MCT1表达位置改变对胶质瘤细胞能量合成、细胞内pH值及凋亡的影响

Effects of MCT1 expression translocation on energy synthesis,pHi,and apoptosis of human glioma cells in vitro

目的研究体外培养U251细胞单羧酸转运蛋白-1(MCT1)被CD147/HAb18G基因工程单克隆抗体改变表达位置后其乳酸外流障碍对细胞能量合成、细胞内pH值(pHi)和凋亡的影响。方法分别以低、高剂量的CD147/HAb18G单抗封闭体外培养U251细胞表面CD147分子,并设立对照组。免疫荧光法检测各组细胞膜上MCT1表达分布改变,分光光度计检测各组细胞内乳酸含量,生物发光法检测细胞内腺嘌呤核苷三磷酸(ATP)含量,连续比色法检测细胞内磷酸果糖激酶(PFK)活性,2′,7′-二(羧乙基)-5(6)-羧基荧光黄(BCECF-AM)荧光探针法检测各组pHi,流式细胞仪检测各组细胞凋亡率。结果CD147/HAb18G单抗作用下,MCT1表达位置由胞膜有效表达转为胞质无效表达且细胞内乳酸浓度增高;细胞内ATP含量下降[对照组(0.831±0.036)×10-8 mmol/L,低剂量组(0.592±0.047)×10-8 mmol/L,高剂量组(0.332±0.042)×10-8 mmol/L,P<0.01];细胞PFK活性下降[对照组(0.855±0.076)mmol.min-1.L-1,低剂量组(0.602±0.057)mmol.min-1.L-1,高剂量组(0.382±0.049)mmol.min-1.L-1,P<0.01];pHi下降(对照组7.27±0.03,低剂量组6.77±0.04,高剂量组6.31±0.02,P<0.01);细胞凋亡率增加[对照组(8.45±1.26)%,低剂量组(16.87±3.26)%,高剂量组(28.92±3.01)%,P<0.01]。结论通过改变MCT1的表达位置,可有效抑制细胞糖酵解乳酸清除、降低糖酵解能量合成,酸化细胞内环境,促进肿瘤凋亡。

Objective To explore the effects of disturbance of lactic acid efflux,occurred by monocarboxylate transporter-1（MCT1） translocation on u251 cells in vitro induced by CD147/HAb18G genetic engineering monoclonal antibody,on energy synthesis,pHi and apoptosis of tumor cells.Methods Low and high dose of CD147/HAb18G monoclonal aitibody were applied to U251 cell in vitro with control group set.The translocation of MCT1 expression was demonstrated by immunofluorescence staining.Intracellular lactate content was detected by spectrophotometer,and ATP content was measured by bioluminescence method.Phosphofructokinase（PFK） activity was checked by continuous colorimetry.pHi was recorded by BCECF-AM fluorescence probe,and apoptopsis rate was detected by flow cytometer.Results Interfered by monoclonal aitibody,the expression of MCT1 was translocated from cell membrane to cytoplasma,intracellular lactate content significantly increased,and ATP content obviously decreased [control group：（0.831±0.036）×10-8 mmol/L,low dose group：（0.592±0.047）×10-8 mmol/L,high dose group：（0.332±0.042）×10-8 mmol/L,P0.01].PFK activity decreased [control group：（0.855±0.076）mmol·min-1·L-1,low dose group：（0.602±0.057）mmol·min-1·L-1,high dose group：（0.382±0.049）mmol·min-1·L-1,P0.01].The difference of pHi was significant among 3 groups [control group：（7.27±0.03）,low dose group：（6.77±0.04）,high dose group：（6.31±0.02）,P0.01].Apoptosis rate increased [control group：（8.45±1.26）%,low dose group：（16.87±3.26）%,high dose group：（28.92±3.01）,P0.01].Conclusion The translocation of MCT1 expression can effectively restrain the clearance of intracellular lactate generated by glycolysis,reduce ATP output,acidate cell microenvironment,and promote cell apoptosis.