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目的:探讨靶向多药耐药蛋白-1(MDR1)基因的RNA干扰(RNAi)对结肠癌细胞MDR1/P-gp依赖的多药耐药性的稳定逆转作用。方法:分别构建含编码#4029MDR1 siRNA和#4123MDR1 siRNA的质粒载体,并转染COLO320DM结肠癌多药耐药细胞,采用G418筛选克隆细胞,经实时定量RT-PCR及Western blotting鉴定阳性克隆细胞。MTT法检测细胞活力并计算各抗肿瘤药的IC50。流式细胞术检测细胞周期并计算PI/AI值。流式细胞仪测定细胞内adriamycin药物累积浓度。结果:阳性克隆细胞(clone#4029和clone#4123)的MDR1mR-NA和P-gp的表达均被抑制。COLO320DM结肠癌亲本细胞adriamycin及vincristine的IC50分别为9.616μmol/L和0.358μmol/L,而clone#4029的IC50分别降至1.094μmol/L和0.023μmol/L(P<0.01),clone#4123的IC50分别降至0.780μmol/L和0.035μmol/L(P<0.01)。COLO320DM结肠癌亲本细胞用adriamycin及vincristine处理后其PI/AI值分别为5.68及9.59,而clone#4029的PI/AI值分别降至2.74及3.59(P<0.01),clone#4123的PI/AI值分别降至2.75及3.24(P<0.01)。COLO320DM结肠癌亲本细胞用10μmol/Ladriamycin处理后细胞内adriamycin累积浓度为27.92,而clone#4029及clone#4123细胞内adriamycin累积浓度分别增加至187.24和215.57(P<0.01)。结论:稳定转染含编码MDR1siRNA的质粒载体能稳定逆转结肠癌细胞MDR1/P-gp依赖的多药耐药性。
AIM: To investigate the reversal of multidrug resistance (MDR1 / P-gp) -dependent multidrug resistance in colon cancer cells by targeting RNA interference (MDR1) targeting multidrug resistance protein-1 (MDR1) gene. METHODS: Plasmid vectors encoding # 4029MDR1 siRNA and # 4123MDR1 siRNA were constructed and transfected into COLO320DM colon cancer multidrug-resistant cells. Clone cells were screened by G418. Positive clones were identified by real-time RT-PCR and Western blotting. Cell viability was measured by MTT method and the IC50 of each antitumor drug was calculated. Cell cycle was measured by flow cytometry and PI / AI values were calculated. Flow cytometry was used to determine the intracellular concentration of adriamycin drug. Results: The expression of MDR1mR-NA and P-gp in the positive clones (clone # 4029 and clone # 4123) were all inhibited. IC50 of adriamycin and vincristine in COLO320DM colon cancer parent cells were 9.616μmol / L and 0.358μmol / L, respectively, while the IC50 of clone # 4029 was decreased to 1.094μmol / L and 0.023μmol / L (P <0.01) IC50 decreased to 0.780μmol / L and 0.035μmol / L (P <0.01). The PI / AI values of COLO320DM colon cancer parent cells treated with adriamycin and vincristine were 5.68 and 9.59, respectively, while the PI / AI values of clone # 4029 decreased to 2.74 and 3.59, respectively (P <0.01) Values decreased to 2.75 and 3.24, respectively (P <0.01). The cumulative concentration of adriamycin in COLO320DM colon cancer parent cells treated with 10μmol / Ladriamycin was 27.92, while the cumulative concentration of adriamycin in clone # 4029 and clone # 4123 increased to 187.24 and 215.57, respectively (P <0.01). CONCLUSION: Stable transfection of plasmid vectors encoding MDR1 siRNA can stably reverse MDR1 / P-gp-dependent multidrug resistance in colon cancer cells.