本文選題：骨髓間充質(zhì)干細(xì)胞(BMSCs) + 細(xì)胞分化��； 參考：《蘇州大學(xué)》2010年碩士論文

【摘要】： 目的惡性膠質(zhì)瘤等原發(fā)腦瘤對人類危害極大,骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)能夠向膠質(zhì)瘤發(fā)生趨化性遷移,并且具有易于獲得、多向分化等優(yōu)點,因而成為治療膠質(zhì)瘤最具希望的治療手段。但是,關(guān)于不同分化狀態(tài)細(xì)胞遷移的研究還較少。本實驗旨在探討成神經(jīng)分化的BMSCs向干細(xì)胞因子(stem cell factor,SCF)的遷移行為。 方法(1)采用Percoll密度梯度分離法分離BMSCs,并在體外擴(kuò)增培養(yǎng),通過免疫細(xì)胞化學(xué)染色的方法檢測表面抗原,以及成脂、成骨誘導(dǎo)分化鑒定。(2)采用抗氧化劑丁羥基茴香醚(BHA)及bFGF聯(lián)合誘導(dǎo)BMSCs向神經(jīng)樣細(xì)胞分化,首先用濃度為10 ng/ml的bFGF預(yù)誘導(dǎo)24 h,之后加入濃度為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)分化過程中BMSCs的形態(tài)變化,并且通過免疫熒光染色方法檢測神經(jīng)細(xì)胞特異性標(biāo)志物nestin、β-III-tubulin和NSE的表達(dá)情況。(3)用Dunn chamber研究成神經(jīng)分化BMSCs向SCF的定向遷移行為,通過德國Leica AF6000活細(xì)胞工作站拍攝遷移圖像,應(yīng)用NIH Image J分析圖像,計算細(xì)胞的遷移速率、遷移效率以及遷移軌跡。并研究了PI3K抑制劑LY294002處理后BMSCs遷移行為的變化。 結(jié)果(1)采用Percoll細(xì)胞分離液密度梯度法,成功分離培養(yǎng)出大鼠骨髓間充質(zhì)干細(xì)胞,免疫熒光染色顯示CD29、CD90、CD106呈陽性,CD34呈陰性,并且能夠分化成脂肪細(xì)胞和成骨細(xì)胞。(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)胞形態(tài)變化較小,仍為長梭形;誘導(dǎo)5 h后細(xì)胞胞體出現(xiàn)回縮、有突觸伸出,形態(tài)改變較大,初步展現(xiàn)出神經(jīng)細(xì)胞的形態(tài)特征;維持培養(yǎng)18h,細(xì)胞分叉稍有增多;維持48 h后,細(xì)胞分叉數(shù)目增多,并且產(chǎn)生二級分叉,類似于成熟神經(jīng)元的形態(tài)。對照組細(xì)胞無明顯的形態(tài)變化,免疫熒光染色顯示誘導(dǎo)的細(xì)胞表達(dá)神經(jīng)細(xì)胞標(biāo)志物nestin、β-III-tubulin以及NSE。(3)Dunn chamber遷移實驗顯示,SCF誘導(dǎo)實驗組細(xì)胞的遷移速率和遷移效率明顯高于對照組,表明SCF對BMSCs具有趨化作用。此外,分化不同狀態(tài)的BMSCs向SCF的趨化遷移程度也不同。另外,PI3K信號通路抑制劑LY294002能夠降低BMSCs的遷移速率和效率。 結(jié)論BMSCs可以分化為神經(jīng)樣細(xì)胞,其對SCF的趨向性遷移與分化狀態(tài)密切相關(guān),并且PI3K信號通路參與該遷移過程。
[Abstract]:Objective malignant glioma and other primary brain tumors are very harmful to human beings. Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) can migrate to glioma, and have the advantages of easy to obtain, multidifferentiation and so on. Therefore, it has become the most promising treatment for glioma. There are few studies on cell migration. The aim of this study was to investigate the migration of BMSCs stem cell factor (SCF) into neural differentiation.
Methods (1) BMSCs was isolated by Percoll density gradient method, and was amplified and cultured in vitro. The surface antigen was detected by immunocytochemical staining, and the differentiation of osteogenesis and osteogenesis induced differentiation. (2) the combination of antioxidant butyl anisole (BHA) and bFGF was used to induce BMSCs to differentiate into neural like cells. First, B with a concentration of 10 ng/ml was used. FGF preinduced 24 h, then Ding Qiangji anisole (BHA) and 2% two methyl sulfoxide (DMSO) were added to 5 h, and the morphological changes of BMSCs were induced in the differentiation process during the induction of differentiation with H-DMEM containing N2, and the expression of neuron specific markers was detected by immunofluorescence staining. (3) Dunn chamber was used to study the directional migration of neurogenic BMSCs to SCF. The migration images were taken by the Leica AF6000 live cell workstation in Germany, and the NIH Image J analysis images were used to calculate the migration rate, migration efficiency and migration path of the cells. The change of BMSCs migration behavior after LY294002 treatment of PI3K inhibitors was also studied.
Results (1) the rat bone marrow mesenchymal stem cells were successfully isolated and cultured by the density gradient method of Percoll cell separation solution. Immunofluorescence staining showed that CD29, CD90, CD106 were positive, CD34 was negative, and could differentiate into adipocytes and osteoblasts. (2) bFGF/BHA induced BMSCs to differentiate into neurolike cells, preinduced 24 h, and cell morphological changes Small, still long spindle shape. After 5 h induction, cell body retracted, synapses protruded, morphologic changes were larger, and the morphological characteristics of nerve cells were shown initially; maintaining 18h, cell bifurcation slightly increased; after maintaining 48 h, the number of cell bifurcations increased and produced two grade bifurcation, similar to the form of mature neurons. The control group cells were not clear. The immunofluorescence staining showed that the induced cell expression nestin, beta -III-tubulin and NSE. (3) Dunn chamber migration experiment showed that the migration rate and migration efficiency of SCF induced experimental group were significantly higher than those of the control group, which indicated that SCF had chemotaxis to BMSCs. Moreover, BMSCs to S in different states was differentiated to S. The degree of chemotactic migration of CF is also different. In addition, PI3K signaling pathway inhibitor LY294002 can reduce the migration rate and efficiency of BMSCs.
Conclusion BMSCs can be differentiated into neuron like cells, which is closely related to the trend migration and differentiation of SCF, and PI3K signaling pathway is involved in the migration process.

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

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 路曉淼;王恩群;;神經(jīng)生長因子對骨髓基質(zhì)細(xì)胞成骨分化影響的研究進(jìn)展[J];安徽醫(yī)藥;2009年02期

2 張文鵬;葉發(fā)剛;y囇澡，

本文編號：1844038