9.4T离体磁共振频谱对间充质干细胞生物标识及成脂分化的研究

发布时间：2018-08-26 13:58

【摘要】：目的：利用9.4T离体型、高分辨率核磁共振频谱探寻人类来源于脐带华尔通胶间充质干细胞代谢生物标识特征,同时定量分析间充质干细胞体外诱导成脂分化后代谢物的变化情况。方法：培养收集第4代融合率约80-85%间充质干细胞和人食道癌EC109细胞系(约7-8×106,样品数分别为n=6和n=2；原代细胞分别来源于汕头大学多学科中心和汕大医学院病理实验室)。采用最终浓度为1μM地塞米松,5001μM异丁基黄嘌呤,60μM吲哚美辛和5μg/mL胰岛素的10% FBS/ LG-DMEM培养基,对间充质干细胞进行14天成脂分化诱导(样品数为n=5)；使用油红0染色及RT-PCR验证成脂分化模型的成功。1H-MRS数据采集使用离体型9.4T高分辨率磁共振频谱仪(Bruker Avance 400MHz)。所获取频谱数据在频率域经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：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、肌酸、谷氨酸、肌醇、醋酸、苏氨酸、甲硫氨酸以及脂肪酸(1.28ppmbiomarker,被认为是神经干细胞特异性标识)。间充质干细胞成脂分化前后可见胆碱复合物、肌酸、谷氨酸,醋酸代谢物均下降,而苏氨酸、甲硫氨酸及脂肪酸却呈上升趋势。定量分析,总胆碱、醋酸、谷氨酸及肌酸分别由6.3±0.68,0.97±0.23,0.3±0.05and 0.1±0.02 mM下降至1.1±0.06(p0.01),0.45±0.1(p0.01),0.16±0.08mM(p0.05)和未能检出水平；而甲硫氨酸、苏氨酸及1.28 ppm脂肪酸浓度从0.03±0.01,0.11±0.02和(0.66±0.17)n mM上升到0.12±0.05(p0.01)and 0.15±0.05(p0.05)和(1.18±0.07)n mM(p0.01)(n代表亚甲基数目)。成脂分化14天后可见细胞变圆,包质内大量脂肪微滴形成,油红0染色脂肪微滴呈橘红色；同时逆转录酶PCR(RT-PCR)可见过氧化物酶体增生物激活受体-r(PPAR-r),酰基辅酶A合成酶(ACS)及脂蛋白脂肪酶(LPL)等基因表达明显增加。结论：离体型高分辨率磁共振频谱能获取间充质干细胞代谢特征；间充质干细胞同样含有被认为是神经干细胞特异性标识的1.28 ppm biomarker,但它不是干细胞所特有的生物标识。根据代谢物变化特点,磁共振频谱能实现间充值干细胞成脂分化的监测和鉴定。
[Abstract]:Objective: to explore the metabolic biomarkers of mesenchymal stem cells derived from human umbilical cord by using 9.4T and high-resolution nuclear magnetic resonance spectroscopy (HNMR). The changes of metabolites of mesenchymal stem cells induced by adipogenic differentiation in vitro were also quantitatively analyzed. Methods: about 80-85% mesenchymal stem cells and EC109 cell lines of human esophageal cancer were collected and collected in the 4th generation (about 7-8 脳 10 ~ 6, the sample numbers were nong6 and nong2, respectively; the primary cells were from the multidisciplinary center of Shantou University and the pathological laboratory of Shantou University Medical College). Mesenchymal stem cells were induced into adipogenic differentiation on 14 days by 10% FBS/ LG-DMEM medium with final concentration of 1 渭 M dexamethasone 5001 渭 M isobutyl xanthine 60 渭 M indomethacin and 5 渭 g/mL insulin. Oil red 0 staining and RT-PCR were used to verify the success of adipogenic differentiation model. The isolated 9.4T high resolution magnetic resonance spectrometer (Bruker Avance 400MHz) was used to collect the data. The acquired spectrum data is processed in the frequency domain by XWINNMR (Bruker GmBH) software, and then analyzed by Mestre-c 4.7 software. Results: the lines of mesenchymal stem cells obtained were of good quality and reproducibility. The main metabolites in the spectrum of mesenchymal stem cells include choline complex, creatine, glutamate, inositol, acetic acid, threonine, methionine and fatty acids (1.28 ppmarker, which is considered to be the specific marker of neural stem cells). Choline complexes, creatine, glutamate and acetic acid metabolites decreased before and after adipogenic differentiation of mesenchymal stem cells, while threonine, methionine and fatty acids increased. Quantitative analysis showed that total choline, acetic acid, glutamate and creatine decreased from 6.3 卤0.680.97 卤0.23n0.30 卤0.05and 0.1 卤0.02 mM to 1.1 卤0.06 (p0.01) 0.45 卤0.45 卤0.16 卤0.08mM (p0.05) and undetectable respectively, while methionine, threonine and 1.28 ppm fatty acid concentrations increased from 0.03 卤0.01g 0.11 卤0.02 and (0.66 卤0.17) n mM) to 0.12 卤0.05 and 0.15 卤0.05p0.05 and 1.18 卤0.07p0.01) (n respectively. After 14 days of adipogenic differentiation, the cells became round, a large number of fat droplets were formed in the envelope, and oil red 0 stained fat droplets were orange red. At the same time, the expression of peroxisome proliferator-activated receptor (PPAR-r), acyl-coA synthase (ACS) and lipoprotein lipase (LPL) were significantly increased by reverse transcriptase PCR (RT-PCR). Conclusion: in vitro high resolution magnetic resonance spectroscopy can obtain the metabolic characteristics of mesenchymal stem cells which also contain 1. 28 ppm biomarker, which is considered to be the specific marker of neural stem cells but it is not a specific biomarker of stem cells. According to the characteristics of metabolites, magnetic resonance spectrum can be used to monitor and identify the adipogenic 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】：2010
【分类号】：R329

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