CD44中和抗体对黑色素瘤B16细胞生长及细胞糖酵解的影响

发布时间：2018-03-03 12:20

本文选题：黑色素瘤　切入点：CD　出处：《医学研究生学报》2017年05期 　论文类型：期刊论文

【摘要】：目的 CD44在肿瘤的生长及糖酵解代谢中起重要作用。文中旨在探讨CD44中和性抗体对鼠黑色素瘤B16细胞生长及糖酵解代谢的影响。方法首先设置对照抗体(50μg/m L)和不同浓度CD44抗体(2、10、50μg/m L)处理B16细胞,24 h后通过免疫印迹检测AKT通路活化情况。随后设置对照抗体(50μg/m L)和CD44抗体(50μg/m L)平行试验使用4μmol/L API-2预处理细胞,处理细胞48 h后通过免疫荧光观察乳酸脱氢酶A(LDHA)表达,比色法检测细胞上清乳酸含量。最后设置对照抗体(50μg/m L)组、API-2(4μmol/L)处理组、CD44抗体(50μg/m L)组和联合处理组(API-2+CD44抗体),作用B16细胞96 h后,通过MTT法检测细胞增殖,AO/EB和Annexin V染色检测细胞死亡及凋亡。结果与对照抗体B16细胞p-AKT水平(1.00±0.25)相比,2、10、50μg/m L CD44抗体呈浓度依赖性增强[2.51±0.32,3.89±0.46,4.07±0.42,P0.01],与对照抗体相比,50μg/m L CD44抗体作用后LDHA表达增强(2.13±0.24)倍。中和CD44后B16细胞LDHA荧光表达增强,且细胞上清乳酸含量升高,与对照抗体相比,CD44抗体(50μg/m L)96 h后细胞上清乳酸含量升高[(35.32±3.24)mmol/L vs(56.34±8.19)mmol/L,P0.01]。联合使用AKT通路抑制剂API-2处理后,CD44抗体所致LDHA表达增强现象明显受到抑制,且对照抗体和CD44抗体组乳酸含量均下降;与对照组相比,CD44抗体细胞上清乳酸含量差异无统计学意义[(20.12±4.35)mmol/L vs(22.65±5.16)mmol/L,P0.05]。MTT检测结果显示,与对照组细胞增殖率[(103±12.91)%]比较,API-2组[(84.87±19.35)%]、CD44抗体组[(71.35±16.23)%]和联合处理组[(41.16±9.15)%]均明显降低(P0.05)。细胞凋亡检测显示,与对照抗体组凋亡率[(5.23±0.96)%]相比,API-2组[(13.65±4.27)%]、CD44抗体组[(19.21±3.53)%]和联合处理组[(43.21±7.87)%]均显著升高(P0.01)。结论体外中和B16细胞CD44功能抑制细胞生长但促进AKT通路介导的糖酵解代谢,抑制AKT通路可增强CD44抗体对肿瘤细胞的杀伤作用。
[Abstract]:Objective CD44 plays an important role in tumor growth and glycolysis metabolism. The aim of this paper is to investigate the effects of CD44 neutralizing antibody on the growth and glycolysis metabolism of mouse melanoma B16 cells. B16 cells were treated with 50 渭 g / mL CD44 antibody for 24 h, then the activation of AKT pathway was detected by Western blotting. Then the control antibody 50 渭 g / mL and CD44 antibody 50 渭 g / mL were used to pretreat B16 cells with 4 渭 mol/L API-2. After 48 h of treatment, the expression of lactate dehydrogenase (LDHA) was observed by immunofluorescence. The content of lactic acid in supernatant was measured by colorimetric method. Finally, the control antibody 50 渭 g / mL) group was treated with 50 渭 g / ml of API-2 antibody and the combined treatment group was treated with 50 渭 g / mL CD44 antibody of API-2, and B16 cells were exposed to API-2 CD44 antibody for 96 h. Cell death and apoptosis were detected by MTT assay with AOP / EB and Annexin V staining. Results compared with the control antibody B16 cell, p-AKT level was increased in a concentration-dependent manner [2.51 卤0.32 卤3.89 卤0.46 卤0.42P0.01] compared with the control antibody B16 cell, the effect of 50 渭 g / mL CD44 antibody was increased in a dose-dependent manner [2.51 卤0.32 卤3.89 卤0.46 卤0.42P0.01]. The expression of LDHA in B16 cells was increased by 2.13 卤0.24) times, and the fluorescence expression of LDHA in B16 cells was increased after neutralization of CD44. The content of lactic acid in supernatant was increased, compared with the control antibody, the lactic acid content in supernatant of CD44 antibody increased after 96 h [35.32 卤3.24 mmol / L vs(56.34 卤8.19 mmol / L P0.01]. The increase of LDHA expression induced by AKT pathway inhibitor API-2 was significantly inhibited. Compared with the control group, the content of lactic acid in the supernatant of CD44 antibody cells was not significantly different from that in the control group [20. 12 卤4. 35 mmol / L vs(22.65 卤5. 16 mmol / L]. Compared with the control group [103 卤12.91%], the cell proliferation rate of the API-2 group [84.87 卤19.35%] and the combined treatment group [71.35 卤16.23%] and the combined treatment group [41.16 卤9.15%] significantly decreased the cell apoptosis. Compared with the control group [5.23 卤0.96 渭 g], the apoptosis rate of the API-2 group [13.65 卤4.27%] and the combined treatment group [19.21 卤3.53%] and the combined treatment group [43.21 卤7.87%] significantly increased the rate of apoptosis. Conclusion the CD44 function of the B16 cells in vitro inhibits the growth of the cells but promotes the glycolytic metabolism mediated by the AKT pathway. Inhibition of AKT pathway can enhance the killing effect of CD44 antibody on tumor cells.
【作者单位】：广东医科大学广东省分子诊断重点实验室;广东医科大学医学检验学院;
【基金】：国家自然科学基金(81503104) 广东医科大学博士启动基金(2XB13070);广东医科大学大学生创新项目(2014ZYDC004,2015ZYDC003)
【分类号】：R739.5

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