本文選題：骨髓間充質(zhì)干細(xì)胞(BMSCs)　切入點(diǎn)：分化　出處：《蘇州大學(xué)》2009年碩士論文

【摘要】： 目的惡性膠質(zhì)瘤是目前最為盛行的原發(fā)腦瘤,干細(xì)胞移植極有希望治療膠質(zhì)瘤。因骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)體內(nèi)體外均顯示很強(qiáng)的膠質(zhì)瘤趨化性,所以應(yīng)用BMSCs治療膠質(zhì)瘤即是很好的細(xì)胞治療手段。然而,關(guān)于細(xì)胞因子參與BMSCs遷移的報(bào)道卻很少,本研究旨在探討成神經(jīng)分化的BMSCs向C6細(xì)胞條件培養(yǎng)基和SDF-1α(stromal cell-derived factor 1α)的遷移行為。 方法本實(shí)驗(yàn)分三個(gè)階段研究成神經(jīng)分化的BMSCs向C6細(xì)胞條件培養(yǎng)基和SDF-1α的遷移。(1)采用Percoll分離法在體外培養(yǎng)并擴(kuò)增BMSCs,通過(guò)免疫熒光染色的方法檢測(cè)表面抗原對(duì)BMSCs進(jìn)行鑒定。(2)采用抗氧化劑誘導(dǎo)方案誘導(dǎo)BMSCs向神經(jīng)樣細(xì)胞分化,即先用濃度為10 ng/ml的bFGF預(yù)誘導(dǎo)24 h,加入濃度為200μM的丁羥基茴香醚(BHA)和2%的二甲基亞砜(DMSO)誘導(dǎo)5 h,再用含有N2的H-DMEM維持48 h。觀察誘導(dǎo)分化過(guò)程中BMSCs的形態(tài)變化,通過(guò)免疫熒光染色檢測(cè)誘導(dǎo)的細(xì)胞表達(dá)神經(jīng)細(xì)胞特異性標(biāo)志物Nestin、β-III-tubulin和NSE的情況。(3)運(yùn)用Dunn chamber研究了分化細(xì)胞在具有濃度梯度的趨化因子中的定向遷移行為。分化不同狀態(tài)BMSCs的遷移通過(guò)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)分化點(diǎn)隨機(jī)抽取40個(gè)細(xì)胞進(jìn)行統(tǒng)計(jì)得到細(xì)胞的遷移速率、遷移效率和誘導(dǎo)5 h的遷移軌跡。接著,我們運(yùn)用Boyden chamber研究了BMSCs在成神經(jīng)分化過(guò)程中的趨化性遷移(Chemotaxis)和隨機(jī)遷移(Chemokinesis)。以正常培養(yǎng)的BMSCs作為對(duì)照,隨機(jī)抽取對(duì)照組和實(shí)驗(yàn)組各10個(gè)視野,細(xì)胞計(jì)數(shù),計(jì)算遷移系數(shù),遷移系數(shù)=實(shí)驗(yàn)組/對(duì)照組。 結(jié)果(1)采用Percoll淋巴細(xì)胞分離液及不連續(xù)密度梯度法,成功分離培養(yǎng)出大鼠骨髓間充質(zhì)干細(xì)胞,可穩(wěn)定傳至20代以上。表型鑒定結(jié)果為CD29、CD71、CD90、CD106呈陽(yáng)性,CD34、CD45呈陰性。(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)胞變成長(zhǎng)梭形;誘導(dǎo)5 h,細(xì)胞即發(fā)生劇烈的形態(tài)改變,出現(xiàn)胞體回縮、突觸伸展等神經(jīng)細(xì)胞的形態(tài)特征;維持48 h,細(xì)胞出現(xiàn)更多的分叉,并出現(xiàn)二級(jí)叉。對(duì)照組細(xì)胞無(wú)明顯的形態(tài)變化,免疫熒光染色顯示誘導(dǎo)的細(xì)胞表達(dá)神經(jīng)前體細(xì)胞標(biāo)志物Nestin、β-III-tubulin和成熟神經(jīng)元標(biāo)志物NSE。對(duì)照組不表達(dá)NSE,誘導(dǎo)組與對(duì)照組比較有統(tǒng)計(jì)學(xué)差異(P0.05)。(3) Dunnchamber分析顯示,C6細(xì)胞條件培養(yǎng)基組和SDF-1α組誘導(dǎo)細(xì)胞的遷移速率和遷移效率明顯高于對(duì)照組,表明C6細(xì)胞條件培養(yǎng)基和SDF-1α對(duì)BMSCs具有趨化作用,單個(gè)細(xì)胞遷移軌跡實(shí)驗(yàn)也證實(shí)了這一結(jié)果。此外,分化不同狀態(tài)的BMSCs向C6細(xì)胞條件培養(yǎng)基和SDF-1α的趨化程度也不同。當(dāng)Boyden上室為L(zhǎng)-DMEM懸浮的細(xì)胞,下室為C6細(xì)胞條件培養(yǎng)基時(shí),分化細(xì)胞的遷移數(shù)目明顯多于對(duì)照;而當(dāng)上下室均為C6細(xì)胞條件培養(yǎng)基時(shí),分化細(xì)胞的隨機(jī)遷移與對(duì)照無(wú)顯著差異。 結(jié)論BMSCs的定向遷移與分化狀態(tài)密切相關(guān),預(yù)誘導(dǎo)24 h分化細(xì)胞的定向遷移最強(qiáng)。
[Abstract]:Objective malignant glioma is the most prevalent primary brain tumor. Stem cell transplantation is very promising in the treatment of glioma.Because bone marrow mesenchymal stem cells (BMSCs) of bone marrow mesenchymal stem cells (BMSCs) have strong chemotaxis in vivo and in vitro, BMSCs is a good cell therapy for glioma.However, there are few reports of cytokines involved in BMSCs migration. This study aims to investigate the migration of neurogenic BMSCs to C6 cell conditioned medium and SDF-1 偽 stromal cell-derived factor 1 偽.Methods in this experiment, neural differentiation of BMSCs into C6 cell conditioned medium and migration of SDF-1 偽 were studied in three stages. BMSCs were cultured and amplified by Percoll in vitro. Surface antigens were detected by immunofluorescence staining for BMSCs.Identification. 2) differentiation of BMSCs into neuronal cells was induced by antioxidant induction.After 24 h preinduction with 10 ng/ml bFGF, 200 渭 M butadiol anisole (BHA) and 2% dimethyl sulfoxide (DMSO) were added for 5 h, and then H-DMEM containing N2 was used for 48 h.The morphological changes of BMSCs during differentiation were observed.The specific markers Nestin, 尾 -III-tubulin and NSE were detected by immunofluorescence staining. Dunn chamber was used to study the migration behavior of differentiated cells in chemokines with concentration gradient.The migration of BMSCs in different differentiation states was captured by Leica AF6000 living cell workstation in Germany. The images were analyzed by NIH Image J. 40 cells were randomly selected from each differentiation point to obtain the migration rate of the cells.Migration efficiency and migration trajectory induced for 5 h.Then, we used Boyden chamber to study the chemotaxis of BMSCs during neuronal differentiation.BMSCs cultured in normal culture was used as control group, 10 visual fields were randomly selected from control group and experimental group, cell count, migration coefficient, migration coefficient = experimental group / control group were calculated.Results 1) Rat bone marrow mesenchymal stem cells were successfully isolated and cultured by Percoll lymphocyte isolate and discontinuous density gradient method.Phenotypic identification showed that CD29, CD71, CD90, CD106, CD34, CD45, negative, and bFGF/BHA were used to induce BMSCs to differentiate into neuron-like cells, and after 24 h preinduction, the cells became long fusiform, and 5 h after induction, the cells showed drastic morphological changes and cell somatic retraction.The morphological features of nerve cells such as synaptic extension were more branched and secondary forks appeared after 48 h.The expression of Nestin, 尾 -III-tubulin and NSE were detected by immunofluorescence staining.The results of Dunnchamber analysis showed that the migration rate and migration efficiency of C6 cells in conditioned medium group and SDF-1 偽 group were significantly higher than those in control group.The results showed that C6 cell conditioned medium and SDF-1 偽 had chemotactic effect on BMSCs, and this result was confirmed by single cell migration trajectory experiment.In addition, the chemotaxis of BMSCs to C6 cell conditioned medium and SDF-1 偽 was also different.When the upper chamber of Boyden was a L-DMEM suspended cell and the lower chamber was a conditioned medium of C6 cells, the migration number of differentiated cells was significantly higher than that of the control, but when the upper and lower compartments were conditioned medium of C6 cells, there was no significant difference between the random migration of differentiated cells and that of the control.Conclusion the directional migration of BMSCs is closely related to the differentiation state, and the directional migration of differentiation cells preinduced 24 h is the strongest.
【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】：2009
【分類號(hào)】：R329

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