本文選題：SKPs　切入點(diǎn)：神經(jīng)元　出處：《華中科技大學(xué)》2011年碩士論文

【摘要】：目的 周圍神經(jīng)損傷是臨床上的常見(jiàn)病、多發(fā)病,嚴(yán)重危害人類的健康。根據(jù)損傷程度(Sunderland分型)及致傷原因可分為多種類型,其傳統(tǒng)的修復(fù)方法(神經(jīng)松解、縫合、移植及植入等)對(duì)神經(jīng)嚴(yán)重?fù)p傷、缺損患者的治療效果不甚理想。尋找比較理想的治療方法一直是創(chuàng)傷外科、手外科等學(xué)科研究的熱點(diǎn)。近年來(lái),組織工程學(xué)技術(shù)作為治療周圍神經(jīng)損傷的一種新方法成為許多學(xué)者研究的對(duì)象。雪旺細(xì)胞(Schwann cell,SCs)來(lái)源于神經(jīng)嵴,是周圍神經(jīng)系統(tǒng)的重要結(jié)構(gòu)和功能細(xì)胞,大量研究證實(shí)其對(duì)周圍神經(jīng)及脊髓損傷的髓鞘再生及功能恢復(fù)有重要作用。人雪旺細(xì)胞來(lái)源于侵入性神經(jīng)活檢,并且其體外培養(yǎng)增殖受限,難以獲得大量子代細(xì)胞,因而其無(wú)法作為種子細(xì)胞應(yīng)用于臨床。骨髓間質(zhì)干細(xì)胞解決了這一難題,但其卻不能產(chǎn)生真正有功能的雪旺細(xì)胞。同樣,胚胎干細(xì)胞、神經(jīng)干細(xì)胞因倫理學(xué)、組織來(lái)源的限制及同種異體移植引發(fā)的嚴(yán)重排斥反應(yīng)而限制了其臨床應(yīng)用。因而,尋找一種合適的種子細(xì)胞成為組織工程治療周圍神經(jīng)損傷的關(guān)鍵。2001年,Toma、Miller等從小鼠背部皮膚中獲得了一種具有多向分化潛能的前體細(xì)胞,將其命名為皮膚來(lái)源前體細(xì)胞(Skin-derived Precursors,SKPs),其可分化產(chǎn)生神經(jīng)元、神經(jīng)膠質(zhì)、平滑肌、脂肪等多種細(xì)胞類型�，F(xiàn)已證實(shí),SKPs來(lái)源胚胎發(fā)育時(shí)期的神經(jīng)嵴,系神經(jīng)嵴來(lái)源的前體細(xì)胞,隨胚胎發(fā)育而遷移,最終主要位于成體皮膚毛囊的真皮乳頭。SKP細(xì)胞體外增殖、分化的培養(yǎng)條件已基本成熟,在體外可大量增殖而保持其分化特性,在戰(zhàn)栗鼠(Shivere mice)周圍神經(jīng)損傷模型中,SKPs及其來(lái)源的雪旺細(xì)胞均可促進(jìn)成髓鞘過(guò)程和神經(jīng)軸突連續(xù)性的恢復(fù)。由此可見(jiàn),SKPs可以產(chǎn)生真正有功能的雪旺細(xì)胞,加之其取材方便,對(duì)機(jī)體損傷小,且能夠進(jìn)行自體移植從而避免排斥反應(yīng),因而SKPs可成為治療周圍神經(jīng)損傷的一種新的、可接受的自體干細(xì)胞移植來(lái)源。 目前對(duì)SKPs的研究雖已取得較多成果,但其增殖及分化的具體調(diào)控機(jī)制尚不清楚。目前胚胎發(fā)育過(guò)程中神經(jīng)嵴形成、遷移及分化的具體機(jī)制已基本明了,其中Notch、Wnt、Sox-10等多種信號(hào)通路及分子參與了神經(jīng)嵴分化的調(diào)節(jié)。既然SKP細(xì)胞來(lái)源于神經(jīng)嵴,且其生物及分化特性與神經(jīng)嵴細(xì)胞極為相似,那么其分化調(diào)控機(jī)制是否也與神經(jīng)嵴細(xì)胞相似呢?對(duì)SKP細(xì)胞分化調(diào)控的研究,將有利于我們?cè)黾悠湎蜓┩?xì)胞分化的數(shù)量,從而為組織工程提供合適的種子細(xì)胞,促進(jìn)周圍神經(jīng)再生及功能恢復(fù)。我們受上述啟發(fā),設(shè)計(jì)了本項(xiàng)實(shí)驗(yàn),初步探討SKPs分化過(guò)程中基因表達(dá)的變化,為進(jìn)一步探討Notch、Wnt等信號(hào)通路在分化調(diào)控中的作用提供實(shí)驗(yàn)依據(jù)。 實(shí)驗(yàn)方法 1.取新生(鼠齡為3d)SPF級(jí)昆明小鼠的背部皮膚,消化、分離,懸浮法培養(yǎng)SKP細(xì)胞,常規(guī)方法進(jìn)行傳代,光鏡下觀察細(xì)胞形態(tài),免疫細(xì)胞染色法檢測(cè)巢蛋白(nestin)的表達(dá),RT-PCR檢測(cè)纖維連接蛋白(Fibronectin)相關(guān)基因的表達(dá)。 2.傳代細(xì)胞分組,分別向神經(jīng)元及雪旺細(xì)胞方向分化,光鏡下觀察,免疫細(xì)胞染色法檢測(cè)神經(jīng)元及雪旺細(xì)胞標(biāo)志物(NeuN、GFAP)的表達(dá),RT-PCR法檢測(cè)神經(jīng)元及雪旺細(xì)胞標(biāo)志物(NFM、CNPase)相關(guān)基因的表達(dá)。 3.用流式細(xì)胞儀分選SKPs,獲得側(cè)群細(xì)胞,然后分別誘導(dǎo)其向神經(jīng)元及雪旺細(xì)胞分化,各組于不同的時(shí)間點(diǎn)收集細(xì)胞后提取總RNA,體外轉(zhuǎn)錄標(biāo)記后通過(guò)Microarray檢測(cè)分析分化過(guò)程中各組細(xì)胞與原代SP細(xì)胞相比基因表達(dá)的差異,其中關(guān)鍵的結(jié)果通過(guò)定量RT-PCR方法給予進(jìn)一步驗(yàn)證。 結(jié)果 1.懸浮法培養(yǎng)的小鼠SKPs生長(zhǎng)良好,增殖旺盛,形成明顯細(xì)胞球,且表達(dá)nestin及Fibronectin。 2. SKPs分化培養(yǎng)過(guò)程中,neuron組及schwann組細(xì)胞形態(tài)有明顯差異,neuron組細(xì)胞表達(dá)NFM和NeuN,而schwann組表達(dá)GFAP及CNPase。 3. SP細(xì)胞分化培養(yǎng)1天后,與原代SP細(xì)胞相比基因表達(dá)差異最明顯。Neuron組有9條基因表達(dá)持續(xù)上調(diào),34條基因表達(dá)持續(xù)下調(diào)。Schwann組共有33條基因表達(dá)上調(diào),27條基因表達(dá)下調(diào)。誘導(dǎo)分化第3、7天后,基因表達(dá)差異不明顯。 結(jié)論 SKPs分化過(guò)程中基因表達(dá)與原代細(xì)胞相比有顯著差異,且主要集中在wnt、notch等信號(hào)通路,說(shuō)明它們對(duì)SKPs分化過(guò)程有重要調(diào)控作用。
[Abstract]:objective
Peripheral nerve injury is a common clinical disease, the incidence of serious harm to human health. According to the degree of injury (Sunderland type) and the causes of injury can be divided into many types, the traditional repair methods (neurolysis, suture, transplantation and implantation) on nerve defect in patients with serious injury, treatment the effect is not very ideal. Finding the ideal method of treatment has been a hot spot in trauma surgery, hand surgery and other subjects. In recent years, a new method of tissue engineering technology for the treatment of peripheral nerve injury has become the research subject of many scholars. The snow (Schwann cell, SCs of Schwann cells derived from the neural crest), is an important structure and the function of the peripheral nervous system cells, many studies have confirmed the injury of peripheral nerve and spinal cord myelin regeneration and functional recovery has an important role. Schwann cells derived from invasive nerve biopsy, and in vitro The proliferation is limited, difficult to obtain a large number of daughter cells, which cannot be used as seed cells for clinical application of bone marrow mesenchymal stem cells. To solve this problem, but it can't generate the real function of Schwann cells. Similarly, embryonic stem cells, neural stem cells for ethics, severe acute rejection and allograft group limited tissue sources caused by limiting its clinical application. Therefore, to find a suitable seed cells for tissue engineering treatment of peripheral nerve injury in.2001, Toma, Miller obtained a multipotent progenitor cells from mouse back skin, named it skin derived precursor cells (Skin-derived Precursors SKPs), which can be differentiated into neurons, glia, smooth muscle, fat and other cell types. It has been confirmed that SKPs during embryonic development of neural crest derived neural crest line to. The precursor cell source, with the development and migration of embryonic, eventually in the proliferation of dermal papilla cells in vitro.SKP skin hair follicle, culture condition differentiation has been basically mature, can proliferate in vitro while maintaining their differentiation characteristics in rats (Shivere mice) at peripheral nerve injury, Schwann cells can be SKPs and origin of the myelination process and promote the recovery of axonal continuity. Thus, SKPs can produce the real function of Schwann cells, coupled with its convenient, small injury to the body, and can be used for autologous transplantation to avoid rejection, so SKPs could be the treatment of peripheral nerve injury is a kind of new, acceptable autogenous the source of stem cell transplantation.
At present the study on SKPs has made many achievements, but the specific regulatory mechanism of proliferation and differentiation is not clear. The current embryonic neural crest formation process, the specific mechanism of migration and differentiation has been basically clear, including Notch, Wnt, Sox-10 and other signaling pathways and molecules involved in the regulation of neural crest differentiation since. SKP cells derived from the neural crest, and its biological characteristics and differentiation and neural crest cells are very similar, so whether the regulatory mechanism of differentiation of neural crest cells is similar with? Research on SKP cell differentiation regulation, will help us to increase the number of Schwann cell differentiation, so as to provide suitable seed cells for tissue engineering, promote the regeneration and functional recovery of peripheral nerve. We are the inspiration, the design of this experiment was to evaluate the changes in gene expression during the differentiation of SKPs, for the further study of Notch, Wnt signal The role of the pathway in the regulation of differentiation provides an experimental basis.
Experimental method
1. newborn (rat age 3D) SPF Kunming mice back skin, digestion, separation, SKP cell suspension culture method, the conventional method of subculture, cell morphology was observed under light microscope, staining of nestin cells (nestin) expression, RT-PCR detection of fibronectin (Fibronectin) gene expression.
2. passage cells were grouped into neurons and Schwann cells. The expression of neurons and Schwann cell markers (NeuN, GFAP) was detected by immuno cell staining. RT-PCR expression was used to detect the expression of neurons and Schwann cell markers (NFM, CNPase) in Xiang Guanji.
3. were sorted by flow cytometry SKPs, obtain the side population cell, and then induced to neurons and Schwann cell differentiation were collected at different time points after cell extraction of total RNA, Microarray detection and analysis in the process of the differentiation of cells and primary SP cells compared gene expression by in vitro transcription after labeling, which is the key the results by quantitative RT-PCR method to give further validation.
Result
In 1. suspension culture, the mice SKPs grew well, proliferated vigorously, formed a clear cell ball, and expressed nestin and Fibronectin.
During the differentiation and culture of 2. SKPs, there were significant differences in cell morphology between group neuron and Schwann group. The cells in group neuron expressed NFM and NeuN, while group Schwann expressed GFAP and CNPase..
3. the differentiation of SP cells cultured for 1 days, compared with the primary SP cell gene expression the most obvious difference between the.Neuron group and 9 genes up-regulated and 34 genes were continued, sustained down-regulation of.Schwann group were upregulated and 33 genes, 27 downregulated genes. Differentiation of the first 3,7 days, gene expression was not significantly different.
conclusion
There was a significant difference in gene expression between SKPs and primary cells, mainly in Wnt, notch and other signal pathways, indicating that they play an important role in regulating SKPs differentiation.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：1715409