低糖浓度耐受对膀胱肿瘤T24细胞细胞学行为及免疫逃逸的影响

EFFECTS OF LOW GLUCOSE TOLERANCE ON CYTOLOGICAL BEHAVIOR AND IMMUNE ESCAPE OF BLADDER CANCER CELL LINE T24

目的探讨低糖培养基筛选的低糖浓度耐受型膀胱肿瘤T24细胞系增殖和迁移能力的变化,以及其免疫检查点PD-L1表达量的变化。方法用不同葡萄糖浓度(2 000、1 500、1 000、500mg/L)的培养基逐代筛选出低糖浓度耐受型膀胱肿瘤T24细胞系。将耐1 000mg/L葡萄糖浓度培养基的T24细胞设为A组,将耐500mg/L葡萄糖浓度培养基的T24细胞设为B组,将普通T24细胞设为C组。通过乳酸浓度检测比较不同组别细胞糖酵解能力的差异。通过细胞划痕实验测定不同组别细胞的迁移能力。通过MTT法检测不同组别细胞的增殖能力。通过荧光定量PCR技术检测不同组别细胞PD-L1mRNA的表达水平。采用Western Blot方法检测不同组别细胞PD-L1蛋白的表达水平。结果乳酸浓度检测结果显示,B组和A组乳酸生成量较C组明显增加(F=30.72,q=7.29、10.88,P<0.05),且B组大于A组(q=3.59,P<0.05)。划痕实验结果显示,B组和A组迁移能力较C组明显增强(F=13.11,q=6.39、6.15,P<0.05),A、B组间比较差异无显著性(P>0.05)。MTT法检测结果显示,B组与A组的膀胱肿瘤增殖能力较C组明显增强(F=63.22,q=9.32、21.99,P<0.05),而且B组大于A组(q=6.42,P<0.05)。荧光定量PCR以及Western Blot方法检测结果证明,无论在mRNA还是蛋白水平,PD-L1的表达量均为B组>A组>C组。结论低糖浓度耐受型膀胱肿瘤细胞系相较于非低糖浓度耐受型膀胱肿瘤细胞系糖酵解、迁移及增殖能力增强,并且其免疫检查点PD-L1的表达亦有明显增加,可能可以作为膀胱肿瘤的潜在治疗靶点。

Objective To investigate the changes in proliferative capacity and migration ability of low-glucose-tolerant bladder cancer cell line T24, which was obtained using a low-glucose medium, as well as the changes in the expression of programmed death-ligand 1 (PD-L1) as the immune checkpoint. Methods Low-glucose-tolerant bladder cancer cell line T24 was generationally obtained using the medium with different glucose concentrations (2 000, 1 500, 1 000, and 500 mg/L). The T24 cells tolerant to the medium with a glucose concentration of 1 000 mg/L were assigned to group A, the cells tolerant to the 500 mg/L medium to group B, and the normal cells to group C. The glycolytic capacity was compared between different groups by lactate concentration test. Besides, the migration ability of different groups was determined by wound healing assay, and the proliferative capacity of different groups was determined by MTT assay. The mRNA expression of PD-L1 in different groups was determined by quantitative real-time PCR (qRT-PCR), and the expression of PD-L1 protein was measured by Western Blot. Results Lactate concentration test showed that the lactate production in groups B and A was significantly higher than that in group C ( F =30.72, q = 7.29,10.88, P <0.05), and group B had significantly higher lactate production compared with group A ( q =3.59, P <0.05). Wound healing assay showed that compared with group C, groups B and A had significantly elevated migration ability ( F =13.11, q =6.39,6.15, P <0.05), but there was no significant difference between groups A and B with respect to this ability ( P >0.05). In the MTT assay, groups B and A showed a significant increase in the proliferative capacity of bladder cancer cells compared with group C ( F =63.22, q =9.32,21.99, P <0.05), and the proliferative capacity in group B was significantly higher than that in group A ( q =6.42, P <0.05). qRT-PCR and Western Blot showed that the mRNA and protein expression of PD-L1 was highest in group B and lowest in group C. Conclusion Compared with other cell lines, the low-glucose-tolerant bladder cancer cell line has enhanced glycolytic capacity, migration ability, and proliferative capacity, as well as increased expression of PD-L1, an immune checkpoint, so it may be a potential therapeutic target for bladder cancer.