本文選題：骨髓間充質(zhì)干細(xì)胞 + 腦源性神經(jīng)營養(yǎng)因子��； 參考：《中南大學(xué)》2010年博士論文

【摘要】： 骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cell, MSCs)是存在于骨髓中由中胚層發(fā)育的一類非造血干細(xì)胞,具有多向分化潛能,不僅能分化為造血細(xì)胞,還能分化成多種造血以外的組織細(xì)胞,特別是中胚層和神經(jīng)外胚層來源的組織細(xì)胞。MSCs在多個領(lǐng)域,如細(xì)胞治療、組織工程、發(fā)育生物學(xué)、基因功能研究、藥物篩選等領(lǐng)域展示了巨大的應(yīng)用前景。 目的：了解大鼠骨髓間質(zhì)干細(xì)胞體外培養(yǎng)特性,探討MSCs的體外分離、培養(yǎng)、擴(kuò)增的規(guī)律和特點(diǎn)。建立SD大鼠骨髓間充質(zhì)干細(xì)胞體外培養(yǎng)體系。 方法：1.原代培養(yǎng),取一月齡SD大鼠股骨和脛骨,采用全骨髓培養(yǎng)法,以L-DMEM完全培養(yǎng)基沖出骨髓過濾后直接接種于培養(yǎng)瓶中,3天后首次換液,以后每3天換液一次。至細(xì)胞融合達(dá)80%以上時,進(jìn)行傳代。2.大鼠骨髓間質(zhì)干細(xì)胞體外擴(kuò)增的研究,取第1、4、7、10、13代細(xì)胞,觀察其細(xì)胞生長曲線及貼壁率。3.大鼠骨髓間質(zhì)干細(xì)胞表面標(biāo)志的鑒定,取第4代細(xì)胞,采用流式細(xì)胞儀檢測。 結(jié)果：1.貼壁篩選法,通過多次換液可以分離、純化MSCs,該法簡單、方便、效果好。2.MSCs在體外培養(yǎng)中有很強(qiáng)的增殖能力,10代以前的細(xì)胞形態(tài)較一致,增殖能力強(qiáng),隨傳代次數(shù)的增加,增殖能力減弱,形態(tài)發(fā)生不規(guī)則變化。細(xì)胞生長的第1、4、7、10、13代的貼壁率變化規(guī)律基本相同,無顯著區(qū)別。傳代后細(xì)胞貼壁率隨時間延長而增高。3.MSCs的表面標(biāo)志抗原呈現(xiàn)多樣性的特征,MSCs不表達(dá)造血細(xì)胞表面抗原CD34、CD45,說明MSCs非造血類細(xì)胞；而其表達(dá)CD90、CD44,說明MSCs的表面標(biāo)志具有非單一性的特性。它表達(dá)了間質(zhì)細(xì)胞、內(nèi)皮細(xì)胞和表皮細(xì)胞的表面標(biāo)志。結(jié)論：本實(shí)驗采用骨髓直接培養(yǎng),貼壁篩選法來分離培養(yǎng)MSCs,方法簡單,效果亦可靠,通過數(shù)代的傳代擴(kuò)增就能獲得高純度、高數(shù)量的MSCs,以滿足組織工程實(shí)驗研究的需要。 目的：1.構(gòu)建pEGFP-N1-BDNF質(zhì)粒。2.獲得到穩(wěn)定表達(dá)BDNF的MSCs,并轉(zhuǎn)染大鼠骨髓間質(zhì)干細(xì)胞(MSCs),觀察其在MSCs內(nèi)的表達(dá)。 方法：用RT-PCR法從大鼠骨髓間充質(zhì)干細(xì)胞中擴(kuò)增出帶有XhoⅠ、BamH工酶切位點(diǎn)的BDNF基因片段,將BDNF基因片段克隆到增強(qiáng)型綠色熒光蛋白(EGFP)基因真核表達(dá)載體pEGFP-N1上,構(gòu)建pEGFP-N1-BDNF質(zhì)粒。通過電轉(zhuǎn)染將pEGFP-N1-BDNF轉(zhuǎn)染入大鼠MSCs。 結(jié)果：pEGFP-N1-BDNF真核表達(dá)載體構(gòu)建成功,熒光顯微鏡下觀察,pEGFP-N1-BDNF表達(dá)載體轉(zhuǎn)染到MSCsl2h后可見BDNF融合蛋白表達(dá)。 結(jié)論：1.成功的構(gòu)建pEGFP-N1-BDNF質(zhì)粒。2.pEGFP-N1-BDNF質(zhì)�？梢赞D(zhuǎn)染真核細(xì)胞HEK293細(xì)胞,證明BDNF-EGFP融合蛋白真核表達(dá)質(zhì)�？梢栽谡婧思�(xì)胞表達(dá)。3.通過電轉(zhuǎn)染和抗性篩選我們獲得到了穩(wěn)定表達(dá)BDNF的MSCs。 目的：1.采用p-硫基乙醇(p-ME)和堿性成纖維細(xì)胞生長因子(bFGF)作為誘導(dǎo)劑,對骨髓間充質(zhì)干細(xì)胞(MSCs)進(jìn)行體外誘導(dǎo)實(shí)驗。探討β-ME和bFGF體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞分化神經(jīng)元細(xì)胞的機(jī)制。2.探討B(tài)DNF基因修飾對MSCs分化神經(jīng)元細(xì)胞的影響。 方法：1.MSCs向神經(jīng)元細(xì)胞誘導(dǎo)分化,取P4代的MSCs和BDNF-MSCs,分別以2×104/ml濃度接種于24孔板內(nèi),待細(xì)胞達(dá)到70%-80%融合時,分別加入p-硫基乙醇(p-ME)和堿性成纖維細(xì)胞生長因子(bFGF),倒置相差顯微鏡下連續(xù)觀察細(xì)胞形態(tài)變化。2.細(xì)胞免疫熒光鑒定,采用細(xì)胞免疫熒光方法,對以上兩組細(xì)胞誘導(dǎo)后5h后檢測神經(jīng)元標(biāo)志物TuJ1的表達(dá)情況,熒光顯微鏡下觀察并拍照記錄。3.MSCs誘導(dǎo)為神經(jīng)元細(xì)胞陽性率比較,熒光顯微鏡下在兩組細(xì)胞隨機(jī)取300個細(xì)胞,計數(shù)TuJ1反應(yīng)陽性細(xì)胞個數(shù),檢驗各組不同時間點(diǎn)的差異,以及每個時間點(diǎn)各組間的差異。 結(jié)果：1.第4代MSCs加入誘導(dǎo)劑后可分化為神經(jīng)元細(xì)胞和星形膠質(zhì)細(xì)胞�？梢姽庀掠^察：前者細(xì)胞體積較小數(shù)量較少,常有數(shù)個短小突起和一個較長的突起,呈典型的雙極、多極或錐形,具有較強(qiáng)的折光性；后者細(xì)胞體積較大,細(xì)胞突起較多且粗,有些形態(tài)呈星狀,有許多突起呈放射狀伸出,平鋪在培養(yǎng)皿上。2.細(xì)胞免疫熒光結(jié)果示細(xì)胞誘導(dǎo)后5h,部分細(xì)胞TuJ1反應(yīng)陽性,為神經(jīng)元細(xì)胞。3.兩組MSCs誘導(dǎo)為神經(jīng)元細(xì)胞陽性率比較,BDNF-MSCs組分化為神經(jīng)元細(xì)胞的陽性率高于MSCs組,并具有統(tǒng)計學(xué)意義。 結(jié)論：1.在體外MSCs在誘導(dǎo)劑bFGF和β-ME的作用下可分化為神經(jīng)元樣細(xì)胞。2.經(jīng)過BDNF基因修飾,可以顯著提高M(jìn)SCs向神經(jīng)元細(xì)胞分化的陽性率。
[Abstract]:Mesenchymal stem cell (MSCs) is a kind of non hematopoietic stem cells developed from mesoderm in bone marrow. It has multiple differentiation potential. It can not only differentiate into hematopoietic cells, but also differentiate into a variety of hematopoietic tissue cells, especially the tissue cells derived from the mesoembryonic and neuroectoderm cells,.MSCs in a number of collar. Domains, such as cell therapy, tissue engineering, developmental biology, gene function research, drug screening and other fields, have shown great application prospects.
Objective: To investigate the culture characteristics of rat bone marrow mesenchymal stem cells in vitro, and to explore the laws and characteristics of the isolation, culture and amplification of MSCs in vitro. The culture system of bone marrow mesenchymal stem cells (MSCs) in SD rats was established.
Methods: 1. primary culture, the femur and tibia of 1 month old SD rats were taken, and all bone marrow culture was used in full bone marrow culture. The bone marrow filtration was directly inoculated in the culture bottle with the L-DMEM complete medium. After 3 days, the first liquid was changed and then changed every 3 days. When the cell fusion was over 80%, the bone marrow mesenchymal stem cells of.2. rats were amplified in vitro. The cells of the 1,4,7,10,13 generation were taken to observe the cell growth curve and the identification of the surface markers of the bone marrow mesenchymal stem cells in.3. rats. The fourth generation cells were taken and the flow cytometry was used to detect the cells.
Results: the 1. adherent wall screening method can be separated and purified by multiple fluid exchange. The method is simple and convenient, and the effect is good..2.MSCs has strong proliferation ability in the culture in vitro. The cell morphology is more consistent and the proliferating ability is strong before the 10 generation, with the increase of the number of passages, the proliferation ability is weakened, and the morphogenesis is irregular. 1,4,7,10, the cell growth of the cell, is 1,4,7,10, The adherence rate of the 13 generation was basically the same, and there was no significant difference. After the passage, the cell adhesion rate increased with time and increased the surface marker antigen of.3.MSCs. MSCs did not express the hematopoietic cell surface antigen CD34, CD45, indicating that MSCs was not hematopoietic cells; and its surface was CD90, CD44, indicating that the surface markers of MSCs were not single. It expresses the surface markers of interstitial cells, endothelial cells and epidermal cells. Conclusion: this experiment uses bone marrow direct culture and wall screening to isolate and culture MSCs. The method is simple, and the effect is reliable. High purity and high quantity of MSCs can be obtained through generation of passages to meet the needs of tissue engineering experimental research.
Objective: 1. to construct pEGFP-N1-BDNF plasmid.2. and obtain stable MSCs expressing BDNF, and transfect rat bone marrow mesenchymal stem cells (MSCs) to observe its expression in MSCs.
Methods: the BDNF gene fragment with Xho I and BamH was amplified from rat bone marrow mesenchymal stem cells by RT-PCR method, and the BDNF gene fragment was cloned into the eukaryotic expression vector pEGFP-N1 of the enhanced green fluorescent protein (EGFP) gene, and the pEGFP-N1-BDNF plasmid was constructed. PEGFP-N1-BDNF was transfected into MSCs. by transfection of pEGFP-N1-BDNF into rat MSCs..
Results: pEGFP-N1-BDNF eukaryotic expression vector was constructed successfully, under fluorescence microscope, the expression of BDNF fusion protein was found after transfection of pEGFP-N1-BDNF expression vector to MSCsl2h.
Conclusion: 1. the successful construction of pEGFP-N1-BDNF plasmid.2.pEGFP-N1-BDNF plasmid can transfect the eukaryotic HEK293 cells. It is proved that the eukaryotic expression plasmid of BDNF-EGFP fusion protein can be expressed in eukaryotic cells by electric transfection and resistance screening, and we have obtained the MSCs. of the stable expression of BDNF.
Objective: 1. using p- sulfur based ethanol (p-ME) and basic fibroblast growth factor (bFGF) as an inducer, bone marrow mesenchymal stem cells (MSCs) were induced in vitro. The mechanism of beta -ME and bFGF in inducing the differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro.2. to explore the effect of BDNF gene modification on MSCs differentiated neuron cells.
Methods: 1.MSCs induced differentiation to neuron cells, took P4 generation MSCs and BDNF-MSCs, and inoculated into 24 orifice plates at 2 x 104/ml concentration respectively. When the cells reached 70%-80% fusion, p- sulfur based ethanol (p-ME) and basic fibroblast growth factor (bFGF) were added to the cell, and the cell morphological changes of.2. cell immunofluorescence were continuously observed under the inverted phase difference microscope. A cell immunofluorescence method was used to detect the expression of the neuron marker TuJ1 after 5h induction in the above two groups. The positive rate of the neuron cells was observed under the fluorescence microscope and recorded by.3.MSCs. Under the fluorescence microscope, 300 cells were taken randomly in two groups, and the number of TuJ1 positive cells was counted, and the number of the positive cells was counted. The differences between groups at different time points were examined, and the differences among groups at each time point were also examined.
Results: 1. the fourth generation of MSCs can differentiate into neuronal cells and astrocytes after adding the inducer. It is observed under the light of light that the former cells are smaller in volume, often have several short protuberances and a long protuberance, and are typical bipolar, multipolar or cone-shaped, with a strong refraction; the latter is larger in volume and more protruding than in the former. The.2. cell immunofluorescence results showed that 5h was induced by cell immunofluorescence on the Petri dish, and the TuJ1 reaction in some cells was positive. The positive rate of MSCs in the.3. two group of neuron cells was compared with the positive rate of neuron cells, and the positive rate of the BDNF-MSCs group to differentiate into neuron cells was higher than that of the MSCs group. And it has statistical significance.
Conclusion: 1. in vitro, MSCs can differentiate into neuron like cells.2. after BDNF gene modification under the action of inducer bFGF and beta -ME, which can significantly increase the positive rate of MSCs to neuronal cell differentiation.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329

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8 周東浩;血清BDNF水平及BDNF單核苷酸多態(tài)性與2型糖尿病的相關(guān)性研究[D];河北醫(yī)科大學(xué);2010年

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10 譚志強(qiáng);BDNF基因修飾骨髓間充質(zhì)干細(xì)胞的生物學(xué)特性及對豚鼠螺旋神經(jīng)元的保護(hù)作用[D];中南大學(xué);2010年

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5 黃月君;母鼠妊娠前慢性應(yīng)激對子代學(xué)習(xí)能力及海馬BDNF、NMDA受體表達(dá)的影響[D];汕頭大學(xué);2010年

6 王保寧;腺病毒介導(dǎo)的CTLA4Ig基因?qū)餐瑢?dǎo)入的BDNF基因在大鼠脊髓表達(dá)的影響[D];河北醫(yī)科大學(xué);2010年

7 樓丹丹;重度抑郁癥患者BDNF基因多態(tài)性與無抽搐電休克治療的相關(guān)性研究[D];重慶醫(yī)科大學(xué);2010年

8 張鈺;D-環(huán)絲氨酸對條件性恐懼消退訓(xùn)練大鼠行為及海馬BDNF及其受體TrkB的影響研究[D];第三軍醫(yī)大學(xué);2011年

9 張鳳久;BDNF基因修飾施萬細(xì)胞結(jié)合組織工程技術(shù)治療周圍神經(jīng)損傷的研究[D];內(nèi)蒙古科技大學(xué)包頭醫(yī)學(xué)院;2010年

10 劉莎;抑郁障礙BDNF系統(tǒng)藥物基因組學(xué)研究[D];山西醫(yī)科大學(xué);2010年

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