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目的:研究T24膀胱癌细胞中Toll样受体4(Toll like receptor-4,TLR4)信号通路活化对B7-H1表达的影响,并探讨其参与膀胱癌免疫逃逸的可能机制。方法:培养膀胱肿瘤T24细胞系,以脂多糖(lipopolysaccharides,LPS)模拟TLR4在体内的激活过程,分别用1μg/mL的LPS刺激T24细胞0、2、4、6、8和12h,流式细胞技术检测T24细胞表面TLR4的表达,RT-PCR技术检测T24细胞中B7-H1mRNA的表达,蛋白质印迹法检测T24细胞中B7-H1蛋白的表达。结果:流式细胞学检测结果显示,以1μg/mL的LPS刺激不同时间T24细胞TLR4表达的阳性率,0h为(3.34±0.86)%,2h为(6.71±1.38)%,4h为(9.71±1.24)%,6h为(11.57±0.91)%,8h为(25.24±5.32)%,12h为(21.67±1.81)%,组间差异均有统计学意义,F=83.049,P<0.05。RT-PCR检测结果显示,LPS刺激0h时B7-H1mRNA的相对表达量为0.86±0.06,2h为1.30±0.11,4h为1.67±0.20,6h为1.99±0.10,8h为2.57±0.23,12h为1.51±0.04,组间差异均有统计学意义,F=84.719,P<0.05。蛋白质印迹检测结果显示,LPS刺激0h时B7-H1蛋白质相对表达量为0.66±0.08,2h为0.65±0.10,4h为0.87±0.11,6h为1.07±0.23,8h为1.45±0.19,12h为1.13±0.06,组间差异均有统计学意义,F=195.258,P<0.05。TLR4表达分别与B7-H1mRNA(r1=0.753)及蛋白(r2=0.836)表达显著正相关,P<0.05。结论:通过LPS激活TLR4信号通路可以上调B7-H1mRNA及蛋白的表达,而这有可能是TLR4信号通路参与膀胱癌免疫逃逸的机制之一。
AIM: To investigate the effect of Toll-like receptor-4 (TLR4) signaling pathway on the expression of B7-H1 in T24 bladder cancer cells and to explore its possible mechanism involved in bladder cancer immune escape. Methods: The bladder cancer T24 cell lines were cultured and lipopolysaccharide (LPS) was used to simulate the activation of TLR4 in vivo. T24 cells were stimulated with 1μg / mL LPS for 0, 2, 4, 6, 8 and 12 h, respectively. Flow cytometry The expression of B7-H1 mRNA in T24 cells was detected by RT-PCR and the expression of B7-H1 protein in T24 cells was detected by Western blotting. Results: The results of flow cytometry showed that the positive rate of TLR4 expression in T24 cells treated with LPS at 1 μg / mL for 3 hours was (3.34 ± 0.86)% at 0h, (6.71 ± 1.38)% at 2h and (9.71 ± 1.24%, 6h was (11.57 ± 0.91)%, 8h was (25.24 ± 5.32)%, and 12h was (21.67 ± 1.81)% respectively. There was significant difference between the two groups (F = 83.049, P <0.05). The results of RT-PCR showed that the relative expression of B7-H1 mRNA at 0h after LPS stimulation was 0.86 ± 0.06, 1.30 ± 0.11 at 2h, 1.67 ± 0.20 at 24h, 1.99 ± 0.10 at 6h, 2.57 ± 0.23 at 8h, 1.51 at 12h ± 0.04, between the two groups were statistically significant, F = 84.719, P <0.05. The results of Western blot showed that the relative expression of B7-H1 protein at 0h was 0.66 ± 0.08, 0.65 ± 0.10 at 2h, 0.87 ± 0.11 at 24h, 1.07 ± 0.23 at 6h, 1.45 ± 0.19 at 8h, 1.13 ± at 12h 0.06, the differences between the groups were statistically significant, F = 195.258, P <0.05. TLR4 expression was positively correlated with B7-H1 mRNA (r1 = 0.753) and protein (r2 = 0.836) respectively, P <0.05. CONCLUSION: Activation of TLR4 signaling pathway by LPS up-regulates the expression of B7-H1 mRNA and protein, which may be one of the mechanisms involved in the TLR4 signaling pathway involved in bladder cancer immune escape.