[A novel chemo-resistant gene MSX2 discovered by establishment of two pancreatic cancer drug resistant cell lines JF305/CDDP and PANC-1/GEM].

Objective: To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines. Methods: The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC(50)), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes. Results: The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day (P<0.05); the proportion of S phase was decreased from (45±2)% to (30±2)% (P<0.05), and the migration ability was enhanced from (32 ±1) cells per field to (158±5) cells per field (P<0.01). The expression of multidrug resistance-related genes MRP2, MDR1, LRP and MSX2 was increased in JF305/CDDP cells (P<0.05). Knockdown of MSX2 in JF305 cells reduced the expression of MRP2, whereas overexpression of MSX2 in PANC-1 cells upregulated MRP2 level (P<0.05). Conclusions: Two stable multidrug resistant cell lines of pancreatic cancer, JF305/CDDP and PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.目的：建立胰腺癌耐药细胞株，探寻新的胰腺癌细胞耐药基因并初步研究其作用机制。 方法：采用大剂量间歇诱导法建立对顺铂(CDDP)和吉西他滨(GEM)耐药的细胞株。采用细胞计数试剂盒8(CCK－8)法检测其半数致死量(IC(50))、耐药指数(R)及交叉耐药情况，并观察其生长差异。应用流式细胞术检测耐药细胞的周期变化，Transwell迁移实验检测其迁移能力的改变，实时荧光定量聚合酶链反应(RT－qPCR)检测细胞中多药耐药相关基因的变化。 结果：成功获得对CDDP耐药的细胞株JF305/CDDP和对GEM耐药的细胞株PANC－1/GEM。JF305/CDDP和PANC－1/GEM细胞株的耐药指数分别为15.3和27.31，并存在交叉耐药。与亲代细胞相比，JF305/CDDP细胞的生长增殖速度减慢，第4天时吸光度(A)值分别为0.60±0.01和1.00±0.06(P＝0.002)；JF305/CDDP细胞的G(1)＋G(2)期细胞比例增加[(70±3)%和(55±3)%]，而S期细胞比例减少[(30±2)%和(45±2)%，P＝0.041]；JF305/CDDP细胞穿过Transwell小室的数量增加[(158±5)个/视野和(32±1)个/视野，P<0.001]；JF305/CDDP细胞中多药耐药基因1(MDR1)、多药耐药相关蛋白2(MRP2)和同源(异型)框基因2(MSX2)的表达量增加到(5.10±0.63)倍、(7.20±0.84)倍和(6.95±0.77倍)。在JF305细胞中，敲降MSX2则MRP2表达降低(0.69±0.17)；在PANC－1细胞中，过表达MSX2则MRP2表达升高(2.1±0.31)。 结论：成功建立了2株稳定的耐药胰腺癌细胞株JF305/CDDP和PANC－1/GEM。发现了新的耐药相关基因MSX2，MSX2可能通过上调MRP2的表达促进胰腺癌细胞耐药。.