人脐带间充质干细胞分化为胰岛素分泌细胞的代谢生物标识的核磁共振频谱研究

发布时间：2018-08-12 16:07

【摘要】：目的: 研究利用9.4T离体型、高分辨率核磁共振频谱(Magnetic Resonance Spectroscopy,MRS)探寻人脐带间充质干细胞(Human Umbilical Cord Wharton’s Jelly-derived Mesenchymal Stem Cells ,huMSCs)和其分化为胰岛素分泌细胞后的特征性代谢物;并且追踪和鉴定huMSCs向胰岛素分泌细胞的分化过程。方法: 收集足月健康剖宫产出生新生儿的脐带并分离培养huMSCs及huMSCs在胰岛细胞培养条件(以含0.1mmol/Lβ-巯基乙醇、10ug/L bFGF的H-DMEM诱导至干细胞形态出现改变,换用含10mmol/L尼克酰胺的H-DMEM继续约6天成胰岛素分泌细胞诱导)下定向诱导其分化。倒置相差显微镜下观察诱导后细胞形态;双硫腙染色鉴定锌离子表达证明分化模型的成功;1H-MRS数据采集使用离体型9.4T高分辨率核磁共振频谱仪(Bruker Avance 400MHz)。所获取频谱数据在频率域经过XWINNMR(Bruker GmBH)软件初步处理后,再应用Mestre-c 4.7软件进行分析。结果: 经药物诱导后huMSCs由长梭形逐渐变为多角形、类圆形部分聚集成团;双硫腙染色实验诱导后的细胞,可见胞浆被染成棕红色,未行诱导的细胞无明显变化,行高分辨率核磁共振频谱获取的间充质干细胞谱线质量及重复性良好。间充质干细胞频谱中可见主要代谢物包括胆碱复合物、谷氨酰胺、乙酸、赖氨酸、亮氨酸、异亮氨酸、缬氨酸、丙氨酸。间充质干细胞在诱导成胰岛样细胞后异亮氨酸、缬氨酸、赖氨酸呈上升趋势,分别从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),谷氨酸呈下降趋势,从0.17±0.058 nmM下降至0.064±0.089 nmM(p0.05),丙氨酸及亮氨酸无明显变化,而诱导后新出现了肌醇。结论:离体型高分辨率核磁共振频谱能获取质量及重复性良好的间充质干细胞代谢物谱线;根据代谢物变化特点,尤其是肌醇的出现等,核磁共振频谱可能用于间充质干细胞成胰岛细胞分化的检测和鉴定。
[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 卤0.54v 0.65 卤0.140.16 卤0.074 nmM to 4.66 卤0.42 (p0.05) 0.31 卤0.052 nmM (p0.05), and glutamate decreased from 0.17 卤0.058 nmM to 0.064 卤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.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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