脂肪组织源性干细胞分步诱导分化为神经元样细胞

发布时间：2018-01-02 08:22

  本文关键词：脂肪组织源性干细胞分步诱导分化为神经元样细胞　出处：《南方医科大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 脂肪组织源性干细胞 分化 神经干细胞 神经元

【摘要】：背景：对于中枢神经系统疾病的治疗,以往主要是以药物控制为主,随着组织工程的研究进展,干细胞移植带来了新的希望。迄今,神经干细胞移植恢复神经系统的正常功能,已取得了令人鼓舞的效果。神经干细胞(Neural stem cells, NSCs)主要分布在海马、大脑皮质、小脑和脊髓等多个部位,其发现、分离和培养为神经系统的疾病的治疗提供了可能,但种子细胞的获取受到限制。随着成体干细胞如骨髓基质干细胞(bone marrow stromal cells, BMSCs)在体外分离、培养及多向诱导分化的成功,有大量关于研究BMSCs向神经元样细胞分化的报道,结果都显示BMSCs可在体、内外分化为神经元样细胞,因此有望成为治疗神经性疾病细胞替代疗法的种子细胞。但是,在临床应用中,BMSCs来源差、干细胞丰度低、有疼痛、创伤、感染等危险因素,因此应用受到一定的限制。脂肪组织源性干细胞(adipose tissue-derived stem cells, ADSCs)是抽吸的脂肪组织经分离培养得到的贴壁的成纤维样细胞克隆,也是一种成体干细胞,与BMSCs一样来源于中胚层,表面抗原与骨髓BMSCs相似,且在在体外的培养条件、生长状态和表达标志物也与BMSCs基本相同,而且还具有来源丰富、取材方便、干细胞丰度高、损伤小等优点,更具有临床价值。 Zuk等研究发现,ADSCs在一定的诱导条件下可向神经元样细胞分化,早期分化的细胞表达巢蛋白(Nesin)和神经元特异性烯醇化酶(NSE),晚期则表达神经微丝(neurofilament, NF)。分化的细胞形态和表达相应的标志物证明这种分化的细胞是神经元。早先报道成体干细胞跨胚层分化为神经元样细胞(neuron-like cells,NLCs)的方法是由Woodbury提出的经典化学诱导法,即用化学试剂作为诱导剂诱导BMSCs向神经元样细胞诱导分化,之后被Deng和Safford证实。但经诱导分化形成的神经元样细胞的形态变化有可能是由细胞毒性引起的细胞骨架变化,而因子直接诱导法是用因子作为诱导剂的另一种诱导方法,虽然避开了DMSO的细胞毒性,但诱导分化率不高。2004年报道大鼠的BMSCs在含有B27,表皮生长因子(EGF),碱性成纤维细胞生长因子(bFGF)的DMEM/F12中可以形成神经球样结构,这种神经球表达神经干细胞的标志物神经Nestin,且能向神经元和胶质细胞方向分化,因此称神经干细胞样细胞。 目前的研究显示,ADSCs的分化受多种因素调控,细胞外环境信号与细胞内基因的有序表达调控着ADSCs的分化。细胞外信号主要包括细胞外基质、相互作用的细胞、信号转导和细胞因子等,对细胞的营养、增殖和分化调控以及神经元细胞的髓鞘形成具有重要作用。目前比较公认的是细胞因子对其分化有调控作用,不同细胞因子可以调控其向不同的方向分化,细胞因子可以由全身血液运输扩散而来,也可以由脂肪组织源性干细胞自身分泌而产生,还可以产生自细胞外基质,细胞因子之间的相互作用尚不很清楚,研究方法主要为体外实验观察细胞因子对脂肪组织源性干细胞分化的调控作用。细胞分化的基因调控是近几年提出的一种新思路,但在这方面的研究还不是很深入。 本研究分为两部分,第一部分工作是对大鼠脂肪干细胞的分离培养,从大鼠附睾部位取材,分离培养的细胞用差速贴壁法达到纯化的目的,对分离纯化的ADSCs进行形态鉴定、成脂诱导鉴定以及流式细胞化学鉴定。第二部分是将分离得到的ADSCs向神经元样细胞诱导分化,本实验主要采取用因子诱导细胞分化的方法,在参考以往用因子诱导分化的方法基础上选择合适的因子以及摸索适合的浓度诱导ADSCs向神经元样细胞诱导分化,细胞的鉴定从细胞的形态和神经元标志物两方面进行。 第一部分大鼠脂肪组织源性干细胞的培养与鉴定 目的：本研究拟从大鼠脂肪组织中分离、培养并鉴定脂肪组织源性干细胞(ADSCs),为下一步诱导分化实验做准备。 方法：1、ADSCs的分离、纯化及传代：取成年120-150g SD雄性大鼠,严格无菌操作分离附睾尾部脂肪垫,小心剔除血管剪碎后用0.15%的Ⅰ型胶原酶于37℃下震荡消化45min；终止消化后1000rpm离心10 min,弃掉上层悬浮的脂肪和中层的培养液,用3 ml含有10%FBS的DMEN/F12培养基重悬下层的细胞并吹打均匀,将细胞悬液用筛网过滤后接种于6 cm的培养皿中,置5%CO2孵箱中培养。细胞培养4h后,有少部分细胞贴壁,将悬浮的细胞重新接种到新的培养皿中继续培养以去除已贴壁的细胞,连续两次；每隔两天给细胞换液一次,待贴壁细胞达到80%-90%融合时按1：3传代,取第4代ADSCs做下一步实验。 2、ADSCs的成脂诱导和脂肪鉴定：取第4代ADSCs,加入含有1μmol/L地塞米松、10 mg/L胰岛素的DMEM/F12成脂诱导培养基,置5%C02、37℃培养箱中培养,每隔两天换液一次；成脂诱导7-10 d后做油红O染色检测脂肪细胞。 3、ADSCs流式细胞术鉴定：采用流式细胞仪,用直接免疫荧光标记检测细胞表面分子,以CD45、CD90、CD29、CD11b、CD106、CD49d为一抗检第4代ADSCs的表达情况。 结果：从大鼠尾部脂肪垫分离的少部分细胞贴壁较快,4h后重新接种细胞去除这部分贴壁细胞后得到的的纯化细胞形态不一,呈短梭型、三角形和扁平状。细胞可以连续传至20代以上,并且增殖稳定,细胞形态更趋向一致,多为长梭型。ADSCs成脂诱导后倒置显微镜下可看到大小不等脂滴,油红O染色阳性。流式细胞术免疫荧光细胞化学鉴定CD106阳性表达率为28.48%、CD45阳性表达率0.45%、D1lb阳性表达率为0.41%、CD49d阳性表达率为0.41%、CD29阳性表达率为98.96%,、CD90阳性表达率82.53%。 结论：可以从大鼠脂肪组织中用酶消化法分离得到ADSCs,细胞在体外能稳定传代,并具有很强的扩增能力,经成脂诱导和流式细胞术鉴定,ADSCs具有干细胞的特征,能继续做下一步诱导实验。 第二部分大鼠脂肪组织源性干细胞向神经元样细胞的诱导分化 目的：探讨分离纯化的ADSCs在体外诱导分化为神经元样细胞的可能性。 方法： 1、ADSCs向NSCs的诱导分化：用含有2%B27、10 ng/ml bFGF、20 ng/mlEGF的DMEM/F12重悬第4代ADSCs,按1×105/ml接种到24孔板中,每天半量换液一次,每3d加入新的因子,待有克隆细胞球形成后进行下一步。 2、NSCs的传代培养：待ADSCs向神经干细胞诱导分化形成的神经球(neurosphere)达20-25个后用2ml TrypLETM消化配合机械吹打使神经球分散成单个细胞,之后500 rmp离心5min,用含2% B27、10 ng/ml bFGF、20 ng/ml EGF的DMEM/F12细胞培养液重悬细胞,置5%CO培养箱中培养。 3、神经干细胞样细胞(NSC-like cells)向神经元的诱导分化：将悬浮的克隆细胞球接种到预先铺有多聚赖氨酸盖玻片的24孔板中,待细胞贴壁后弃掉原有培养基,换为含有10 ng/ml GDNF、10ng/ml BDNF、1μmol/L RA的DMEM/F12培养基中,1d后换成含有10 ng/ml GDNF、10ng/ml BDNF、1μmol/L RA、2% B27的Neurobasal培养基,每3d换液一次。 4、诱导后的NSC-like cells和NLCs的表型鉴定：用免疫荧光检测法检测NSC-like cells的Nestin表达及neuron-like cells的Nestin、MAP2、NeuN和β-tubμlinⅢ的表达情况。 结果：ADSCs高密度(105/ml)接种后,部分细胞悬浮生长,3d后悬浮的单个细胞抱成小球,半量换液后小球逐渐变大,显微镜下观察细胞球的折光度高,尤其是周边的折光度最高,有绒状突起,细胞球吹打均匀后离心传代后3天又可成球,可连续传到第五代,Nestin表达阳性。NSC-like cells向NLCs诱导分化后神经球贴壁生长,倒置显微镜下观察,有少部分细胞由中央向周边迁出,细胞胞体折光性较强,少数细胞周边有2-3个较短的突起,细胞胞体立体感强,类似于神经元的形态,低倍镜下观察类神经元样细胞排列交织成网。Nestin、MAP2、NeuN和β-tubμlinⅢ表达都阳性。 结论：通过本实验分离纯化的ADSCs在体外可以通过分步诱导法,用细胞因子作为诱导剂向神经元样细胞诱导分化。第一步向神经干细胞样细胞诱导分化后的细胞Nestin高表达,说明诱导出的细胞的确具有神经干细胞的特征；.第二步继续向神经元样细胞诱导分化后Nestin仍有少许表达,但表达量降低,而MAP2、NeuN和β-tubμlinⅢ表达都阳性,说明诱导后的细胞的确具有神经元样细胞的特征,因此说明ADSCs具有一定的可塑性,在体外有可能分化为神经元。
[Abstract]:Background: for treatment of diseases of the central nervous system, the past is mainly to drug control, with the development of tissue engineering, stem cell transplantation has brought new hope. So far, the normal function of neural stem cell transplantation on recovery of nerve system, has achieved encouraging results. The neural stem cells (Neural stem cells, NSCs) is mainly distributed in the hippocampus, cerebral cortex, cerebellum and spinal cord and other parts, it found that provides a possible treatment for isolation and culture of nerve system disease, but the seed cells get restricted. With adult stem cells such as bone marrow stromal stem cells (bone marrow stromal cells, BMSCs) in vitro. Culture and multi-directional differentiation, there are a lot of research on BMSCs differentiation into neuron like cells, results showed that BMSCs in vivo, and differentiated into neuron like cells, so it is expected to be Seed cells for the treatment of neurodegenerative diseases for cell replacement therapy. However, in clinical application, BMSCs source, stem cells have low abundance, pain, trauma, infection and other risk factors, so it limits the application of adipose tissue derived stem cells. (adipose tissue-derived stem cells, ADSCs) is the suction of adipose tissue by cultured fibroblast like cell clones obtained by adherent, is also a kind of adult stem cells and BMSCs derived from the mesoderm, surface antigen and bone marrow was similar to that of BMSCs, and in vitro culture conditions, growth status and expression of markers are also the same as BMSCs, but also has rich resources, convenient. Stem cell abundance is high, has the advantages of little injury, has more clinical value.
Zuk found that ADSCs can differentiate into neuron like cells under induction conditions, early differentiation (Nesin) cells expressed nestin and neuron specific enolase (NSE), the late expression of neurofilament (neurofilament, NF). The cell morphology and expression of differentiation markers proved that the corresponding differentiation cells are neurons. Earlier reports of adult stem cell differentiation into neuron like cells (neuron-like, cells, NLCs) induced by classical chemical method is put forward by Woodbury, which uses chemical reagents to induce BMSCs differentiation into neuron like cells induced by Deng and Safford, after being confirmed. But the morphological changes of neuron like cell differentiation may be the formation of cytoskeleton changes caused by cell toxicity, and direct factor method is used as the induction factor inducing another induction method, while avoiding The cytotoxicity of DMSO, but the rate of differentiation is not high.2004 reports of BMSCs in rats with B27, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) DMEM/F12 could form neurosphere like structures, the neurospheres express neural stem cell markers of neural Nestin, and can differentiate into neurons and glial cells, so that neural stem cell like cells.
The present study shows that differentiation is regulated by a variety of factors of ADSCs signal and the extracellular environment in order to regulate the expression of ADSCs gene differentiation. Extracellular signal includes extracellular matrix, cell interaction, signal transduction and cell factor, nutrition for cells, proliferation and differentiation of neuronal cells and myelin regulation the formation plays an important role. The most accepted is cytokines on the differentiation regulation of different cytokines can regulate the differentiation in different directions, cytokines can by systemic blood transport and diffusion, can also be produced by adipose tissue derived stem cells secreted, can also produce extracellular self matrix interactions between cytokines is not clear, the main research methods for in vitro study of cytokines on the differentiation of adipose tissue derived stem cell regulation. The gene regulation of cell differentiation is a new idea in recent years, but the research in this field is not very deep.
This study is divided into two parts, the first part is on isolation and culture of rat adipose derived stem cells, derived from rat epididymis parts, cells with differential adhesion method to achieve the purpose of purification culture and morphological identification of purified ADSCs, adipogenic identification and chemical identification of flow cytometry. Second is part of the isolated ADSCs differentiation into neuron cells, this experiment mainly adopts the method of cell differentiation factor induced differentiation factor, based on the reference of previous on the selection of suitable factors and find suitable concentration to induce ADSCs differentiation into neuron like cells, cells identified by cell morphology and neuronal markers in two aspects.
The first part of the culture and identification of adipose tissue derived stem cells in rats
Objective: to isolate and identify the adipose tissue derived stem cells (ADSCs) from rat adipose tissue and prepare for the next induction of differentiation.
Methods: 1 ADSCs were isolated, purified and passaged: adult 120-150g male SD rats, strict aseptic operation separation of epididymal fat pad, carefully cut out blood vessels with type 0.15% to 37 DEG C shock digestion digestion was terminated after 1000rpm 45min; 10 min centrifugal liquid culture, discard the upper suspension the fat and the middle, with 3 ml 10%FBS containing DMEN/F12 medium weight hanging lower cells and army uniform, the cell suspensions by sieve were inoculated in 6 cm culture dish and cultured in 5%CO2 incubator. After 4H cell culture, a small part of adherent cells will re suspended cells to continue to develop in order to remove the inoculated adherent cell culture dishes in the new, two consecutive times; every two days to the cells was changed once, the adherent cells reached 80%-90% fusion 1:3 according to the passage, the fourth generation of ADSCs to do the next experiment.
2, ADSCs adipogenic and fat identification: the fourth generation of ADSCs, with 1 mol/L dexamethasone, 10 mg/L insulin DMEM/F12 adipogenic induction medium, 5%C02,37 deg.c incubator, every two days for a liquid; adipogenic 7-10 d after oil red O staining, fat cell.
3, ADSCs flow cytometry identification: flow cytometry and direct immunofluorescence labeling were used to detect cell surface molecules. The expression of fourth generation ADSCs was detected by CD45, CD90, CD29, CD11b, CD106 and CD49d.
Results: a few cells isolated from rat tail fat pad adherent quickly again after 4H cells were inoculated to remove this part of adherent cells obtained after purification of cell morphology, were short spindle shaped, triangular and flat cells. Can be continuously passaged over 20 times, and the proliferation of the cells were more stable. Consistency, were long spindle type.ADSCs can see different size of lipid droplets after fat induced under the inverted microscope. Oil red O staining. Flow cytometry and immunofluorescence staining were positive expression rate of CD106 was 28.48%, CD45 positive rate was 0.45%, the positive expression rate of D1lb was 0.41%, the positive expression rate of CD49d was 0.41% CD29, the positive rate was 98.96%, and the positive expression rate of CD90 82.53%.
Conclusion: ADSCs can be isolated from rat adipose tissue by enzyme digestion. The cells can be passaged steadily in vitro, and have strong amplification ability. After identification of adipogenesis and flow cytometry, ADSCs has the characteristics of stem cells, and can continue to do further induction experiments.
Induction and differentiation of adipose derived stem cells from rats to neuron like cells in the second part
Objective: To investigate the possibility of isolation and purification of ADSCs to induce differentiation into neuron like cells in vitro.
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