體—低親和力神經(jīng)生長因子受體的抗體p75及神經(jīng)巢蛋白nestin染色陽性,表明培養(yǎng)的細(xì)胞為軀干神經(jīng)嵴干細(xì)胞。生長曲線計算結(jié)果顯示軀干神經(jīng)嵴干細(xì)胞的倍增時間為34.2h,細(xì)胞呈克隆性生長;在10%血清誘導(dǎo)下,軀干神經(jīng)嵴干細(xì)胞自然分化為神經(jīng)元、神經(jīng)膠質(zhì)細(xì)胞和平滑肌樣細(xì)胞,免疫細(xì)胞化學(xué)染色顯示細(xì)胞分別呈MAP2、GFAP、α-SMA陽性。將傳代的軀干神經(jīng)嵴干細(xì)胞球在雪旺細(xì)胞條件培養(yǎng)液中培養(yǎng)發(fā)現(xiàn)24h后細(xì)胞貼壁,約36h可見細(xì)胞遷出,部分細(xì)胞胞體較飽滿,突起細(xì)長,大體形態(tài)與成熟神經(jīng)元相似;部分細(xì)胞有二個或三個突起,呈梭形,形態(tài)類似雪旺細(xì)胞。隨著培養(yǎng)時間延長,突起束狀排列,顯微鏡觀察可見在神經(jīng)元的軸突局部呈雙軌樣結(jié)構(gòu),也有少數(shù)多個突起的細(xì)胞胞體呈不規(guī)則形。對照組中神經(jīng)嵴干細(xì)胞球貼壁較少,細(xì)胞生長狀況較差,隨培養(yǎng)時間延長遷出的細(xì)胞逐漸減少,形態(tài)變化不大,至培養(yǎng)1周,細(xì)胞大多死亡。通過細(xì)胞免疫熒光染色和圖像分析觀察到在雪旺細(xì)胞條件培養(yǎng)液中,軀干神經(jīng)嵴干細(xì)胞分化的神經(jīng)元與膠質(zhì)細(xì)胞比例為0.75:1,可分化為大量的神經(jīng)膠質(zhì)細(xì)胞。MTT分析顯示:軀干神經(jīng)嵴干細(xì)胞在條件培養(yǎng)液中存活及增殖活性顯著增加(P＜0.01)。培養(yǎng)4周后發(fā)現(xiàn)部分軸突局部出現(xiàn)陽性表達MBP,表明分化形成的神經(jīng)膠質(zhì)細(xì)胞向成髓鞘雪旺細(xì)胞方向分化。 結(jié)論:本實驗成功分離培養(yǎng)了軀干神經(jīng)嵴干細(xì)胞并探討了其生物學(xué)特性,并且在雪旺細(xì)胞條件培養(yǎng)液中培養(yǎng)時發(fā)現(xiàn)有利于軀干神經(jīng)嵴干細(xì)胞存活和增殖,其分化形成的神經(jīng)膠質(zhì)細(xì)胞可進一步誘導(dǎo)分化形成成髓鞘雪旺細(xì)胞,并表達功能性雪旺細(xì)胞的主要蛋白MBP。
[Abstract]:Peripheral nerve injury is a common clinical disease, the incidence of sensory and motor disorders. The innervation area after nerve injury often leads to muscle atrophy, paralysis, and even left permanently disabled. Finding the ideal method of treatment has been a hot research topic of trauma surgery and rehabilitation medicine. In recent years, bring new hope for progress in tissue engineering technology for the treatment of peripheral nerve injury, the scaffold and seed cells is the key technology of tissue engineering.
Schwann cells (Schwann cell SCs) is the main structure and function of peripheral nerve system cells derived from the neural crest stem cells. After peripheral nerve injury, it can guide the directional axonal growth and secretion of various neurotrophic factors, micro environment to improve nerve regeneration, promote the recovery and regeneration of peripheral nerve function is recognized. However, autologous seed cells derived SCs under appropriate conditions is difficult to obtain a large number of offspring of Schwann cells; allogeneic SCs immune reaction, it is necessary to find more effective seed cells.
The neural crest is from early vertebrate embryonic neural tube located between dorsal lateral migration of neural tube and ectoderm cells with two longitudinal. Neural crest cells are capable of self-renewal and differentiation into neurons, glial cells, smooth muscle cells and other various types of cells, neural crest stem cells (also known as neuralcrest stem cell, NCSC). Since the trunk neural tube regional migration out of the neural crest stem cells, the trunk neural crest stem cells (trunk neural crest stem cell, TNCSC), can differentiate into most of the peripheral nervous system neurons and glial cells of the peripheral nervous system, is the base, the trunk neural crest stem cells applied to nerve tissue engineering has a good application prospect. The research results show that the cells can differentiate into neural lineages and neural crest stem cells, osteogenic differentiation of multicellular system direction, but due to the neural crest Stem cells require strict culture in vitro, and the cells with high purity have some difficulties. Therefore, there are few reports about the influencing factors and mechanisms that promote their differentiation into mature Schwann cells, and the interaction between Schwann cells and NCSC.
It is a urgent problem to explore how to get the trunk neural stem cells and increase the number of Schwann cells differentiated, and provide suitable seed cells for tissue engineering, so as to promote peripheral nerve regeneration and functional recovery.
Based on the above concept, we design this experiment: isolated from pregnant 10.5d Wistar rat embryonic neural tube, the trunk neural tube explants were cultured, purified neural crest stem cells, p75 and nestin immunocytochemical double staining to identify the cultured neural crest stem cells, proliferation and the colony formation ability, biological characteristics of natural differentiation were observed; the trunk neural crest stem cells into Schwann cells were cultured in liquid culture conditions, using MTT analysis, cell counting, immunocytochemical staining were used to observe neural crest stem cell survival, proliferation, differentiation and function of Schwann cell case and to obtain a sufficient amount of Schwann cells in vitro and to provide experimental basis for cell transplantation in the repair of peripheral nerve injury.
The experimental results show that the neural tube explants cultured 24h cells after the self-organizing block edge to move out, after 48h cells to neural tube tissue as the center of radiate distribution were fusiform, small polygonal, round cell body, larger nucleus, immunocytochemical staining results showed that antibody p75 and nestin nestin neural crest stem - cell specific antibodies of low affinity nerve growth factor receptor staining showed that cultured cells, neural crest stem cells. The results showed that the trunk neural crest stem cell doubling time for the growth curve of 34.2h cells showed clonal growth; in 10% serum induction. The trunk neural crest stem cells differentiated into neurons, glial cells and smooth muscle cells, immunocytochemistry staining showed that the cells were respectively MAP2, GFAP, alpha -SMA positive. The trunk neural crest stem cells. The ball training found that 24h cells adherent fluid in Schwann cells, about 36h cells to move out, some cells are plump, slender projections, gross morphology of mature neurons and similar parts; cells have two or three projections, spindle shaped, with similar morphology of Schwann cells. With longer incubation time. Bundles of axons in the microscope visible part of a track like structure, there are a plurality of protrusions cells were irregular. In the group of neural crest stem cells adherent ball less control, cell growth status is poor, with training time to move out of the cells gradually decreased, morphological changes little, to 1 weeks of culture, most of the cells died. By immunofluorescence staining and image analysis were observed in Schwann cell conditioned medium, the trunk neural crest stem cell differentiation of neurons and glial cell ratio of 0.75:1, can be divided into For a large number of glial cells.MTT analysis showed that the cells increased significantly in survival and proliferation in liquid culture conditions of trunk neural crest stem (P < 0.01). After 4 weeks of culture, found that some local axonal MBP expression, suggesting that glial cells differentiated into medullary sheath Schwann cell differentiation.
Conclusion: the successful isolation and culture of neural crest stem cell and explore its biological characteristics in this experiment, and in the Schwann cell conditioned medium in culture found in favor of the trunk neural crest stem cell survival and proliferation, glial cell differentiation may further differentiate into myelinating Schwann cells, and expression of MBP. protein the function of Schwann cells

【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

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