【摘要】：目的：利用9.4T離體型、高分辨率核磁共振頻譜探尋人類來(lái)源于臍帶華爾通膠間充質(zhì)干細(xì)胞代謝生物標(biāo)識(shí)特征,同時(shí)定量分析間充質(zhì)干細(xì)胞體外誘導(dǎo)成脂分化后代謝物的變化情況。 方法：培養(yǎng)收集第4代融合率約80-85%間充質(zhì)干細(xì)胞和人食道癌EC109細(xì)胞系(約7-8×106,樣品數(shù)分別為n=6和n=2；原代細(xì)胞分別來(lái)源于汕頭大學(xué)多學(xué)科中心和汕大醫(yī)學(xué)院病理實(shí)驗(yàn)室)。采用最終濃度為1μM地塞米松,5001μM異丁基黃嘌呤,60μM吲哚美辛和5μg/mL胰島素的10% FBS/ LG-DMEM培養(yǎng)基,對(duì)間充質(zhì)干細(xì)胞進(jìn)行14天成脂分化誘導(dǎo)(樣品數(shù)為n=5)；使用油紅0染色及RT-PCR驗(yàn)證成脂分化模型的成功。1H-MRS數(shù)據(jù)采集使用離體型9.4T高分辨率磁共振頻譜儀(Bruker Avance 400MHz)。所獲取頻譜數(shù)據(jù)在頻率域經(jīng)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：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、肌酸、谷氨酸、肌醇、醋酸、蘇氨酸、甲硫氨酸以及脂肪酸(1.28ppmbiomarker,被認(rèn)為是神經(jīng)干細(xì)胞特異性標(biāo)識(shí))。間充質(zhì)干細(xì)胞成脂分化前后可見(jiàn)膽堿復(fù)合物、肌酸、谷氨酸,醋酸代謝物均下降,而蘇氨酸、甲硫氨酸及脂肪酸卻呈上升趨勢(shì)。定量分析,總膽堿、醋酸、谷氨酸及肌酸分別由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)和未能檢出水平；而甲硫氨酸、蘇氨酸及1.28 ppm脂肪酸濃度從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代表亞甲基數(shù)目)。成脂分化14天后可見(jiàn)細(xì)胞變圓,包質(zhì)內(nèi)大量脂肪微滴形成,油紅0染色脂肪微滴呈橘紅色；同時(shí)逆轉(zhuǎn)錄酶PCR(RT-PCR)可見(jiàn)過(guò)氧化物酶體增生物激活受體-r(PPAR-r),�；o酶A合成酶(ACS)及脂蛋白脂肪酶(LPL)等基因表達(dá)明顯增加。 結(jié)論：離體型高分辨率磁共振頻譜能獲取間充質(zhì)干細(xì)胞代謝特征；間充質(zhì)干細(xì)胞同樣含有被認(rèn)為是神經(jīng)干細(xì)胞特異性標(biāo)識(shí)的1.28 ppm biomarker,但它不是干細(xì)胞所特有的生物標(biāo)識(shí)。根據(jù)代謝物變化特點(diǎn),磁共振頻譜能實(shí)現(xiàn)間充值干細(xì)胞成脂分化的監(jiān)測(cè)和鑒定。
[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 鹵0.680.97 鹵0.23n0.30 鹵0.05and 0.1 鹵0.02 mM to 1.1 鹵0.06 (p0.01) 0.45 鹵0.45 鹵0.16 鹵0.08mM (p0.05) and undetectable respectively, while methionine, threonine and 1.28 ppm fatty acid concentrations increased from 0.03 鹵0.01g 0.11 鹵0.02 and (0.66 鹵0.17) n mM) to 0.12 鹵0.05 and 0.15 鹵0.05p0.05 and 1.18 鹵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.
【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】：2010
【分類號(hào)】：R329

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