顺铂诱导脑胶质瘤U87细胞的PD-L1表达及耐药机制的研究

Study on Expression of PD-L1 and Resistance Mechanism on Glioma U87 Cells Induced by Cisplatin

关键词:  脑胶质瘤  U87细胞  顺铂  PD-L1  耐药性

Key words:  glioma  U87 cells  cisplatin  PD-L1  tolerance

摘要:

目的 研究PD-L1的表达对U87细胞的顺铂(cisplatin，CDDP)耐药性的影响。方法 建立U87对CDDP耐药细胞株(U87/CDDP)，用不同浓度的CDDP处理24 h后，CCK-8检测U87/CDDP细胞活力以验证耐药性。成功建立U87/CDDP细胞株后转染PD-L1 shRNA，用CCK-8、BrdU染色、流式细胞仪检测U87/CDDP细胞的增殖和凋亡。并分别用qRT-PCR检测PD-L1、Bax、caspase-3、Survivin mRNA的表达，Western blotting检测PD-L1、Bax、caspase-3、cleaved-caspase-3和P53蛋白的表达。结果CCK-8结果表明，U87/CDDP细胞成功建立。用相同浓度的CDDP处理24 h后，转染PD-L1 shRNA的U87/CDDP细胞的细胞增殖率比未转染的U87/CDDP细胞低，细胞凋亡率高。同时，与对照组细胞相比，在转染PD-L1 shRNA并给予CDDP处理的U87/CDDP细胞中，Bax、caspase-3 mRNA和Bax、cleaved-caspase-3和P53蛋白的表达显著增加，PD-L1、Survivin mRNA和PD-L1蛋白的表达显著降低。结论 通过抑制U87细胞中PD-L1的表达可以逆转脑胶质瘤U87细胞对CDDP的耐药性。

Abstract:

OBJECTIVE To investigate the effect of PD-L1 expression on the cisplatin(CDDP) resistance of U87 cells. METHODS U87 CDDP drug-resistant cell line(U87/CDDP) was established and the cell viability was estimated by CCK-8 to verify drug resistance after treated with different concentration of CDDP for 24 h. Then the U87/CDDP cells were transferred PD-L1 shRNA, and CCK-8, BrdU staining, flow cytometry was performed to detected the proliferation and apoptosis. Meanwhile, the mRNA expression of PD-L1, Bax, caspase-3, Survivin was detected by qRT-PCR, while the protein expression of PD-L1, Bax, caspase-3, cleaved-caspase-3, P53 was detected by Western blotting. RESULTS The result of CCK-8 showed that U87/CDDP was established successfully. After treated with same concentration of CDDP 24 h, the proliferation of PD-L1 shRNA-U87/CDDP cells was decreased and the apoptosis was increased compared with non transfected U87/CDDP cells. Meanwhile, compared to the control group, the mRNA expression of Bax, caspase-3 and the protein expression of Bax, cleaved-caspase-3 and P53 was significantly increased, while the mRNA expression of PD-L1, Survivin and the protein expression of PD-L1 was significantly decreased in PD-L1 shRNA transfected and CDDP treated U87/CDDP cells. CONCLUSION The tolerance of CDDP on U87 cells can reverse by down-regulating the expression of PD-L1.