【作者】 李霞；

【导师】 李锋； 贾薇；

【作者基本信息】 石河子大学 ， 肿瘤学（专业学位）， 2017， 硕士

【摘要】 目的:干扰MAP3K3表达和/或阻断NF-κB信号通路,检测MAP3K3在卵巢癌细胞中的表达,结合NF-κB信号通路相关因子及凋亡相关蛋白的表达变化,分析和探讨MAP3K3介导NF-κB信号通路在卵巢癌化疗抵抗中的作用。方法:分别用特异性干扰MAP3K3表达质粒和MAP3K3真核表达质粒转染内源性高表达和/或低表达MAP3K3的卵巢癌细胞系SKOV3和OV2008,采用Western Blot和qRT-PCR检测转染效率;运用MTT方法检测干扰前后卵巢癌细胞对顺铂、紫杉醇和TNF-α敏感性的变化,TUNEL法检测细胞凋亡;通过使用NF-κB信号通路特异性阻断剂QNZ,观察顺铂处理后NF-κB信号通路关键因子和凋亡相关因子表达的变化情况。结果:(1)MAP3K3蛋白和mRNA在SKOV3细胞中内源性高表达,在OV2008细胞中低表达,OV2008细胞对顺铂的敏感性显著高于SKOV3细胞;(2)在SKOV3中敲低MAP3K3表达后,顺铂诱导细胞凋亡的数目显著增加,细胞对顺铂敏感性增强,生长速度减慢,同时NF-κB信号通路相关因子p-P65、p-IκBα和抗凋亡蛋白Bcl-2表达下调,促凋亡蛋白cleaved-caspase3、BAX表达上调;(3)在OV2008中过表达MAP3K3后,顺铂诱导细胞凋亡的数目显著减少,细胞对顺铂的敏感性减弱,生长加快,同时NF-κB信号通路关键因子p-P65、p-IκBα和抗凋亡蛋白Bcl-2表达上调,促凋亡蛋白cleaved-caspase3、BAX表达下调;(4)在SKOV3中抑制MAP3K3表达后细胞对紫杉醇和TNF-α的敏感性增强;在OV2008中过表达MAP3K3后细胞对紫杉醇和TNF-α的敏感性减弱;阻断NF-κB信号通路,两株细胞对紫杉醇和TNF-α的敏感性增强。结论:MAP3K3在卵巢癌细胞系中表达水平的差异可能与卵巢癌细胞的化疗抵抗相关。MAP3K3高表达促进了NF-κB信号通路激活以及下游抗凋亡蛋白的表达,抑制促凋亡蛋白表达,降低了化疗药物对卵巢癌细胞的诱导凋亡能力,提示MAP3K3可能是通过介导NF-κB信号通路促进卵巢癌细胞发生化疗抵抗。更多还原

【Abstract】 Objective: Interfering MAP3K3 expression and / or blocking NF-κB signal pathway,to detect MAP3K3 expression in ovarian carcinoma cells,combined with NF-κB signal pathway-related factors and apoptosis-related protein expression changes and analysis the role of MAP3K3 mediated NF-κB signal pathway in chemotherapy resistance of ovarian carcinoma.Methods: MAP3K3 specific interference expression plasmid and MAP3K3 eukaryotic expression plasmid were transfected into SKOV3 and OV2008 cells respectively.The transfection efficiency of MAP3K3 was detected by Western Blot and q RT-PCR.MTT assay was used to detect the sensitivity of cells to cisplatin,paclitaxel and TNF-α.TUNEL was used to detect cell apoptosis.The expression of NF-κB signaling pathway-related factors and the apoptosis-related factors were observed before and after treatment with NF-κB signal-specific blocker QNZ.Results:(1)MAP3K3 protein and m RNA were highly expressed in SKOV3 cells and were lower in OV2008 cells.The sensitivity of OV2008 cells to cisplatin was significantly higher than SKOV3 cells.(2)After knockdown of MAP3K3 in SKOV3,the expression of p-P65,p-IκBα and anti-apoptotic protein Bcl-2 were down-regulated,and the expression of pro-apoptotic protein cleaved-caspase3 and BAX were up-regulated.(3)After overexpression of MAP3K3 in OV2008,the number of cisplatin-induced apoptosis was significantly decreased,the sensitivity of cisplatin to cells was weakened and the growth of cells was accelerated,with the expression of cleaved-caspase3 and BAX were down-regulated,the expression of p-P65,p-IκBα and anti-apoptotic protein Bcl-2 were up-regulated.(4)The sensitivity of cells to paclitaxel and TNF-α was increased in SKOV3 cells after knockdown of MAP3K3.The sensitivity of cells to paclitaxel and TNF-α was decreased after overexpression of MAP3K3 in OV2008.The sensitivity of both cells to paclitaxel and TNF-α was enhanced after blocking NF-κB signaling pathway.Conclusion: Differential expression level of MAP3K3 in ovarian carcinoma cell lines may be related to the cell chemotherapy resistance.The high expression of MAP3K3 promotes the activation of NF-κB signaling pathway and the expression of downstream antiapoptotic protein,inhibits the expression of pro-apoptotic protein and reduces the ability of chemotherapeutic drugs induced apoptosis,suggesting that MAP3K3 may promotes chemotherapy resistance in ovarian carcinoma cells by mediating NF-κB signaling pathway.更多还原