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目的:探讨葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)缺陷对A375细胞凋亡的影响及其可能的机制。方法:以A375-WT和A375-G6PDΔ细胞为模型,用Real-time PCR检测G6PD mRNA表达,蛋白质印迹法检测G6PD、Bcl-2、Bcl-xL、STAT5和P-STAT5蛋白的表达,紫外分光光度法测定G6PD酶活性,Heochst 33342/PI双染流式细胞仪检测细胞凋亡,免疫组化观察STAT5蛋白核迁移。结果:A375-WT和A375-G6PDΔ细胞中G6PD mRNA分别为0.545±0.13和0.207±0.03,降低了62.02%,t=-5.854,P=0.028;G6PD蛋白分别为0.975±0.16和0.227±0.10,降低了76.72%,t=-21.593,P=0.002;G6PD酶的比活性分别为(0.088±0.023)和(0.024±0.008)U/mg;降低了72.23%,t=-7.390,P=0.018;A375-G6PDΔ细胞的凋亡率为(8.62±1.67)%,比A375-WT细胞的(2.37±0.78)%升高了3.64倍,t=-12.163,P=0.007;A375-G6PDΔ细胞抗凋亡蛋白Bcl-2的表达量为0.245±0.037,比A375-WT细胞的0.578±0.073降低了57.61%,t=-16.021,P=0.004;Bcl-xL的表达量为0.138±0.019,比A375-WT细胞的0.287±0.067降低了51.92%,t=-5.377,P=0.033;A375-G6PDΔ细胞的P-STAT5/STAT5比值为0.72±0.201,比A375-WT细胞的2.28±0.367降低了68.4%(P=0.004),细胞核内STAT5表达为0.051±0.012,比A375-WT细胞核的STAT5蛋白(0.093±0.018)降低了45%(P=0.007),STAT5核迁移减少。结论:转录因子STAT5磷酸化降低、活性下降是G6PD缺陷所诱发的人黑色素瘤A375细胞凋亡的重要因素之一,为黑色素瘤发生及治疗的研究提供了新的思路。
Objective: To investigate the effect of glucose-6-phosphate dehydrogenase (G6PD) deficiency on A375 cell apoptosis and its possible mechanism. Methods: The expression of G6PD mRNA was detected by Real-time PCR using A375-WT and A375-G6PDΔ cells. The expressions of G6PD, Bcl-2, Bcl-xL, STAT5 and P-STAT5 were detected by Western blotting. The activity of G6PD was determined by Helast 33342 / PI double staining flow cytometry. The nuclear translocation of STAT5 protein was observed by immunohistochemistry. Results: The G6PD mRNA in A375-WT and A375-G6PDΔ cells were 0.545 ± 0.13 and 0.207 ± 0.03, respectively, decreased by 62.02%, t = -5.854 and P = 0.028, respectively. The G6PD protein was decreased by 0.975 ± 0.16 and 0.227 ± 0.10 (0.088 ± 0.023) and (0.024 ± 0.008) U / mg, respectively, with a decrease of 72.23%, t = -7.390, P = 0.018; The apoptotic rate of G6PDΔ cells was (8.62 ± 1.67)%, which was 3.64 times higher than that of (2.37 ± 0.78)% of A375-WT cells, t = -12.163, P = 0.007. The expression of Bcl-2 was 0.245 ± 0.037, which was 57.61% lower than that of 0.578 ± 0.073 in A375-WT cells, t = -16.021, P = 0.004. The expression of Bcl-xL was 0.138 ± 0.019, The P-STAT5 / STAT5 ratio of A375-G6PDΔ cells was 0.72 ± 0.201, which was 68.4% lower than that of A375-WT cells (2.28 ± 0.367, P = 0.004). The expression of STAT5 in the nucleus was 0.051 ± 0.012, which was 45% lower than that of STAT5 protein in the nucleus of A375-WT (0.093 ± 0.018) (P = 0.007). The nuclear translocation of STAT5 was decreased. Conclusion: The decrease of phosphorylation of STAT5 and the decrease of its activity are one of the important factors in the apoptosis of human melanoma A375 cells induced by G6PD deficiency, which provides a new idea for the study on the occurrence and treatment of melanoma.