【作者】 谭雪梅；

【导师】 郑辉；

【作者基本信息】 暨南大学 ， 生理学， 2010， 硕士

【摘要】 目的：本研究观察ERK1/2抑制剂联合5-FU对转移性恶性黑素瘤的治疗效果,从分子水平初步探讨其作用机理,为临床化疗用药和进一步完善恶性黑素瘤手术后的辅助治疗提供实验依据。方法：B16细胞用DMEM高糖培养液体外常规培养,培养液含10%新生牛血清,0.1g／L青霉素、0.1g／L链霉素。实验分为四个组：A组单用ERK1／2抑制剂；B组单用5-FU；C组ERK1／2抑制剂联合5-FU；D组为空白对照组。用MTT法观察ERK1／2抑制剂联合5-FU对B16细胞增殖的抑制作用,显微镜下观察细胞形态学的变化,流式细胞仪检测细胞周期的变化和凋亡率,用RT-PCR观察ERK1／2抑制剂联合5-FU对Bcl-2和Caspase-9的mRNA表达的影响。结果：1 ERK1/2抑制剂联合5-FU抑制B16细胞的增殖,呈剂量依赖关系,与单用药组和对照组相比,差异有统计学意义(p<0.05)。2 ERK1/2抑制剂联合5-FU改变细胞周期,呈剂量依赖关系,与单用药组和对照组相比,差异有统计学意义(p<0.05)。3镜下观察细胞形态,结果显示：未经药物作用的细胞呈梭形,形态饱满,单用药组形态改变不明显,而联合用药组细胞皱缩,破裂,呈不规则形,有的细胞两端出现细长的伪足,脱落细胞增多,明显抑制了B16细胞的生长,细胞出现明显的形态学改变。4两药联合促进细胞凋亡,呈剂量依赖关系,凋亡率从(15.56±0.73)%提高到(29.86±1.72)%,与单用药组和对照组相比,差异有统计学意义(p<0.05)。5联合用药组与单用药组和对照组相比,下调基因Bcl-2的表达和上调Caspase-9的表达,差异有统计学意义(p<0.05)。结论：本实验结果表明,ERK1/2抑制剂联合5-FU抑制B16细胞的增殖,促进细胞凋亡,有显著的联合抗癌作用,其抗癌作用可能与下调抗凋亡基因Bcl-2和上调Caspase-9有关。ERK1/2抑制剂联合5-FU代表一种新的抗癌策略,会降低细胞毒性和增强肿瘤抑制,这为临床化疗用药和进一步完善恶性黑素瘤手术后的辅助治疗提供了实验依据。更多还原

【Abstract】 Objective:This study was to observe the effects of combination of the ERK1/2 inhibitor and 5-FU at different concentrations on malignant melanoma and their antitumor mechanisms. It can provide experimental evidence for the clinical chemotherapy drug and improve adjuvant therapy of malignant melanoma after surgeryMethod:B16 melanoma cells were routinely cultivated in DMEM medium containing 10% new-born calf serum,0.1 g/L benzylpenicillin and 0.1 g/L xanthomycin.The study was divided into four experimental groups which was group A, group B, group C and group D. Group A was applied to only the ERK1/2 inhibitor, group B only 5-FU, group C combination of the ERK1/2 inhibitor and 5-FU and group D the control group.MTT assays was used to detect the inhibitory effects of proliferation on the B16 cells.Fow cytometry was used to detect the cell cycle and the rate of apoptosis.Take picture under microscope and reverse transcription-polymerase chain reaction was used to determine the mRNA levels of Bcl-2 and Caspase-9.Results:1 Combination of the ERK1/2 inhibitor and 5-FU showed a dose-dependent inbititio--n of B16 cell proliferation, compared to the effeccts of each agent alone and the control group(P<0.05).2 Combination of the ERK1/2 inhibitor and 5-FU showed a dose-dependent changes in cell phase, compared to the effeccts of each agent alone and the control group (p< 0.05).3 Microscopic observation showed that the control group were spindle, plump shape and only one drug had little change.The combination group had cell shrinkage, cracking, irregular shape, exfoliated cells increased and some cells appeared pseudo--podia at both ends of slender.This indicated the combination significantly inhibited the growth of B16 cells and cells showed significantly obvious morphological hanges.4 Combination of the ERK1/2 inhibitor and 5-FU potentiated the apoptosis and promoted the rate of apoptosis, compared to the effects of each agent alone and the control group(p<0.05).5 Combination of the ERK1/2 inhibitor and 5-FU potentiated a reduction in gene bcl-2 expression and upregulation of Caspase-9 expression.The difference was statistic-ally significant(P＜0.05).Conclusion:These results indicated that combination of the ERK1/2 inhibitor and 5-FU can inhibit the proliferation of B16 cells,accelerate apoptosis and join anti-cancer effect. Anti-cancer mechanism may be associated with activation of apoptosis, reduced gene bcl-2 expression and the upregulation of Caspase-9 expression.So the combination therapy of the inhibitor of ERK1/2 and 5-FU represent a novel anti-cancer strategy, which will reduce the toxicity and enhance tumor suppression.lt can provide experimental evidence for the clinical chemotherapy drug and improve adjuvant therapy of malignant melanoma after surgery.更多还原