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目的探讨吉西他滨耐药胰腺癌细胞系的耐药机制。方法应用免疫组织化学方法和逆转录PCR法(RT-PCR),对吉西他滨耐药细胞系的耐药机制进行了研究。选用沙尔威辛(SAL)进行逆转耐药实验,采用了RT-PCR法、流式细胞仪和MTT法,评估SAL逆转耐药的效果。结果免疫组织化学法显示,耐药细胞SW1990-GEM的P-糖蛋白(P-gp)表达为弱阳性,其亲本细胞SW1990为阴性。多药耐药相关蛋白(MRP)在两细胞系表达均为阴性。SW1990-GEM的多药耐药基因(mdr-1)在mRNA水平上的转录比其亲本细胞系SW1990增强;脱氧胞苷激酶(dCK)基因的转录减少;核苷酸还原酶(RR)基因的转录增强。经过SAL作用后,mdr-1在mRNA水平上的转录减少。流式细胞仪显示,经过4,30,60,90nmol/L SAL作用后,细胞内罗丹明123的蓄积累分别增加1.04,1.16,1.39,1.72倍。用4nmol/L SAL进行逆转吉西他滨耐药实验,未接受SAL处理的对照组对吉西他滨的IC50为203.72μmol/L;经SAL作用1h后再加吉西他滨组的IC50为121.36μmol/L(P=0.219);经SAL作用24h后再加吉西他滨组的IC50为121.64μmol/L(P=0.167)。结论胰腺癌细胞对吉西他滨耐药的原因与dCK减少有关,同时也与mdr-1和RR升高相关。SAL能够下调SW1990-GEM耐药细胞的mdr-1和P-gp表达,但低浓度的SAL不能逆转SW1990-GEM对吉西他滨的耐药。
Objective To investigate the drug resistance mechanism of gemcitabine-resistant pancreatic cancer cell lines. Methods The mechanism of resistance in gemcitabine-resistant cell lines was studied by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Salicylic acid (SAL) was used to reverse the drug resistance test, RT-PCR method, flow cytometry and MTT method to assess the effect of SAL reversal of drug resistance. Results Immunohistochemistry showed that P-glycoprotein (P-gp) expression in SW1990-GEM cells was weakly positive and SW1990 was negative in parental cells. Multidrug resistance-associated protein (MRP) was negative in both cell lines. Transcription of multidrug resistance gene (mdr-1) of SW1990-GEM at the mRNA level was enhanced compared with that of its parental cell line SW1990; transcription of deoxycytidine kinase (dCK) gene was reduced; transcription of nucleotide reductase (RR) Transcriptional enhancement. After SAL, mdr-1 mRNA transcription decreased. Flow cytometry showed that accumulation of intracellular rhodamine 123 increased by 1.04, 1.16, 1.39 and 1.72 times, respectively, after treated with 4, 30, 60 and 90 nmol / L SAL. Gemcitabine resistance test was performed in 4 nmol / L SAL. The IC50 of gemcitabine in control group without SAL treatment was 203.72 μmol / L. The IC50 of gemcitabine group after SAL treatment for 1 h was 121.36 μmol / L (P = 0.219) The IC50 of gemcitabine group treated with SAL for 24 hours was 121.64μmol / L (P = 0.167). Conclusions The reason of pancreatic cancer cells resistance to gemcitabine is related to the decrease of dCK and the increase of mdr-1 and RR. SAL down-regulated the expression of mdr-1 and P-gp in SW1990-GEM cells, but the low concentrations of SAL did not reverse the resistance of SW1990-GEM to gemcitabine.