【作者】 张亚男；

【导师】 刘竞；

【作者基本信息】 中南大学 ， 临床医学， 2014， 硕士

【摘要】 背景与目的：多发性骨髓瘤(multiple myeloma, MM)是一种以骨髓中单克隆浆细胞大量增生为特征的恶性疾病。因其多发耐药株的形成及低瘤负荷的持续存在,临床疗效不甚理想,目前MM仍然是不可治愈的,寻找新的治疗靶点迫在眉睫。而近年来研究发现,MM的发生发展与mTORC2信号通路以及分子伴侣途径有着密切的联系,并且两种途径都与AKT蛋白的活性相关联,本研究探讨共同抑制mTORC2信号通路和热休克蛋白90对蛋白AKT表达的影响及其后续对多发性骨髓瘤细胞凋亡的影响,为MM临床药物的治疗提供一个新的思路。方法：MTT法检测不同浓度雷帕霉素、17-烯丙胺-17-脱甲氧格尔德霉素(17-AAG)及二者联合使用后不同作用时间(0h、8h、24h、48h)对多发性骨髓瘤细胞株U266、KM3细胞增殖的影响。光学显微镜联合台盼蓝染色观察细胞形态及活细胞比率。流式细胞术检测细胞凋亡及细胞周期分布。Western Blotting检测目标蛋白AKT的表达。结果：雷帕霉素、17-AAG及二者联用后均可抑制多发性骨髓瘤细胞U266、KM3的增殖,尤以联用后抑制作用最为明显(P<0.05)；雷帕霉素使细胞周期阻滞在G1期,17-AAG亦使细胞周期阻滞在G1期,尤其是作用48h时周期阻滞明显(P<0.0001)；单用雷帕霉素或17-AAG时均可降低蛋白AKT的表达并在一定程度上诱导细胞凋亡(P<0.0001)；联合使用雷帕霉素与17-AAG时可显著降解AKT,明显诱导细胞凋亡,凋亡率明显高于任一单药(P<0.0001)。结论：1.共同抑制mTORC2和HSP90的活性在多发性骨髓细胞中可降解蛋白质AKT；2.共同抑制mTORC2和HSP90的活性降解了AKT后可在体外诱导多发性骨髓瘤细胞凋亡。更多还原

【Abstract】 Background and Objective:Multiple myeloma (MM) is a malignant disease which characterized by the proliferation of monoclonal plasma cells in bone marrow. Due to the tumor cells’ long proliferation time and their resistance to multidrug chemotherapy, the clinical curative effect is unimpressive, thus MM is still considered to be an incurable disease.And recent study found that the occurrence and development of MM has closely connection with mTORC2pathways and chaperon pathways.Both of them can regulate AKT.This study explores apoptosis of multiple myeloma cells and its mechanism by the common inhibition of mTORC2signaling pathway and heat shock protein90.It will probably provide a new potential way for clinical treatment of MM.Methods:The effect of Rapamycin,17-AAG and the combination of them on proliferation of multiple myeloma cell line U266and KM3were assessed using MTT in different time (0hour,8hour,24hour,48hour). Cell morphology was observed by optical microscopy and cell viability was determined by the trypan blue dye exclusion.Cell apoptosis and cell cycle distribution were measured by Flow cytometry.The specific protein AKT was detected by Western Blotting.Results:Rapamycin,17-AAG and the combination of them can suppress the proliferation of multiple myeloma cell line U266and KM3, especially the combination of Rapamycin and17-AAG synergistically inhibited proliferation (P<0.05); Rapamycin induced G1arrest both at24hours and48hours,17-AAG also induced G1arrest, especially at48hours(P<0.0001); Rapamycin,17-AAG alone can decrease the expression of AKT and induce myeloma cell apoptosis to some extent (P<0.0001); Inhibition of mTORC2and chaperon pathways can destabilize AKT and induce myeloma cell apoptosis, the apoptosis rate was significantly higher than that of any single drug (P<0.0001).Conclusion:1. Inhibition of mTORC2and chaperon pathways can destabilize AKT in multiple myeloma cells;2. Inhibition of mTORC2and chaperon pathways will induce apoptosis of multiple myeloma cells in vitro.更多还原