Serum metabolomic profiling of acute complete spinal cord transection in macaques

doi:10.3969/j.issn.2095-4344.2017.36.014

Abstract

Abstract:

BACKGROUND: Non-targeted metabolomic profiling is used to uncover metabolic changes and to identify relationships between metabolites and spinal cord injury, which contributes to further understanding the pathophysiological process and mechanisms of secondary spinal cord injury.
OBJECTIVE: To detect and analyze the serum metabolite changes after complete spinal cord transaction in macaques, explore its relationship with the pathophysiological progress of spinal cord injury, and screen the potential biomarkers.
METHODS: Five adult macaques were selected, in which the models of complete spinal cord transaction were established. The serum metabolic features were detected using a non-targeted metabolic profiling strategy based on gas chromatography time-of-flight mass spectrometry at 1 day before modeling, 3 hours (superacute phase) and 3 days (acute phase) after modeling. After compared with the spectrometry profiling, recognizing the metabolites, searching for differential metabolites and the related metabolic pathways, the pathophysiological process and mechanisms were analyzed.
RESULTS AND CONCLUSION: Three hundred and fifty-eight chromatographic peaks were obtained for subsequent data analysis. Fourteen metabolites, including low-molecular-weight organic acid, amino acids, fatty acid and carbohydrate, were identified as differential metabolites. To conclude, the acute phase of complete spinal cord transection is closely related to some metabolic pathways, such as amino acid metabolism, tricarboxylic acid cycle and pyruvate metabolism.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Gas Chromatography-Mass Spectrometry, Spinal Cord Injuries, Metabolism, Tissue Engineering

CLC Number:

R318

Tang Tao1, Cheng Jian-hua2, Yang Shu-guang1, Gao Jian1, Huang Tao1, Wang Bin1, Dong Fang-ting2, Liu Shao-jun1. Serum metabolomic profiling of acute complete spinal cord transection in macaques[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(36): 5818-5823.

Figures/Tables 2

2.1 样本数量分析研究共检测猕猴血清标本15份，分别获取于脊髓全横断损伤造模前1 d(正常对照)、脊髓全横断损伤后3 h(超急性期)和脊髓全横断损伤后3 d(急性期)。 2.2 代谢物分析和血浆总离子流色谱不同时间点采集的猕猴血清，经过衍生和GC-TOF MS分析得到总离子流色谱图(图1)，经与ChromaTOF software中代谢产物库中的质谱图进行比对分析，再通过本实验室自建的标准物质的质谱图进行比对，共识别出358个谱峰，其中与谱库匹配谱峰196个，包括小分子有机酸、脂肪酸、氨基酸、糖类及胺类物质。从图1中可以看到，在不同时间点，有些谱峰存在较大差异，这些谱峰所代表的代谢物即为差异代谢物。 2.3 多元数据处理结果首先对3个采样点的血清数据进行主成分分析(PCA)，来观察样品的聚集、离散及离群点，如PCA得分图(图2)所示，正常对照组与脊髓损伤3 d组数据聚集程度较高，脊髓损伤3 h组聚集度相对较低，可能与脊髓全横断损伤后超急性期代谢变化个体差异有关。脊髓损伤3 h组和脊髓损伤3 d组能和正常对照组明显区分，而脊髓损伤3 h组与脊髓损伤3 d组数据有部分重叠。提示脊髓全横断损伤会造成体内代谢的显著改变。所有数据均在95%可信区间内，未见异常值。 2.4 脊髓全横断损伤血清代谢差异物的确认对3个采样时间点的血清数据进行正交偏最小二乘判别分析(OPLS-DA)，绘制OPLS-DA 得分图和载荷图，如图3所示，显示3组血清数据可明显区分，并从相应的载荷图中查找导致这种差异的代谢物。同理分别对3个采样时间点的血清数据两两比较，进行正交偏最小二乘判别分析(OPLS-DA)，绘制OPLS-DA 得分图和载荷图，并查找代谢差异物。进一步对3时间点的代谢数据进行方差分析并进行组间比较，Tukey的事后检验验证上述结果。进而确定潜在的生物标志物(表1)。与正常对照相比脊髓全横断损伤猕猴血清在能量代谢、脂代谢、糖代谢、氨基酸代谢、核酸代谢等代谢通路中的中间产物有变化。 2.5 通路分析将显著性差异代谢物进行代谢通路分析(metabolic pathway analysis，MetPA)用以评估相关代谢通路[16](图4)。X轴代表通路拓扑学分析计算的通路影响(pathway impact)值，Y轴代表通路富集分析计算的-log(p)值，与损伤相关的通路应同时具有高的通路影响值与-log(p)值(低p值)。可见脊髓横断损伤后猕猴血清显著代谢变化物在三羧酸循环，甘氨酸、丝氨酸和苏氨酸代谢，丙氨酸，天门冬氨酸和谷氨酸代谢以及丙酮酸代谢通路中起到重要作用。另外氨酰tRNA生物合成通路、半胱氨酸和蛋氨酸代谢与亚油酸代谢通路也发生显著改变。"

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