皮肤来源前体细胞的分离、培养及其分化过程中基因表达变化的初步研究

发布时间：2018-04-05 15:32

本文选题：SKPs　切入点：神经元　出处：《华中科技大学》2011年硕士论文

【摘要】：目的周围神经损伤是临床上的常见病、多发病,严重危害人类的健康。根据损伤程度(Sunderland分型)及致伤原因可分为多种类型,其传统的修复方法(神经松解、缝合、移植及植入等)对神经严重损伤、缺损患者的治疗效果不甚理想。寻找比较理想的治疗方法一直是创伤外科、手外科等学科研究的热点。近年来,组织工程学技术作为治疗周围神经损伤的一种新方法成为许多学者研究的对象。雪旺细胞(Schwann cell,SCs)来源于神经嵴,是周围神经系统的重要结构和功能细胞,大量研究证实其对周围神经及脊髓损伤的髓鞘再生及功能恢复有重要作用。人雪旺细胞来源于侵入性神经活检,并且其体外培养增殖受限,难以获得大量子代细胞,因而其无法作为种子细胞应用于临床。骨髓间质干细胞解决了这一难题,但其却不能产生真正有功能的雪旺细胞。同样,胚胎干细胞、神经干细胞因伦理学、组织来源的限制及同种异体移植引发的严重排斥反应而限制了其临床应用。因而,寻找一种合适的种子细胞成为组织工程治疗周围神经损伤的关键。2001年,Toma、Miller等从小鼠背部皮肤中获得了一种具有多向分化潜能的前体细胞,将其命名为皮肤来源前体细胞(Skin-derived Precursors,SKPs),其可分化产生神经元、神经胶质、平滑肌、脂肪等多种细胞类型。现已证实,SKPs来源胚胎发育时期的神经嵴,系神经嵴来源的前体细胞,随胚胎发育而迁移,最终主要位于成体皮肤毛囊的真皮乳头。SKP细胞体外增殖、分化的培养条件已基本成熟,在体外可大量增殖而保持其分化特性,在战栗鼠(Shivere mice)周围神经损伤模型中,SKPs及其来源的雪旺细胞均可促进成髓鞘过程和神经轴突连续性的恢复。由此可见,SKPs可以产生真正有功能的雪旺细胞,加之其取材方便,对机体损伤小,且能够进行自体移植从而避免排斥反应,因而SKPs可成为治疗周围神经损伤的一种新的、可接受的自体干细胞移植来源。目前对SKPs的研究虽已取得较多成果,但其增殖及分化的具体调控机制尚不清楚。目前胚胎发育过程中神经嵴形成、迁移及分化的具体机制已基本明了,其中Notch、Wnt、Sox-10等多种信号通路及分子参与了神经嵴分化的调节。既然SKP细胞来源于神经嵴,且其生物及分化特性与神经嵴细胞极为相似,那么其分化调控机制是否也与神经嵴细胞相似呢?对SKP细胞分化调控的研究,将有利于我们增加其向雪旺细胞分化的数量,从而为组织工程提供合适的种子细胞,促进周围神经再生及功能恢复。我们受上述启发,设计了本项实验,初步探讨SKPs分化过程中基因表达的变化,为进一步探讨Notch、Wnt等信号通路在分化调控中的作用提供实验依据。实验方法 1.取新生(鼠龄为3d)SPF级昆明小鼠的背部皮肤,消化、分离,悬浮法培养SKP细胞,常规方法进行传代,光镜下观察细胞形态,免疫细胞染色法检测巢蛋白(nestin)的表达,RT-PCR检测纤维连接蛋白(Fibronectin)相关基因的表达。 2.传代细胞分组,分别向神经元及雪旺细胞方向分化,光镜下观察,免疫细胞染色法检测神经元及雪旺细胞标志物(NeuN、GFAP)的表达,RT-PCR法检测神经元及雪旺细胞标志物(NFM、CNPase)相关基因的表达。 3.用流式细胞仪分选SKPs,获得侧群细胞,然后分别诱导其向神经元及雪旺细胞分化,各组于不同的时间点收集细胞后提取总RNA,体外转录标记后通过Microarray检测分析分化过程中各组细胞与原代SP细胞相比基因表达的差异,其中关键的结果通过定量RT-PCR方法给予进一步验证。结果 1.悬浮法培养的小鼠SKPs生长良好,增殖旺盛,形成明显细胞球,且表达nestin及Fibronectin。 2. SKPs分化培养过程中,neuron组及schwann组细胞形态有明显差异,neuron组细胞表达NFM和NeuN,而schwann组表达GFAP及CNPase。 3. SP细胞分化培养1天后,与原代SP细胞相比基因表达差异最明显。Neuron组有9条基因表达持续上调,34条基因表达持续下调。Schwann组共有33条基因表达上调,27条基因表达下调。诱导分化第3、7天后,基因表达差异不明显。结论 SKPs分化过程中基因表达与原代细胞相比有显著差异,且主要集中在wnt、notch等信号通路,说明它们对SKPs分化过程有重要调控作用。
[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.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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