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摘要:目的
分析人正常肝细胞（HL-7702）、不同转移潜能的肝细胞癌细胞系（SMMC-7721、HCCLM3）以及肝癌门静脉癌栓细胞系（CSQT2）的糖代谢流量及相关基因表达。
方法利用稳定同位素13C标记的葡萄糖与谷氨酰胺作为营养源，结合酶活实验，对四种细胞进行糖代谢流量解析。
结果MTT与Transwell实验证实了HL-7702、SMMC-7721、HCCLM3、CSQT2细胞的增殖与迁移能力依次增加。酶活实验与标记实验结果均显示，糖酵解与三羧酸循环的代谢活性在四种细胞中依次增加，具体表现为葡萄糖消耗与乳酸产生的增加，代谢流量比率在多种糖代谢产物中的上升，尤其是在CSQT2细胞中达到最高。
结论随着肝癌细胞的恶性程度及转移能力的增加，糖酵解与三羧酸循环的代谢活性增加。代谢流量分析可能用以判断肝癌的发生发展与转移。
Abstract:ObjectiveTo analyze glucose metabolic flux and related gene expression of human normal liver cells (HL-7702) ,hepatoma cell lines with different metastasis degree (SMMC-7721,HCCLM3) and hepatic portal vein tumor thrombus cell line CSQT2.
MethodsStable isotope 13C labeled glucose and glutamine were used as the nutrient source. Label assay and enzymes activity assay were employed to analyze glucose metabolic activity and flux of the four cell lines.
ResultsMTT and Transwell assay confirmed the proliferation and migration abilities were increased successively in HL-7702,SMMC-7721,HCCLM3 and CSQT2 cells. Label assay and enzyme activity assay showed that metabolic activity in glycolysis and tricarboxylic acid cycle(TCA cycle) was successively increased in the four cells,mainly reflected by the increase of glucose consumption and lactate production,as well as metabolic flux and ratio of glucose metabolism in hepatoma cell lines compared with normal liver cells,and reached the highest level in CSQT2 cell line.
ConclusionThe metabolic pathway of glycolysis and TCA cycle is activated as malignant and metastasis degrees are increased in liver cancer cell lines. Metabolic flux analysis may help to diagnose the development and metastasis of liver cancer.
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图 1 四种细胞系的增殖(A)与迁移(B)能力
Figure 1 Proliferation(A) and migration(B) abilities of four cell lines
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图 2 糖酵解与三羧酸循环代谢通路图(A) 和标记实验的代谢流量分析示意图(B)
Figure 2 Metabolic pathways and metabolites of glycolysis and tricarboxylic acid(TCA) cycle(A) and Sketch map of metabolic flux analysis of label assay(B)
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图 3 13C-glucose标记实验检测四种细胞系糖酵解通路的代谢流量
Figure 3 Metabolic flux of glycolysis in four cell lines detected by 13C-glucose label assay
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图 4 13C-glutamine标记实验检测四种细胞系三羧酸循环的代谢流量
Figure 4 Metabolic flux of TCA cycle in four cell lines detected by 13C-glutamine label assay
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图 5 四种细胞的葡萄糖消耗曲线(A)与乳酸产生曲线(B)
Figure 5 Glucose consumption curve(A) and lactate production curve(B) of four cell lines
表 3 细胞中13C标记的碳原子在各代谢物中的比例 (%)
Table 3 Proportion of carbon labeled from 13C in metabolites from cells (%)
下载: 导出CSV
表 4 培养液中13C标记的碳原子在各代谢物中的比例 (%)
Table 4 Proportion of carbon labeled from 13C in metabolites from medium (%)
下载: 导出CSV
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