【摘要】：目的: 研究利用9.4T離體型、高分辨率核磁共振頻譜(Magnetic Resonance Spectroscopy,MRS)探尋人臍帶間充質(zhì)干細(xì)胞(Human Umbilical Cord Wharton’s Jelly-derived Mesenchymal Stem Cells ,huMSCs)和其分化為胰島素分泌細(xì)胞后的特征性代謝物;并且追蹤和鑒定huMSCs向胰島素分泌細(xì)胞的分化過(guò)程。 方法: 收集足月健康剖宮產(chǎn)出生新生兒的臍帶并分離培養(yǎng)huMSCs及huMSCs在胰島細(xì)胞培養(yǎng)條件(以含0.1mmol/Lβ-巰基乙醇、10ug/L bFGF的H-DMEM誘導(dǎo)至干細(xì)胞形態(tài)出現(xiàn)改變,換用含10mmol/L尼克酰胺的H-DMEM繼續(xù)約6天成胰島素分泌細(xì)胞誘導(dǎo))下定向誘導(dǎo)其分化。倒置相差顯微鏡下觀察誘導(dǎo)后細(xì)胞形態(tài);雙硫腙染色鑒定鋅離子表達(dá)證明分化模型的成功;1H-MRS數(shù)據(jù)采集使用離體型9.4T高分辨率核磁共振頻譜儀(Bruker Avance 400MHz)。所獲取頻譜數(shù)據(jù)在頻率域經(jīng)過(guò)XWINNMR(Bruker GmBH)軟件初步處理后,再應(yīng)用Mestre-c 4.7軟件進(jìn)行分析。 結(jié)果: 經(jīng)藥物誘導(dǎo)后huMSCs由長(zhǎng)梭形逐漸變?yōu)槎嘟切�、�?lèi)圓形部分聚集成團(tuán);雙硫腙染色實(shí)驗(yàn)誘導(dǎo)后的細(xì)胞,可見(jiàn)胞漿被染成棕紅色,未行誘導(dǎo)的細(xì)胞無(wú)明顯變化,行高分辨率核磁共振頻譜獲取的間充質(zhì)干細(xì)胞譜線質(zhì)量及重復(fù)性良好。間充質(zhì)干細(xì)胞頻譜中可見(jiàn)主要代謝物包括膽堿復(fù)合物、谷氨酰胺、乙酸、賴氨酸、亮氨酸、異亮氨酸、纈氨酸、丙氨酸。間充質(zhì)干細(xì)胞在誘導(dǎo)成胰島樣細(xì)胞后異亮氨酸、纈氨酸、賴氨酸呈上升趨勢(shì),分別從3.44±0.54、0.65±0.14、0.16±0.074 nmM上升至4.66±0.42(p0.05)、1.05±0.21(p0.05)、0.31±0.052 nmM(p0.05),谷氨酸呈下降趨勢(shì),從0.17±0.058 nmM下降至0.064±0.089 nmM(p0.05),丙氨酸及亮氨酸無(wú)明顯變化,而誘導(dǎo)后新出現(xiàn)了肌醇。 結(jié)論:離體型高分辨率核磁共振頻譜能獲取質(zhì)量及重復(fù)性良好的間充質(zhì)干細(xì)胞代謝物譜線;根據(jù)代謝物變化特點(diǎn),尤其是肌醇的出現(xiàn)等,核磁共振頻譜可能用于間充質(zhì)干細(xì)胞成胰島細(xì)胞分化的檢測(cè)和鑒定。
[Abstract]:Objective: to explore the characteristic metabolites of human umbilical cord mesenchymal stem cells (Human Umbilical Cord Wharton's Jelly-derived Mesenchymal Stem Cells) and their differentiation into insulin-secreting cells by 9.4T (Magnetic Resonance spectroscopic Mrs. The differentiation of huMSCs into insulin-secreting cells was traced and identified. Methods: umbilical cord of newborns born with full term and healthy cesarean section were collected, and huMSCs were isolated and cultured in islet cells (induced by H-DMEM containing 0.1mmol/L 尾 -mercaptoethanol 10ugr / L bFGF to change the morphology of stem cells. H-DMEM containing 10mmol/L nicotinamide continued to induce insulin-secreting cells for about 6 days. The morphology of induced cells was observed under inverted phase contrast microscope, and the expression of zinc ion was identified by dithizone staining. The successful 1H-MRS data acquisition of the differentiation model was carried out by using an isolated 9.4T high resolution nuclear magnetic resonance spectrometer (Bruker Avance 400MHz). The acquired spectrum data is processed by XWINNMR (Bruker GmBH) software in frequency domain, and then analyzed by Mestre-c 4.7 software. Results: after drug induction, huMSCs gradually changed from long fusiform to polygonal shape, and round parts gathered into clusters, the cytoplasm of cells induced by dithizone staining was brownish red, but no obvious changes were observed in uninduced cells. The quality and reproducibility of the lines of mesenchymal stem cells obtained by high resolution nuclear magnetic resonance spectroscopy were good. The main metabolites in the spectrum of mesenchymal stem cells include choline complex, glutamine, acetic acid, lysine, leucine, isoleucine, valine, alanine. After mesenchymal stem cells were induced into islet like cells, isoleucine, valine and lysine increased from 3.44 鹵0.54v 0.65 鹵0.140.16 鹵0.074 nmM to 4.66 鹵0.42 (p0.05) 0.31 鹵0.052 nmM (p0.05), and glutamate decreased from 0.17 鹵0.058 nmM to 0.064 鹵0.089 nmM (p0.05), while alanine and leucine showed no significant change. After induction, inositol appeared. Conclusion: in vitro high resolution nuclear magnetic resonance spectroscopy can obtain good quality and reproducibility of the metabolites of mesenchymal stem cells, according to the characteristics of metabolites, especially the appearance of inositol, and so on. Nuclear magnetic resonance spectroscopy may be used to detect and identify islet cell differentiation of mesenchymal stem cells.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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