【作者】 林怡；

【导师】 吴丽贤；

【作者基本信息】 福建医科大学 ， 药理学， 2015， 硕士

【摘要】 目的研究新型化合物XN51对急性粒细胞白血病(AML)细胞增殖抑制作用及其干扰热休克蛋白90(Hsp90)功能的关系。方法(1)采用台盼蓝染色活细胞计数和MTT方法检测XN51在体外抑制AML细胞增殖作用。(2)采用流式细胞术检测XN51对AML细胞周期时相、线粒体膜电位(MMP)和细胞凋亡的影响。(3)蛋白免疫印迹(Western blot)探讨XN51对AML细胞周期和细胞凋亡相关蛋白表达的影响。(4)孔雀绿磷钼酸铵——无机磷检测法检测XN51对HSP90蛋白ATP酶活性的影响。(5)蛋白免疫印迹法检测XN51对AML细胞Hsp90客户蛋白Akt和CDK2表达的影响。结果(1)台盼蓝染色实验和MTT实验结果表明XN51具有明显抑制AML细胞HL-60和NB4增殖的作用,半数抑制率浓度IC50分别为(3.49±0.02)μmol·L-1和(2.52±0.04)μmol·L-1,且其体外抑制AML细胞增殖的活性比Nov提高了近100倍。(2)流式细胞术结果表明XN51明显增加HL-60和NB4细胞S期细胞的比例,使AML细胞周期阻滞在细胞周期S期;XN51能显著降低AML细胞线粒体膜电位水平;随着XN51浓度的增加,HL-60和NB4细胞的凋亡比例明显增加;XN51破坏了AML细胞线粒体的稳定性和增加AML细胞的凋亡率表明XN51通过激活细胞线粒体凋亡途径诱导AML细胞发生程序性死亡或坏死;XN51处理AML细胞诱导Hsp70蛋白表达,增加细胞内Cleaved parp、Cleaved Caspase3和Cleaved Caspase9蛋白蛋白含量,而下调p-Akt和CDK2蛋白的表达水平。(3)孔雀绿磷钼酸铵——无机磷实验结果表明XN51和Hsp90抑制剂GA(阳性对照药)均能显著抑制Hsp90 ATPase酶活性,表现为使Hsp90内源荧光发生规律性淬灭。通过Stern—Volmer方程计算得出XN51对Hsp90ATPase作用的动态淬灭速率常数Kq值为(4.45±0.70x1012)L?mol-1?s-1远大于各类淬灭剂对生物大分子最大扩散控制的碰撞动态淬灭速率常数Kq(2.0x1010L?mol-1?s-1),表明XN51是通过与Hsp90形成复合物引起Hsp90内源荧光发生静态淬灭达到体外抑制Hsp90 ATPase的催化活性的作用;XN51通过抑制Hsp90分子伴侣功能,使Akt蛋白不能形成正确稳定的活性构象而被蛋白酶体降解,达到抑制AML细胞增殖作用;XN51通过抑制Hsp90分子伴侣功能,使CDK2蛋白不能形成正确稳定的构象结构而被蛋白酶体降解,使AML细胞周期阻滞在S期;XN51在抑制Hsp90功能的同时使Hsp90的辅伴侣分子Hsp70蛋白表达上调。结论新型化合物XN51通过与Hsp90结合,干扰Hsp90分子伴侣功能,使参与细胞周期和增殖相关的Hsp90客户蛋白Akt和CDK2不能正确折叠修饰形成有效稳定的生物学构象而失活,依赖蛋白酶体通路在细胞内被降解清除,使AML细胞周期进程不能通过S期细胞周期检测点,诱导AML细胞阻滞在细胞周期的S期;XN51通过破坏线粒体膜电位的稳定性,激活AML细胞线粒体凋亡通路,激活AML细胞内Caspase级联反应,诱导AML细胞发生凋亡,抑制AML细胞增殖。更多还原

【Abstract】 Aim To investigate the relationship between the effect of the novel compound XN51 on acute myelocytic leukemia(AML) cells and the heat shock protein 90(Hsp90) chaperone function in AML cells.Methods(1) To test the proliferation inhibition effect of XN51 on AML cells using trypan blue staining and MTT methods.(2) To test the effect of XN51 on cell cycle process, mitochondrial membrane potential(MMP) and cell apoptosis ratio in AML cells by Flow cytometry.(3) To exame the effect of XN51 on protein level by Westernblot.(4) To test the effect of XN51 on Hsp90 ATPase activity by malachite green ammonium phosphomolybdate- inorganic phosphorus assay in vitro.(5) To detect the expression level of Hsp90’s client proteins Akt and CDK2 after XN51 treatment by Westernblot.Results(1) XN51 could obviously inhibite the proliferation of AML cells, and the proliferation inhibition activity was nearly 100 folds greater than that of the Nov.(2) XN51 could induce cell cycle S phase arrest, decrease mitochondrial membrane potential and increase the apoptosis ratio of AML cells. Weternblot data illustrated that XN51 could upregulate the protein level of Cleaved parp, Cleaved Caspase3 and Cleaved Caspase9 but downregulate expression level of p- Akt and CDK2.(3) XN51 could combine with Hsp90 in vitro, with Hsp90 endogenous fluorescence statically quenching, resulting in inhibition of Hsp90 ATPase activity; XN51 leaded to the degradation of Akt protein by proteasome pathway because of forming abnormal and instability molecule structure without assistance of heat shock protein 90 molecular chaperone, resulting in proliferation inhibition of AML cells; In the same way, CDK2 protein could not form the correct and stable conformation structure leading to AML cells cycle arrest in S phase; Additionally,XN51 induced expression of Hsp70 after inhibiting the chaperone function of Hsp90.Conclusion XN51 inhibited the activity of the Hsp90 molecular chaperone through combining with Hsp90 statically, resulting in dysfunction and degradation of Akt and CDK2 proteins without forming correct and stable conformation, finally induced AML cell cycle arrest in S phase, increased the apoptosis proportion of AML cells, and inhibited the proliferation of AML cells.更多还原