口腔鳞癌相关成纤维细胞糖代谢的初步研究

Glucose metabolic alteration in oral carcinoma-associated fibroblasts

目的通过对口腔鳞癌相关成纤维细胞(carcinoma-associate fibroblasts,CAFs)糖代谢相关指标的检测,探讨肿瘤微环境CAFs糖代谢的可能方式并分析其作用和意义。方法培养CAFs和癌旁正常成纤维细胞(normal fibroblasts,NFs),詹纳斯绿B染色观察线粒体的数量及分布,JC-1荧光染色观察线粒体膜电位的改变。培养1、2、3、4 d,通过定磷法用可见分光光度计检测细胞中ATP的含量,葡萄糖氧化酶-过氧化物酶法检测细胞摄取葡萄糖的能力,乳酸酶法检测细胞分泌乳酸情况。结果与NFs组相比,CAFs线粒体詹纳斯绿B和JC-1荧光染色强度明显下降,显示CAFs线粒体活性下降。随着培养时间的延长,CAFs较NFs组摄取葡萄糖增多,ATP产能却减少,分泌乳酸增加,差异均有统计学意义(P<0.05);以培养第4天差异更明显,CAFs和NFs组培养基葡萄糖浓度分别为(10.85±1.42)、(16.04±1.09)mmol/L,产生ATP浓度分别为(42.99±4.64)、(57.37±2.75)μmol/g,产生乳酸浓度分别为(12.1±0.81)、(7.06±0.95)mmol/L。结论口腔鳞癌CAFs细胞糖代谢方式发生了改变,以糖酵解为主。

Objective To explore the possible ways of glucose metabolism in carcinoma-associated fibroblasts （CAFs） by detecting the related indexes of metabolism, and analyze its function and significance in tumorstroma interplay. Methods After the CAFs and paracancerous normal fibroblasts （NFs） were cultured, the amount and distribution of mitochondria were observed by Janus green B staining. The mitochondrial membrane potential was tested with specific fluorescent dye JC-1 staining. The phosphomolybdic acid colorimetry was used to measure the ATP content of the cells. Glucose oxidase-peroxidase reagent was used to detect the intracellular glucose level. The change of lactate concentration in 2 groups of cells was detected by lactic acid assay kit. Results Compared with the NFs group, the mitochondrial activity was lower in the CAFs group, according to the results of Janus green B staining and JC-1 fluorescence staining. As the time elapsed, the ability of glucose-uptaking and lactate secretion were increased in CAFs group, while ATP production was declined, with significant difference when compared with the NFs group （P〈0.05）. At the fouth day, glucose concentration were 10.85±1.42 mmol/L in the CAFs group and 16.04±1.09 mmol/L in the NFs group, while, ATP concentration was 42.99±4.64 and 57.37±2.75 μmol/g, respectively, and lactic acid concentration were 12.1±0.81 and 7.06±0.95 mmol/L, separately. Conclusion Oral squamous CAFs have glucose metabolic alteration, mainly through glycolysis.