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目的探讨沉默多药耐药基因(multi-drug resistance 1,MDR1)对大肠癌耐药细胞株LoVo/5-氟尿嘧啶(fluorouracil,Fu)细胞耐药性的逆转作用。方法将MDR1逆转的大肠癌耐药细胞株LoVo/5-Fu细胞分为EASYshRNA-MDR1(实验组)、shRNA-control(对照质粒组)和normal(未转染组),3组采用MTT法检测细胞对氟尿嘧啶的敏感性,采用流式细胞仪检测细胞凋亡情况,采用RT-PCR法检测细胞MDR1mRNA表达水平,采用ELISA法检测细胞P-糖蛋白水平,比较3组半数抑制浓度(half maximal inhibitory concentration,IC50)、耐药指数(resistance index,RI)、MDR1mRNA和P糖蛋白表达水平、对氟尿嘧啶的敏感性相对逆转率和细胞凋亡率。结果实验组IC50[(2.197±0.245)g/L]、RI(4.980)、MDR1 mRNA表达水平[(7.380±1.501)×107拷贝/μg]、P-糖蛋白表达水平[(46.569±4.200)%]低于未转染组[(7.283±0.461)g/L、15.221、(8.891±1.362)×108拷贝/μg、(97.810±1.921)%]和对照质粒组[(7.012±0.027)g/L、14.928、(7.346±1.201)×108拷贝/μg、(95.301±2.334)%](P<0.05),对5-Fu的敏感性相对逆转率(72.2%)、细胞凋亡率[(20.904±0.493)%]高于未转染组[0、(6.215±0.721)%]和对照质粒组[3.2%、(6.580±0.188)%](P<0.05);未转染组与对照质粒组各指标比较差异均无统计学意义(P>0.05)。结论 RNA干扰可明显抑制P-糖蛋白表达,降低LoVo/5-Fu细胞对药物的耐药性,通过促进细胞凋亡和在mRNA环节中表达而降低耐药性。
Objective To investigate the reversal effect of multi-drug resistance 1 (MDR1) on drug resistance of multidrug-resistant human LoVo / fluorouracil (Fu) cells. Methods The LoVo / 5-Fu cell line of multidrug-resistant human colorectal cancer cell line MDR1 was divided into EASY shRNA-MDR1 (experimental group), shRNA-control group (normal transfected group) and normal (untransfected group) The sensitivity of cells to fluorouracil was determined by flow cytometry. The expression of MDR1 mRNA was detected by RT-PCR. The level of P-glycoprotein was detected by ELISA. The half maximal inhibitory concentrations concentration, IC50), resistance index (RI), MDR1mRNA and P-glycoprotein, relative sensitivity to fluorouracil, and apoptosis rate. Results The expression of P-glycoprotein in the experimental group [(2.197 ± 0.245) g / L], RI (4.980), MDR1 mRNA expression level [(7.380 ± 1.501) × 107 copies / ] Was lower than that of untreated group [(7.283 ± 0.461) g / L, 15.221, (8.891 ± 1.362) × 108 copies / μg, (97.810 ± 1.921)%] and control plasmid group [(7.012 ± 0.027) g / L , 14.928 (7.346 ± 1.201) × 108 copies / μg, (95.301 ± 2.334)%〕 (P <0.05), the relative reversal of sensitivity to 5-Fu was 72.2%, and the apoptosis rate was 20.904 ± 0.493)%] was higher than that in the untransfected group [0, (6.215 ± 0.721)%] and control plasmid group [3.2%, (6.580 ± 0.188)%] There was no significant difference between the indexes (P> 0.05). Conclusion RNAi can significantly inhibit the expression of P-glycoprotein, reduce the drug resistance of LoVo / 5-Fu cells and decrease the drug resistance by promoting apoptosis and expression in mRNA.