【作者】 余美春；

【导师】 原林；

【作者基本信息】 南方医科大学 ， 人体解剖学， 2011， 硕士

【摘要】 背景：原林教授提出的筋膜学“两系统理论”认为：人体由非特异性结缔组织筋膜支架所构成的支持与储备系统和被该筋膜支架所包绕的功能系统所构成。支持与储备系统以干细胞为核心,可为功能系统的更新提供细胞补充,并为功能系统的各种功能细胞的更新、代谢提供一个较为稳定的内部环境和干细胞源。疏松结缔组织是支持与储备系统重要的组成部分,其中的脂肪源干细胞(adipose-derived stem cells, ADSCs)是机体重要的干细胞储备之一。支持与储备系统使生物维持较长的生命周期和维持机体稳定的内环境。疏松结缔组织中的间充质干细胞(Mesenchymal stem cells, MSCs)构成了支持与储备系统的主要物质基础。该学说的“损伤修复机制”认为,机体在受外界刺激作用而损伤时,通过动员支持与储备系统(机体非特异性的结缔组织)中未分化的干细胞,经增殖,不断为功能系统提供源源不断的干细胞源,并分化成各种定向干细胞、进而分化成功能细胞来修复机体损伤,维持机体的稳定。有相关文献报道,在人体受到创伤时,间充质干细胞通过增殖、分化来修复损伤。骨损伤作为一种常见的创伤,可能具有相同或相似的损伤修复机制。ADSCs是目前干细胞的研究热点之一。作为一种新的MSCs,其具备了成体干细胞(Adult stem cells, ASCs)的特征,具有自我增殖和多向分化的能力。能在一定的条件下分化为成骨细胞、脂肪细胞、软骨细胞、骨骼肌细胞和神经细胞等。近期的研究发现,和骨髓干细胞(Bone marrow stem cell, BMSCs)相比,ADSCs具有取材手术小、可重复进行、酶消化分离程序简单等优点,可以作为新的成体干细胞来源。ADSCs是一种能够自我更新,并且具有多种分化潜能的新型干细胞,可用于急性或慢性组织损伤的修复与再生,诱导后移植到骨缺损部位可修复骨缺损。成骨细胞(Osteoblast, OB)主要由MSCs分化而来,其发育分化过程主要经历四个阶段,即细胞增殖、基质分泌、基质成熟和矿化形成等。该过程受一系列细胞因子、信号分子和周围环境因素等的影响和控制,包括骨形态发生蛋白(bone morphogenetic protein, BMP)、转化生长因子2β(transforming growth factor-p,TGF2-p)、钙黏附蛋白(cadherins)等。其中BMP具有非常重要的作用,通过信号转导,具有诱导干细胞成骨的作用。BMP属于转化生长因子-β(TGF-β)超家族的一员,在成骨细胞的发育过程中具有非常重要的作用,能够促进骨的形成。而骨形成的关键在于基质干细胞向成骨细胞分化,BMP在诱导MSCs成骨、修复骨损伤过程中具有非常重要的作用。BMP及其下游的级联蛋白组成的信号转导系统通过影响相关转录因子,如：核心结合因子α(Core-binding factorα1, Cbfα1), Osterix (OSX), Dlx5/Msx2等的表达,或是直接作用于成骨细胞特异性蛋白,如碱性磷酸酶(alkaline phosphatase, ALP),骨钙素(osteocalcin, OCN)的mRNA,从而促进成骨细胞的分化,刺激骨形成。BMPs信号转导过程是：BMPs首先与Ⅱ型受体和Ⅰ型受体结合,Ⅰ型受体磷酸化Drosophila mothers against decapentaplegic protein (Smads), Smads进入核内与转录因子相互作用影响相关蛋白的转录。Smads可分为受体调节Smads(R-Smads:Smad1、2、3、5、8和9)、共同介导者Smad (Co-Smad:Smad4)和抑制性Smads(I-Smads:Smad6、7)。其中Smad1、Smad5和Smad8,可能还有Smad9涉及BMPs的信号传导。与Smad1和Smad5相关的转录因子有核心结合蛋白A1 (core binding factor A1, CBFA1)、鸟氨酸脱羧酶拮抗酶(ornithine decarboxylase antizyme, OAZ)、Smad相互作用蛋白-1(smad- interacting protein 1, SIP1)、蟾蜍腹侧形成同源框蛋白-2(xenopus ventralizing homeobox protein-2, Xvent-2)、激活蛋白-1 (activating protein-1, AP-1)、生长抑素(sandostatin, Ski)、抗增殖蛋白(antip roliferative proteins, Tob)、同源结构域转录因子-8(homeodomain-containing transcription factor-8, Hoxc-8)等。其中CBFA1与TGF-β和BMP-2信号传导有关,能与Smad1、2、3、5相互作用,在骨形成中起重要作用。目的MSCs的多组织来源性,与筋膜结缔组织支架的全身分布性具有一致性,其横向、纵向分化的生物学特性,与筋膜结缔组织对机体的支持储备作用也是一致的。ADSCs是筋膜中未分化干细胞的一种储备形式。本实验采用大鼠筋膜结缔组织皮下脂肪作为组织来源,分离培养ADSCs,并对其表型鉴定,探讨筋膜结缔组织中对机体产生支持储备作用的细胞学基础；研究ADSCs是否表达与骨损伤修复密切相关的BMP信号通路相关分子；骨损伤后,ADSCs在成骨修复损伤过程中其表面BMP受体和Smads的表达是否增加,BMP信号通路相关分子的表达是否增加。1、通过细胞分离培养判断非特异性筋膜结缔组织中是否存在未分化的间充质干细胞,以及这种干细胞的相关特性；2、通过RT-PCR技术观察ADSCs中BMP信号通路相关分子的表达,以证实ADSCs是否表达BMP信号通路相关分子；3.探索骨损伤后,在修复损伤的过程中自体ADSCs表面BMP受体和Smads的表达变化。方法1、取2月龄Wistar雌性大鼠腹股沟部皮下脂肪组织,采用酶消化法分离、培养脂肪源干细胞。通过细胞形态学、功能学、流式细胞术鉴定非特异性筋膜结缔组织中的脂肪源干细胞细胞是否符合间充质干细胞的特性以及部分表面标志,以验证非特异性筋膜结缔组织中是否含有间充质干细胞。2、将第4代ADSCs经成骨诱导和成脂肪诱导分化培养后,分别进行相应的碱性磷酸酶、茜素红和油红O染色,以鉴定ADSCs的分化潜能。同时取培养的第四代ADSCs进行流式细胞术检测部分表面抗原,证明ADSCs的干细胞特性。3、提取培养的第4代ADSCs的总RNA进行RT-PCR,检测BMP信号通路相关分子mRNA的表达。4、30只雌性Wistar大鼠随机分为空白对照组(A组5只)、实验组(B组25只)。A组不做处理,直接取腹股沟脂肪垫进行ADSCs培养。B组制造骨缺损模型。分别于造模后第1、3、7、14、21天,取腹股沟脂肪组织进行ADSCs培养,每次均取5只。原代培养第7天,提取ADSCs(?)总RNA,进行RT-qPCR检测骨形态发生蛋白受体(Bone morphogenetic protein receptor, BMPRs)和Smads的mRNA表达变化。结果1、光学显微镜下观察贴壁ADSCs形态多呈梭形、多边形,细胞大小均匀；2、流式细胞术鉴定ADSCs的表面抗原显示CD29、CD90、CD106呈阳性,CD11b、CD45、CD49d呈阴性；3、ADSCs在成脂诱导培养14天后,油红O染色显示特异性脂滴,而阴性对照孔中未观察到红染的脂滴。成骨诱导培养21天后,茜素红染色显示矿化结节呈特异性的粉红色,阴性对照孔中未见被染成粉红色的钙结节；4、RT-PCR检测结果显示ADSCs中BMP信号通路相关分子Bmprla、Bmpr1b、Bmpr2; Smad1、Smad5、Smad8的mRNA均阳性表达。5、RT-qPCR检测显示,大鼠在骨缺损刺激后,ADSCs中Bmprla、Bmpr1b、Bmpr2和Smad1、Smad5、Smad8的mRNA表达量增加。结论1、通过本实验,笔者认为在发育成熟Wister大鼠的筋膜结缔组织的脂肪中存在多能干细胞,而且可通过消化-贴壁-传代的程序分离得到这些ADSCs;为筋膜结缔组织对机体发挥着支持与储备作用(干细胞储备)提供实验支持,即筋膜结缔组织中的ADSCs为机体损伤修复提供细胞来源。2、ADSCs具有成体间充质干细胞特性,具有多向分化潜能,经定向诱导培养后可向成骨和成脂肪分化；ADSCs可能存在BMP信号通路。3、ADSCs表达BMP受体,表达BMP信号通路相关分子,骨损伤可引起大鼠ADSCs中BMP信号通路相关分子表达增加。综上所述,本研究分别采用了细胞分离培养、流式细胞术,成脂、成骨诱导培养及检测等技术手段,从ADSCs形态特征,成脂、成骨诱导、表面标志等方面探讨ADSCs。证实在发育成熟大鼠的结缔组织中存在间充质干细胞,提示分布于全身的筋膜结缔组织支架很可能就是机体内在的“干细胞库”。这种干细胞易于分离培养,具有良好的增殖、分化能力。笔者认为ADSCs是筋膜中间充质干细胞的一种主要细胞储备形式,更重要的是筋膜结缔组织对机体的支持与储备起着细胞储备作用,为机体损伤修复提供干细胞支持。通过RT-qPCR技术,从分子水平检测证实ADSCs表达与骨损伤修复密切相关的BMP信号通路相关分子,而且在骨损伤可引起大鼠ADSCs中BMP信号通路相关分子表达增加。提示骨损伤后,在骨损伤修复过程中,ADSCs可能参与修复损伤。机体可能通过产生某种活性物质作用,如细胞因子,炎症介质等,动员自身“干细胞库”在机体正常更新、损伤后的修复中发挥支持储备作用,为机体修复损伤提供干细胞来源。进一步验证了“筋膜学”理论所提及的损伤修复机制。更多还原

【Abstract】 Background:Professor Lin Yuan has put forward a new hypothesis——fasciaology. In fasciaology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of undifferentiated cells of unspecialized connective tissues. The other is the functional system, which is consisted of differentiated functional cells and is enclosed by the supporting-storing system.That is "two system theory". The undifferentiated stem cells in the supporting-storing system incessantly differentiate into functional cells. The supporting-storing system throughout the body regulates the functional and living status of differentiated cells and provides a stable internal environment for the survival of these cells. Loose connective tissue is a an important part of the supporting-storing system, and adipose-derived stem cells (ADSCs) are main stem cells reserved in this system. Injury-repair mechanism of faciology regard that living body can mobilize the undifferentiated stem cells in the supporting-storing system and these cells proliferate and differentiate incessantly into functional cells for repairing the damage, when the living body suffer from any damage. It was reported, when humam body confront any injury, mesenchymal stem cells (MSCs) can repair the injury via proliferation, migration and differentiation. As a common injury, bone trauma may have the same or similar damage repair mechanisms. In recent years, interest has rapid grown in the research of ADSCs. Being one kind of mesenchymal stem cells, ADSCs have the capacity to selfrenew and differentiate to many different cell types. Mesenchymal stem cells can be isolated from several organs, such as bone marrow, fat, umbilical cord blood, peripheral blood and skeletal muscles. ADSCs derived from adipose tissue, which can differentiate to multiple cell phenotype under appropriate culturing conditions, including osteoblast, adipocyte, chondrocyte, skeletal muscle cell and neurocyte. Bone marrow mesenchymal stem cells were first identified and are one of the most widely used stem cell sources. Therapeutic potential of transplantation of them is invigorating. Compared with bone marrow mesenchymal stem cells, ADSCs do have an equal potential to differentiate into multiple cell phenotype. However, the simple surgical procedure, the easy and repeatable access to the subcutaneous adipose tissue, and the uncomplicated enzyme-based isolation procedures make ADSCs most attractive for researchers. Being a new source of therapeutic stem cells, ADSCs should be given more attention to be used in repairing acute or chronic tissue injury and tissue regeneration. transplantation to bone defect site can repair bone defects after ADSCs were induced into osteogenesis differentiation.Osteoblasts (OB) mainly derived from mesenchymal stem cells, their differentiation and development into bone tissue through four major stages, namely cell proliferation, matrix secretion, matrix maturation and mineralization. The process is affected and controled by a series of cytokines, signaling molecules and the surrounding environment. including bone morphogenetic protein (BMPs), transforming growth factor-β(TGF2-β), cadherin proteins and so on. BMPs has a very important role in the process through signal transduction. BMPs belongs to transforming growth factor-β(TGF-β) superfamily, and can promote bone formation. The key of the bone formation is stromal stem cells to differentiate into osteoblasts. BMPs has a very important role in the differentiation of MSCs into bone and repair process of bone injury. The systems that were composed of BMPs and its downstream cascade proteins of signal transduction affect the expression of related transcription factors, such as Cbfa1, Osx, Dlx5/Msx2, or influence directly on the mRNA of the osteoblast-specific proteins ALP and OCN, thus, promote osteoblast differentiation and stimulate bone formation. The transduction of BMPs Signaling pathways are it binding first with typeⅡreceptor and typeⅠreceptor; typeⅠreceptor make phosphorylation of Smads; Smads enter the nucleus and interact with transcription factors, affecting the transcription of related proteins. Smads can be divided into receptor-regulated Smads (R-Smads:Smad 1,2,3,5,8 and 9), the common mediator Smad (Co-Smad:Smad4) and inhibitory Smads (I-Smads:Smad6,7). Of which, Smadl, Smad5 and Smad8, probable Smad9 were involved in the signal transduction of BMPs signaling pathway. Smadl and Smad5 related transcription factors have core binding protein A1 (CBFA1), Smad-interacting protein 1 (SIP1), ornithine decarboxylase ant izyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), somatostatin (Ski), anti-proliferation protein (Tob), homeodomain transcription factor -8 (Hoxc-8) and so on. Nevertheless, CBFA1 is involved TGF-βand BMP-2 signal transduction. It interact with Smadl,2,3,5, playing important role in bone formation.ObjectiveWhether ADSCs express BMP signaling pathway related molecules which closely related with the repair of bone damage; when bone injury, whether the expression of BMP receptors and Smads in ADSCs are increased in the process of repairing bone damage. That provide further evidence to the theory of "injury-repair mechanism" of fasciaology.1. To determine whether there are undifferentiated mesenchymal stem cells in nonspecific connective tissue by isolating subcutaneous adipose tissue and culturing cells;2. To verify whether ADSCs express the BMP signaling pathway molecules by RT-PCR;3. To explore the change of the expression of BMP receptors and Smads in autologous ADSCs after bone injury in the repair process. Methods1. To detach the ADSCs from the 2 month old female Wistar rats’inguinal adipose tissue; to cultivate the cells in vitro by the enzyme digestion; to identify whether ADSCs in non-specific fascia connective tissue consistent with the characteristics of MSCs by observing their formation, means of growth, morphological, functional study, flow cytometry and biological markers on the cells’membrane; To judge whether connective tissue contains ADSCs.2. To induce passage 4th ADSCs differentiation into adipogenic and osteogenesis, in order to conform their differentiation potential. After ADSCs were cultured under adipogenic and osteogenic condition, The morphological characterization of the inductive cells was observed via alizarin red and alkaline phosphatase staining for mineralization nodules and oil red O staining for lipid accumulation. Meanwhile, to detect part of the surface antigens of passage 4th ADSCs by flow cytometry so that identify the stem cell characteristics of ADSCs.3.The total RNA are extracted from passage 4th ADSCs for RT-PCR, to detect the expression of mRNA of BMP signaling pathway related molecules.4.30 female Wistar rats were randomly divided into the control group (group A 5 rats), the experimental group (group B 25 rats). group A did not take any treatment, and cut directly the inguinal fat pads for culture ADSCs. group B manufacturing bone defect model. After modeling, we cut the inguinal adipose tissue of the rats for culturing ADSCs at day 1,3,7,14,21 respectively,5 rats each time. The total RNA were extracted from ADSCs of primary culture for 7 days; to detect the expression change of mRNA of BMPRs and Smads by RT-qPCR.Results1. ADSCs can be extracted from the rats’fat tissue. In primary culture, the isolated ADSCs show a fibroblast-like morphology.The two passage cells were adherent and have a spindle-shaped morphology in the days 3rd; the mostly adherent ADSCs presented spindle shape or polygon, cell size uniformity under optical microscope;2. Flow cytometric analysis of surface antigens demonstrated that ADSCs were uniformly expressed CD29, CD90 and CD106, and negative for CD 49d, CD11b and CD45;3. The undifferentiated cells were cultured for 14 days under adipogenic conditions to induced the formation of lipid-filled vesicles that were stained red by oil-red-O staining and were presented characteristics of adipocytes, while the red staining of lipid droplets were not observed in the negative control wells.. Induction of osteogenic differentiation of the cells for 21 days resulted in the deposition of mineralized nodules that were stained red by Alizarin red staining and were presented the characteristics of osteoblasts. negative control wells were not dyed pink calcium nodules;4. RT-PCR results showed that ADSCs can express positively mRNA of the BMP signaling pathway related molecules Bmprla, Bmprlb, Bmpr2; Smadl, Smad5, Smad8;5. RT-qPCR analysis showed that, after stimulation of bone defects, the expression of mRNA of Bmpr1a, Bmpr1b, Bmpr2 and Smad1, Smad5, Smad8 were increased in rat’s autogeneic ADSCs.Conclusion1. Through this experiment, the author consider that there are multipotent stem cell in adult Wister rat’s adipose tissue of the connective tissue. Furthermore, ADSCs can be successfully obtained by the digested-adherent-passage programs. The experimental support are provided to the view that fascia connective tissue of the body play a supporting-storing role for the functional system(stem cell reserve). In other word, ADSCs existed in fascia connective tissue suplly stem cells source to repair body damage.2. ADSCs with the characteristics of mesenchymal stem cells and multipotent differentiation potential, can be induced into the osteogenic and adipogenic differentiation.3. ADSCs can express BMP receptors and BMP signaling pathway related molecules, bone injury can induce increase of expression of BMP signaling pathway molecule in ADSCs In summary, this study investigate ADSCs from morphological features, adipogenic and osteogenic differentiation, surface markers etc., by using cells isolation and culture, flow cytometry, adipogenic, osteogenic induction culture and detection techniques. The experiment conform there are MSCs in adult rat’s connective tissue and show fascia connective tissue distributed all over the body is probably the inherent pool of stem cell. The stem cells are easy isolation and culture, and have a good proliferation and differentiation capabilities. We consider ADSCs are main reserve stype of MSCs. more importantly, the fascia connective tissue plays the role of support and reserve, and supply stem cells to repair injury.The experiment vetify not only ADSCs express positively BMP signaling pathway related molecules related closely with the repair of bone injury, but also the expression of BMP signaling pathway related molecules are raised in rats bone defect model. The results suggest, after bone injury, ADSCs may be involved in repairing damage. The organism may mobilize the inherent pool of stem cell to provide stem cells to repairing damage. The experiment validate partly "the damage repairing mechanisms" of fasciology.更多还原