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该实验旨在探讨曲古霉素A(trichostatin A,TSA)对胰腺癌PANC-1细胞株侵袭转移力的影响。分别通过短时间低浓度TSA(0.1~0.2μmol/L)处理胰腺癌细胞株PANC-1 24 h,以及长期低浓度递增法建立TSA耐药株(PANC-1-TSA)。采用CCK8(cell counting kit-8)测得PANC-1、PANC-1-TSA的IC50分别为(0.51±0.09)μmol/L、(78±5)μmol/L,耐药株耐药指数为153。Transwell侵袭小室实验显示,0.1μmol/LTSA处理后,胰腺癌PANC-1细胞侵袭力未见明显改变,而耐药株PANC-1-TSA侵袭力较亲本株明显增强。实时荧光定量PCR(RT-qPCR)结果显示,耐药株PANC-1-TSA转移相关基因MMP1/2/7/9/14/15及TIMP1/2 mRNA的表达均明显上升,尤以MMPs mRNA明显。在倒置显微镜观察到耐药细胞形态上发生上皮型向间叶型转化(epithelial mesenchymal transition,EMT),进一步通过RT-qPCR及蛋白免疫印迹(Western blot)实验证实耐药株发生EMT,其中上皮型标志物表达下降及间叶型标志物表达上升。该实验显示,耐药细胞株PANC-1-TSA侵袭力增强,侵袭、转移相关基因表达明显增加。耐药株形成的过程中,发生上皮–间叶转化(EMT),EMT可能参与了胰腺癌PANC-1-TSA细胞株侵袭能力的增强。
The purpose of this experiment was to investigate the effect of trichostatin A (TSA) on the invasion and metastasis of pancreatic cancer cell line PANC-1. The pancreatic cancer cell line PANC-1 was treated with low concentration of TSA (0.1-0.2 μmol / L) for 24 h, and the long-term low concentration increment method was used to establish the TSA resistant strain (PANC-1-TSA). The IC50 of PANC-1 and PANC-1-TSA were (0.51 ± 0.09) μmol / L and (78 ± 5) μmol / L respectively with cell counting kit-8 and the resistance index of drug- . Transwell invasion chamber experiments showed that the invasiveness of pancreatic cancer PANC-1 cells was not significantly changed after treatment with 0.1μmol / L TSA, but the invasiveness of PANC-1-TSA was significantly higher than that of the parental strains. Real-time quantitative PCR (RT-qPCR) showed that the expression of MMP1 / 2/7/9/14/15 and TIMP1 / 2 mRNA in PANC-1-TSA drug- . Epithelial mesenchymal transition (EMT) occurred in the resistant cell morphology observed by inverted microscope, and EMT was further confirmed by RT-qPCR and Western blot. The epithelial type Decreased expression of markers and increased expression of mesenchymal markers. The experiment showed that the invasiveness of the drug-resistant cell line PANC-1-TSA is enhanced, and the expression of genes involved in invasion and metastasis is obviously increased. Epithelial-mesenchymal transition (EMT) occurs during the formation of drug-resistant strains, and EMT may be involved in the enhancement of invasiveness of pancreatic cancer cell line PANC-1-TSA.