發(fā)布時(shí)間：2018-01-10 01:12

  本文關(guān)鍵詞：血小板源性生長(zhǎng)因子對(duì)成神經(jīng)分化的間充質(zhì)干細(xì)胞定向遷移研究　出處：《蘇州大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 分化 PDGF Rac1 遷移

【摘要】： 目的目前惡性膠質(zhì)瘤是最為盛行的原發(fā)性腦瘤,傳統(tǒng)的方法,如化療,放療難以從根本上對(duì)其進(jìn)行治療,但由于骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)在體內(nèi)外均顯示出很強(qiáng)的趨瘤性,所以BMSCs有望成為膠質(zhì)瘤治療的優(yōu)選種子細(xì)胞。然而,關(guān)于分化的BMSCs細(xì)胞是否有同樣的趨化性以及BMSCs向膠質(zhì)瘤趨化性遷移機(jī)制研究的卻很少,本研究旨在探討成神經(jīng)分化的BMSCs向膠質(zhì)瘤分泌的趨化因子之一血小板源性生長(zhǎng)因子(platelet-derived growth factor, PDGF)的遷移行為及其分子機(jī)制。 方法本實(shí)驗(yàn)分四部分進(jìn)行研究,其中前三部分研究成神經(jīng)分化的BMSCs向PDGF的遷移,最后一部分研究的是PDGF引起的BMSCs遷移的分子機(jī)制。各個(gè)部分內(nèi)容具體為:(1)采用Percoll密度梯度離心法體外分離培養(yǎng)大鼠BMSCs,通過免疫組織化學(xué)染色和多向分化的方法對(duì)培養(yǎng)的細(xì)胞進(jìn)行鑒定。(2)采用加入抗氧化劑的方法誘導(dǎo)BMSCs向神經(jīng)樣細(xì)胞分化,即先用10 ng/ml的bFGF預(yù)誘導(dǎo)24 h,再加入200μM的丁羥基茴香醚(BHA)和2%的二甲基亞砜(DMSO)誘導(dǎo)5 h,最后用含有10μl/ml N2的H-DMEM維持培養(yǎng)48 h。觀察誘導(dǎo)分化過程中BMSCs的形態(tài)變化,通過免疫熒光染色檢測(cè)誘導(dǎo)細(xì)胞的神經(jīng)細(xì)胞特異性標(biāo)志物nestin、β-III-tubulin和NSE的表達(dá)情況。(3)運(yùn)用Dunn chamber研究成神經(jīng)分化不同階段BMSCs向膠質(zhì)瘤分泌的因子之一PDGF的趨化性遷移。細(xì)胞的遷移通過德國(guó)Leica AF6000活細(xì)胞工作站拍攝得到圖像,應(yīng)用NIH Image J軟件分析圖像,每個(gè)狀態(tài)隨機(jī)抽取30個(gè)細(xì)胞進(jìn)行統(tǒng)計(jì)得到細(xì)胞的遷移速率、遷移效率和單個(gè)細(xì)胞的遷移軌跡。(4) Rac1對(duì)PDGF誘導(dǎo)的BMSCs遷移行為的影響通過實(shí)時(shí)顯微攝像系統(tǒng)進(jìn)行研究,而PI3K信號(hào)通路對(duì)該趨化性遷移行為的作用通過實(shí)時(shí)顯微攝像系統(tǒng)和Dunn chamber兩種方法進(jìn)行探索。兩種方法均用德國(guó)Leica AF6000活細(xì)胞工作站拍攝得到圖像,應(yīng)用NIH Image J分析圖像,每個(gè)狀態(tài)隨機(jī)抽取30個(gè)細(xì)胞進(jìn)行統(tǒng)計(jì)得到細(xì)胞的遷移速率和效率。 結(jié)果(1)采用Percoll密度梯度離心法成功分離培養(yǎng)出大鼠BMSCs,可穩(wěn)定傳至20代以上。免疫組織化學(xué)鑒定結(jié)果為CD29、CD90、CD106陽性,CD34陰性;多向性分化實(shí)驗(yàn)證明其既可以成骨分化又可以成脂分化,證實(shí)實(shí)驗(yàn)所培養(yǎng)的細(xì)胞具備BMSCs的基本特征。(2)用bFGF/BHA誘導(dǎo)BMSCs向神經(jīng)樣細(xì)胞分化,預(yù)誘導(dǎo)24 h,細(xì)胞胞體出現(xiàn)收縮,細(xì)胞邊緣變得不規(guī)整;誘導(dǎo)5 h,細(xì)胞胞體進(jìn)一步收縮,折光性增強(qiáng),伸出細(xì)長(zhǎng)的突起,并且多為兩極突起,小部分細(xì)胞出現(xiàn)死亡并脫落;維持48 h細(xì)胞出現(xiàn)更多的分叉,并出現(xiàn)二級(jí)分叉;對(duì)照組細(xì)胞形態(tài)上則無顯著性變化。免疫熒光染色結(jié)果顯示誘導(dǎo)的細(xì)胞表達(dá)神經(jīng)前體細(xì)胞標(biāo)志物nestin,未成熟神經(jīng)元標(biāo)志β-III-tubulin和成熟神經(jīng)元標(biāo)志物NSE。(3) Dunn chamber分析顯示,BMSCs能夠趨化PDGF定向遷移,細(xì)胞的遷移速率和遷移效率均明顯高于對(duì)照組,單個(gè)細(xì)胞遷移軌跡也證實(shí)了這一結(jié)果。成神經(jīng)分化不同時(shí)期的細(xì)胞對(duì)PDGF的趨化能力不同:未誘導(dǎo)的BMSCs、預(yù)誘導(dǎo)24 h和誘導(dǎo)5 h的分化細(xì)胞的遷移速率得到明顯提高,而未誘導(dǎo)的BMSCs、維持18 h和維持48 h的分化細(xì)胞的遷移效率顯著升高。(4)實(shí)時(shí)顯微攝像系統(tǒng)分析顯示,提高Rac1的總量或者激活其表達(dá)水平會(huì)提高PDGF誘導(dǎo)的BMSCs的遷移速率,反之則會(huì)顯著性的降低遷移速率,但對(duì)細(xì)胞的遷移效率均無顯著性的影響;同時(shí)PI3K信號(hào)通路的抑制劑LY294002的加入會(huì)降低遷移速率,Dunn chamber獲得同樣的結(jié)果,說明Rac1和PI3K信號(hào)通路均參與PDGF誘導(dǎo)的BMSCs的遷移。 結(jié)論BMSCs可以趨向PDGF遷移,其對(duì)PDGF的趨向性遷移與其分化狀態(tài)密切相關(guān),在此過程中有Rac1和PI3 K信號(hào)通路的參與。
[Abstract]:The purpose of the malignant glioma is the most prevalent primary brain tumors, traditional methods, such as chemotherapy, radiotherapy is fundamentally to treatment, but because of bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) in vivo showed strong tumor tendency, so BMSCs is expected to become the preferred treatment for glioma seed cells. However, whether on the differentiation of BMSCs cells have the same chemotaxis and BMSCs to glioma migration mechanism research is seldom, this study aims to investigate the neural differentiation of BMSCs glioma cells to secrete chemotactic factor of platelet-derived growth factor (platelet-derived growth factor, PDGF) the migration behavior and its molecular mechanism.
Methods the study was divided into four parts, the first three parts research into neural differentiation of BMSCs to PDGF migration, the last part is the study of the molecular mechanism of PDGF induced BMSCs migration. Each part content is as follows: (1) using Percoll density gradient centrifugation in vitro cultured rat BMSCs were identified. In cultured cells by immunohistochemistry staining and differentiation. (2) by adding antioxidants to induce BMSCs to differentiate into neuron like cells. That is using bFGF pre 10 ng/ml followed by 24 h, adding 200 M Ding Qiangji ANISOL (BHA) two DMSO and 2% (DMSO 5) induced by H, finally with 10 l/ml N2 to maintain the H-DMEM to observe the morphological changes induced by BMSCs during the differentiation of cultured 48 h. markers of neural cell specific detection of nestin cells induced by immunofluorescence staining, -III-tubulin beta and NSE table Respectively. (3) using Dunn chamber differentiation into neurons in different stages of BMSCs secretion to glioma factor PDGF chemotactic migration. Cell migration through the German Leica AF6000 live cell station captured images, image analysis using NIH Image J software, 30 cells each state random migration statistics the rate of cell migration, the migration trajectory of efficiency and single cells. (4) the effect of Rac1 on the migration behavior of BMSCs induced by PDGF were investigated by real-time imaging system, and the PI3K signaling pathway to explore the chemotactic migration to function by real-time microscopic imaging system and Dunn chamber two. Two methods cell live by German Leica AF6000 workstation captured images, using NIH Image J image analysis, 30 cells were randomly selected from each state statistics obtained moving speed move cells Rate and efficiency.
Results (1) cultured rat BMSCs isolated by Percoll density gradient centrifugation, stably passaged more than 20 passages. Immunohistochemical identification results for CD29, CD90, CD106 positive, CD34 negative; differentiation experiment shows that it can not only make the osteogenic differentiation and adipogenic differentiation, confirmed the basic characteristics of experimental training the cells with BMSCs. (2) bFGF/BHA induced BMSCs differentiated into neuron like cells induced by pre 24 h, the cell body contraction, cell edge becomes irregular; induced by 5 h, the cell body further contraction, strong refraction, slender processes, and two processes, die and fall off the emergence of a small number of cells; 48 h cells maintain more divergent, and the emergence of the two split; control group cells had no significant changes. Immunofluorescence staining showed that the neural precursor cell marker nestin expression induced by the cell, not Mature neuronal markers -III-tubulin and beta neuronal marker NSE. (3) Dunn chamber analysis showed that BMSCs can chemoattract PDGF directional migration, migration rate and migration efficiency of cells were significantly higher than the control group, single cell migration path also confirmed this result. Cells in different periods after differentiation of PDGF chemotactic God different: not induced by BMSCs, the pre induction 24 h and induced the migration rate of differentiated cells of 5 h has been significantly improved, but not induced by BMSCs, to maintain 18 h and maintain the migration efficiency of 48 h cells increased significantly. (4) real-time image system analysis, improve the total amount of Rac1 or activation its expression level will increase the migration rate of PDGF induced by BMSCs, otherwise it will reduce the migration rate significantly, but had no significant effect on cell migration efficiency; at the same time, the PI3K signaling pathway inhibitor LY294002 The addition of Dunn chamber has the same result, indicating that both Rac1 and PI3K signaling pathways are involved in the migration of PDGF induced BMSCs.
Conclusion BMSCs tends to migrate to PDGF, and its tendency to migrate to PDGF is closely related to its differentiation state. In this process, there are Rac1 and PI3 K signaling pathways involved.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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