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目的探讨三磷酸腺苷结合转运蛋白G家族成员2(ATP-binding cassette superfamily G member 2,ABCG2)抑制剂维拉帕米对体内外胰腺癌SW1990细胞侵袭转移能力影响及其机制。方法以终浓度分别为0、12.5、25、50、100和200μmol/L维拉帕米处理SW1990细胞24、48和72h后,以CCK-8法检测维拉帕米对SW1990细胞增殖抑制影响;RT-PCR和蛋白质印迹法检测维拉帕米对体内外SW1990细胞ABCG2mRNA及蛋白表达水平的影响;Transwell小室侵袭迁移实验及划痕实验分析维拉帕米处理后细胞侵袭迁移能力的改变。将SW1990细胞接种至裸鼠皮下,对比观察维拉帕米干扰前后肿瘤细胞在裸鼠体内的成瘤情况;免疫组化分析裸鼠肿瘤组织中ABCG2的表达。结果 CCK-8检测结果显示,浓度为25~100μmol/L维拉帕米对胰腺癌SW1990细胞的抑制呈现明显的剂量及时间依赖性。划痕实验结果显示,细胞平均迁移率比较,划痕24h维拉帕米组为(19.2±2.04)%,对照组为(36.8±2.25)%,t=-17.23,P<0.001;48h维拉帕米组为(43.7±3.14)%,对照组为(78.4±2.67)%,t=-23.85,P<0.001。Transwell侵袭实验结果显示,维拉帕米组平均穿膜细胞数为46.6±3.3,明显少于对照组的90.2±2.7,t=-47.2,P<0.001;迁移实验结果显示,维拉帕米组平均穿膜细胞数为61.4±2.8,亦少于对照组的110.3±3.5,t=-39.5,P<0.001;蛋白质印迹法及RT-PCR结果显示,维拉帕米能够明显降低体内外SW1990细胞ABCG2蛋白及mRNA的表达水平。裸鼠成瘤实验结果显示,细胞接种后10d左右可见肿瘤结节,50d时维拉帕米组裸鼠肿瘤体积为(521.6±48.5)mm3,小于对照组的(1 496.6±73.1)mm3,t=-38.6,P<0.001;维拉帕米组瘤质量(0.53±0.18)g,明显低于对照组(1.61±0.45)g,t=-22.49,P<0.001。免疫组化结果显示,维拉帕米能明显降低SW1990细胞ABCG2的表达。结论维拉帕米能明显抑制胰腺癌SW1990细胞在体内外的侵袭和转移,其机制可能与维拉帕米下调ABCG2的表达有关。
Objective To investigate the effect of verapamil on the invasion and metastasis of pancreatic cancer cell line SW1990 in vitro and in vivo and its mechanism. Methods SW1990 cells were treated with 0, 12.5, 25, 50, 100 and 200 μmol / L verapamil for 24,48 and 72 h, respectively. The effects of verapamil on the proliferation of SW1990 cells were determined by CCK-8 assay. The effects of verapamil on the expression of ABCG2 mRNA and protein in SW1990 cells in vitro and in vivo were detected by RT-PCR and Western blotting. The invasion and migration of verapamil were assayed by Transwell invasion assay and scratch assay. SW1990 cells were inoculated subcutaneously into nude mice. The tumorigenicity of the tumor cells in nude mice before and after verapamil interference was observed. The expression of ABCG2 in the tumor tissue was detected by immunohistochemistry. Results The results of CCK-8 assay showed that verapamil at a concentration of 25-100 μmol / L exhibited a dose-and time-dependent inhibition of pancreatic cancer cell line SW1990. Scratch test results showed that the average cell migration rate compared with scratch 24h verapamil group was (19.2 ± 2.04)%, the control group was (36.8 ± 2.25)%, t = -17.23, P <0.001; 48h Villa Pam group (43.7 ± 3.14)%, control group (78.4 ± 2.67)%, t = -23.85, P <0.001. The results of Transwell invasion assay showed that the average number of transmembrane cells in the verapamil group was 46.6 ± 3.3, which was significantly lower than that in the control group (90.2 ± 2.7, t = -47.2, P <0.001). The migration assay showed that the verapamil group The mean number of transmembrane cells was 61.4 ± 2.8, which was also lower than that of the control group (110.3 ± 3.5, t = -39.5, P <0.001). Western blotting and RT-PCR results showed that verapamil could significantly reduce SW1990 cells ABCG2 protein and mRNA expression levels. The results of tumor formation in nude mice showed that the tumor nodules could be seen about 10 days after inoculation. The tumor volume of verapamil group (521.6 ± 48.5) mm3 on day 50 was smaller than that of control group (1 496.6 ± 73.1) mm3, t = -38.6, P <0.001; The tumor mass of verapamil group (0.53 ± 0.18) g was significantly lower than that of the control group (1.61 ± 0.45) g, t = -22.49, P <0.001. The results of immunohistochemistry showed that verapamil could significantly reduce the expression of ABCG2 in SW1990 cells. Conclusion Verapamil can significantly inhibit the invasion and metastasis of pancreatic cancer cell line SW1990 in vitro and in vivo. The mechanism may be related to the down-regulation of ABCG2 expression by verapamil.