本文關(guān)鍵詞：脂肪組織源性干細(xì)胞分步誘導(dǎo)分化為神經(jīng)元樣細(xì)胞　出處：《南方醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 脂肪組織源性干細(xì)胞 分化 神經(jīng)干細(xì)胞 神經(jīng)元

【摘要】：背景：對于中樞神經(jīng)系統(tǒng)疾病的治療,以往主要是以藥物控制為主,隨著組織工程的研究進(jìn)展,干細(xì)胞移植帶來了新的希望。迄今,神經(jīng)干細(xì)胞移植恢復(fù)神經(jīng)系統(tǒng)的正常功能,已取得了令人鼓舞的效果。神經(jīng)干細(xì)胞(Neural stem cells, NSCs)主要分布在海馬、大腦皮質(zhì)、小腦和脊髓等多個部位,其發(fā)現(xiàn)、分離和培養(yǎng)為神經(jīng)系統(tǒng)的疾病的治療提供了可能,但種子細(xì)胞的獲取受到限制。隨著成體干細(xì)胞如骨髓基質(zhì)干細(xì)胞(bone marrow stromal cells, BMSCs)在體外分離、培養(yǎng)及多向誘導(dǎo)分化的成功,有大量關(guān)于研究BMSCs向神經(jīng)元樣細(xì)胞分化的報道,結(jié)果都顯示BMSCs可在體、內(nèi)外分化為神經(jīng)元樣細(xì)胞,因此有望成為治療神經(jīng)性疾病細(xì)胞替代療法的種子細(xì)胞。但是,在臨床應(yīng)用中,BMSCs來源差、干細(xì)胞豐度低、有疼痛、創(chuàng)傷、感染等危險因素,因此應(yīng)用受到一定的限制。脂肪組織源性干細(xì)胞(adipose tissue-derived stem cells, ADSCs)是抽吸的脂肪組織經(jīng)分離培養(yǎng)得到的貼壁的成纖維樣細(xì)胞克隆,也是一種成體干細(xì)胞,與BMSCs一樣來源于中胚層,表面抗原與骨髓BMSCs相似,且在在體外的培養(yǎng)條件、生長狀態(tài)和表達(dá)標(biāo)志物也與BMSCs基本相同,而且還具有來源豐富、取材方便、干細(xì)胞豐度高、損傷小等優(yōu)點,更具有臨床價值。 Zuk等研究發(fā)現(xiàn),ADSCs在一定的誘導(dǎo)條件下可向神經(jīng)元樣細(xì)胞分化,早期分化的細(xì)胞表達(dá)巢蛋白(Nesin)和神經(jīng)元特異性烯醇化酶(NSE),晚期則表達(dá)神經(jīng)微絲(neurofilament, NF)。分化的細(xì)胞形態(tài)和表達(dá)相應(yīng)的標(biāo)志物證明這種分化的細(xì)胞是神經(jīng)元。早先報道成體干細(xì)胞跨胚層分化為神經(jīng)元樣細(xì)胞(neuron-like cells,NLCs)的方法是由Woodbury提出的經(jīng)典化學(xué)誘導(dǎo)法,即用化學(xué)試劑作為誘導(dǎo)劑誘導(dǎo)BMSCs向神經(jīng)元樣細(xì)胞誘導(dǎo)分化,之后被Deng和Safford證實。但經(jīng)誘導(dǎo)分化形成的神經(jīng)元樣細(xì)胞的形態(tài)變化有可能是由細(xì)胞毒性引起的細(xì)胞骨架變化,而因子直接誘導(dǎo)法是用因子作為誘導(dǎo)劑的另一種誘導(dǎo)方法,雖然避開了DMSO的細(xì)胞毒性,但誘導(dǎo)分化率不高。2004年報道大鼠的BMSCs在含有B27,表皮生長因子(EGF),堿性成纖維細(xì)胞生長因子(bFGF)的DMEM/F12中可以形成神經(jīng)球樣結(jié)構(gòu),這種神經(jīng)球表達(dá)神經(jīng)干細(xì)胞的標(biāo)志物神經(jīng)Nestin,且能向神經(jīng)元和膠質(zhì)細(xì)胞方向分化,因此稱神經(jīng)干細(xì)胞樣細(xì)胞。 目前的研究顯示,ADSCs的分化受多種因素調(diào)控,細(xì)胞外環(huán)境信號與細(xì)胞內(nèi)基因的有序表達(dá)調(diào)控著ADSCs的分化。細(xì)胞外信號主要包括細(xì)胞外基質(zhì)、相互作用的細(xì)胞、信號轉(zhuǎn)導(dǎo)和細(xì)胞因子等,對細(xì)胞的營養(yǎng)、增殖和分化調(diào)控以及神經(jīng)元細(xì)胞的髓鞘形成具有重要作用。目前比較公認(rèn)的是細(xì)胞因子對其分化有調(diào)控作用,不同細(xì)胞因子可以調(diào)控其向不同的方向分化,細(xì)胞因子可以由全身血液運輸擴(kuò)散而來,也可以由脂肪組織源性干細(xì)胞自身分泌而產(chǎn)生,還可以產(chǎn)生自細(xì)胞外基質(zhì),細(xì)胞因子之間的相互作用尚不很清楚,研究方法主要為體外實驗觀察細(xì)胞因子對脂肪組織源性干細(xì)胞分化的調(diào)控作用。細(xì)胞分化的基因調(diào)控是近幾年提出的一種新思路,但在這方面的研究還不是很深入。 本研究分為兩部分,第一部分工作是對大鼠脂肪干細(xì)胞的分離培養(yǎng),從大鼠附睪部位取材,分離培養(yǎng)的細(xì)胞用差速貼壁法達(dá)到純化的目的,對分離純化的ADSCs進(jìn)行形態(tài)鑒定、成脂誘導(dǎo)鑒定以及流式細(xì)胞化學(xué)鑒定。第二部分是將分離得到的ADSCs向神經(jīng)元樣細(xì)胞誘導(dǎo)分化,本實驗主要采取用因子誘導(dǎo)細(xì)胞分化的方法,在參考以往用因子誘導(dǎo)分化的方法基礎(chǔ)上選擇合適的因子以及摸索適合的濃度誘導(dǎo)ADSCs向神經(jīng)元樣細(xì)胞誘導(dǎo)分化,細(xì)胞的鑒定從細(xì)胞的形態(tài)和神經(jīng)元標(biāo)志物兩方面進(jìn)行。 第一部分大鼠脂肪組織源性干細(xì)胞的培養(yǎng)與鑒定 目的：本研究擬從大鼠脂肪組織中分離、培養(yǎng)并鑒定脂肪組織源性干細(xì)胞(ADSCs),為下一步誘導(dǎo)分化實驗做準(zhǔn)備。 方法：1、ADSCs的分離、純化及傳代：取成年120-150g SD雄性大鼠,嚴(yán)格無菌操作分離附睪尾部脂肪墊,小心剔除血管剪碎后用0.15%的Ⅰ型膠原酶于37℃下震蕩消化45min；終止消化后1000rpm離心10 min,棄掉上層懸浮的脂肪和中層的培養(yǎng)液,用3 ml含有10%FBS的DMEN/F12培養(yǎng)基重懸下層的細(xì)胞并吹打均勻,將細(xì)胞懸液用篩網(wǎng)過濾后接種于6 cm的培養(yǎng)皿中,置5%CO2孵箱中培養(yǎng)。細(xì)胞培養(yǎng)4h后,有少部分細(xì)胞貼壁,將懸浮的細(xì)胞重新接種到新的培養(yǎng)皿中繼續(xù)培養(yǎng)以去除已貼壁的細(xì)胞,連續(xù)兩次；每隔兩天給細(xì)胞換液一次,待貼壁細(xì)胞達(dá)到80%-90%融合時按1：3傳代,取第4代ADSCs做下一步實驗。 2、ADSCs的成脂誘導(dǎo)和脂肪鑒定：取第4代ADSCs,加入含有1μmol/L地塞米松、10 mg/L胰島素的DMEM/F12成脂誘導(dǎo)培養(yǎng)基,置5%C02、37℃培養(yǎng)箱中培養(yǎng),每隔兩天換液一次；成脂誘導(dǎo)7-10 d后做油紅O染色檢測脂肪細(xì)胞。 3、ADSCs流式細(xì)胞術(shù)鑒定：采用流式細(xì)胞儀,用直接免疫熒光標(biāo)記檢測細(xì)胞表面分子,以CD45、CD90、CD29、CD11b、CD106、CD49d為一抗檢第4代ADSCs的表達(dá)情況。 結(jié)果：從大鼠尾部脂肪墊分離的少部分細(xì)胞貼壁較快,4h后重新接種細(xì)胞去除這部分貼壁細(xì)胞后得到的的純化細(xì)胞形態(tài)不一,呈短梭型、三角形和扁平狀。細(xì)胞可以連續(xù)傳至20代以上,并且增殖穩(wěn)定,細(xì)胞形態(tài)更趨向一致,多為長梭型。ADSCs成脂誘導(dǎo)后倒置顯微鏡下可看到大小不等脂滴,油紅O染色陽性。流式細(xì)胞術(shù)免疫熒光細(xì)胞化學(xué)鑒定CD106陽性表達(dá)率為28.48%、CD45陽性表達(dá)率0.45%、D1lb陽性表達(dá)率為0.41%、CD49d陽性表達(dá)率為0.41%、CD29陽性表達(dá)率為98.96%,、CD90陽性表達(dá)率82.53%。 結(jié)論：可以從大鼠脂肪組織中用酶消化法分離得到ADSCs,細(xì)胞在體外能穩(wěn)定傳代,并具有很強(qiáng)的擴(kuò)增能力,經(jīng)成脂誘導(dǎo)和流式細(xì)胞術(shù)鑒定,ADSCs具有干細(xì)胞的特征,能繼續(xù)做下一步誘導(dǎo)實驗。 第二部分大鼠脂肪組織源性干細(xì)胞向神經(jīng)元樣細(xì)胞的誘導(dǎo)分化 目的：探討分離純化的ADSCs在體外誘導(dǎo)分化為神經(jīng)元樣細(xì)胞的可能性。 方法： 1、ADSCs向NSCs的誘導(dǎo)分化：用含有2%B27、10 ng/ml bFGF、20 ng/mlEGF的DMEM/F12重懸第4代ADSCs,按1×105/ml接種到24孔板中,每天半量換液一次,每3d加入新的因子,待有克隆細(xì)胞球形成后進(jìn)行下一步。 2、NSCs的傳代培養(yǎng)：待ADSCs向神經(jīng)干細(xì)胞誘導(dǎo)分化形成的神經(jīng)球(neurosphere)達(dá)20-25個后用2ml TrypLETM消化配合機(jī)械吹打使神經(jīng)球分散成單個細(xì)胞,之后500 rmp離心5min,用含2% B27、10 ng/ml bFGF、20 ng/ml EGF的DMEM/F12細(xì)胞培養(yǎng)液重懸細(xì)胞,置5%CO培養(yǎng)箱中培養(yǎng)。 3、神經(jīng)干細(xì)胞樣細(xì)胞(NSC-like cells)向神經(jīng)元的誘導(dǎo)分化：將懸浮的克隆細(xì)胞球接種到預(yù)先鋪有多聚賴氨酸蓋玻片的24孔板中,待細(xì)胞貼壁后棄掉原有培養(yǎng)基,換為含有10 ng/ml GDNF、10ng/ml BDNF、1μmol/L RA的DMEM/F12培養(yǎng)基中,1d后換成含有10 ng/ml GDNF、10ng/ml BDNF、1μmol/L RA、2% B27的Neurobasal培養(yǎng)基,每3d換液一次。 4、誘導(dǎo)后的NSC-like cells和NLCs的表型鑒定：用免疫熒光檢測法檢測NSC-like cells的Nestin表達(dá)及neuron-like cells的Nestin、MAP2、NeuN和β-tubμlinⅢ的表達(dá)情況。 結(jié)果：ADSCs高密度(105/ml)接種后,部分細(xì)胞懸浮生長,3d后懸浮的單個細(xì)胞抱成小球,半量換液后小球逐漸變大,顯微鏡下觀察細(xì)胞球的折光度高,尤其是周邊的折光度最高,有絨狀突起,細(xì)胞球吹打均勻后離心傳代后3天又可成球,可連續(xù)傳到第五代,Nestin表達(dá)陽性。NSC-like cells向NLCs誘導(dǎo)分化后神經(jīng)球貼壁生長,倒置顯微鏡下觀察,有少部分細(xì)胞由中央向周邊遷出,細(xì)胞胞體折光性較強(qiáng),少數(shù)細(xì)胞周邊有2-3個較短的突起,細(xì)胞胞體立體感強(qiáng),類似于神經(jīng)元的形態(tài),低倍鏡下觀察類神經(jīng)元樣細(xì)胞排列交織成網(wǎng)。Nestin、MAP2、NeuN和β-tubμlinⅢ表達(dá)都陽性。 結(jié)論：通過本實驗分離純化的ADSCs在體外可以通過分步誘導(dǎo)法,用細(xì)胞因子作為誘導(dǎo)劑向神經(jīng)元樣細(xì)胞誘導(dǎo)分化。第一步向神經(jīng)干細(xì)胞樣細(xì)胞誘導(dǎo)分化后的細(xì)胞Nestin高表達(dá),說明誘導(dǎo)出的細(xì)胞的確具有神經(jīng)干細(xì)胞的特征；.第二步繼續(xù)向神經(jīng)元樣細(xì)胞誘導(dǎo)分化后Nestin仍有少許表達(dá),但表達(dá)量降低,而MAP2、NeuN和β-tubμlinⅢ表達(dá)都陽性,說明誘導(dǎo)后的細(xì)胞的確具有神經(jīng)元樣細(xì)胞的特征,因此說明ADSCs具有一定的可塑性,在體外有可能分化為神經(jīng)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.
Method錛，

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