【作者】 杨倩；

【导师】 王征； 张峰；

【作者基本信息】 天津大学 ， 药学， 2022， 硕士

【摘要】 卵巢癌是女性生殖系统的高度恶性肿瘤,在妇科恶性肿瘤中死亡率最高。目前在临床治疗卵巢癌中,虽然化学药物作用机制精确,疗效显著,但易导致耐药,其中尤其以抗凋亡B细胞淋巴瘤2(B-cell lymphoma 2,BCL-2)蛋白家族的上调为主。卵巢癌细胞SKOV3中的蛋白髓样细胞白血病蛋白1(Myeloid leukemia 1,MCL-1)是BCL-2家族的一种,由于其半衰期短等特点,使其抑制剂不能完全发挥其抑制作用,进而导致的凋亡逃逸被认为是癌症发展的结果之一,促进了肿瘤细胞对化疗的耐药性,严重阻碍了化疗药物的抗癌发展。基于上述问题,本课题设计了可以抑制卵巢癌细胞耐药的脂质体来共包载抗肿瘤药物。将细胞凋亡诱导剂辛伐他汀和铁死亡诱导剂伏立诺他共包载于脂质体的水溶性内腔和脂溶性膜材中。辛伐他汀可抑制MCL-1的表达,诱导卵巢癌细胞凋亡,阻止细胞的耐药。伏立诺他,一方面作为组蛋白去乙酰化酶抑制剂,通过稳定MCL-1的结构,增敏辛伐他汀对MCL-1的抑制作用;另一方面,伏立诺他可通过溶质载体家族7成员11(Recombinant Solute Carrier Family 7,Member11,SLC7A11)通路诱导卵巢癌细胞发生铁死亡,实现了辛伐他汀和伏立诺他的协同抗肿瘤作用。通过动态光散射(DLS)和透射电镜(TEM)对脂质体进行表征和分析,所制备的载药脂质体具有合适的粒径分布、较高的包封率和良好的稳定性。采用细胞毒性实验验证了辛伐他汀与伏立诺他的协同作用;结果表明,3个药物比例的联合指数值均小于1,药物具有协同作用且药物比例为1:1时协同效果最好。MCL-1的蛋白免疫印迹分析及多种细胞内因子检测结果显示,伏立诺他可增敏辛伐他汀对卵巢癌细胞MCL-1的抑制作用,促进卵巢癌细胞凋亡。此外,通过定量分析细胞内铁死亡标记物,包括谷胱甘肽、还原型烟酰胺腺嘌呤二核苷酸磷酸、过氧化脂质及降解产物(丙二醛)、前列腺素E2,以及SLC7A11的蛋白印迹分析,课题发现伏立诺他还可以通过SLC7A11促进铁死亡,进而实现了凋亡和铁死亡的联合抗癌作用。综上所述,本课题针对MCL-1过表达的卵巢癌,构建了MCL-1抑制剂辛伐他汀和铁死亡诱导剂伏立诺他共递送的脂质体传递系统,并在体外验证了该载药系统具有较好的安全性和显著增强体外抑瘤活性的作用,为卵巢癌的联合治疗提供了更多可能性。更多还原

【Abstract】 Ovarian cancer(OC)is a highly malignant tumor of female reproductive system,with the highest mortality rate among gynecological malignant tumors.Currently,in the clinical treatment of OC,although chemical drugs have precise mechanism of remarkable efficacy,they are prone to drug resistance,which seriously hinders the development of chemotherapy drugs against the cancer.Apoptosis escape is considered to be one of the results of cancer development,which promotes the resistance of cancer cells to chemotherapy.Based on these problems,this project designed liposomes that can inhibit drug resistance of ovarian cancer cells to co-encapsulate anti-tumor drugs.Simvastatin,an apoptosis inducer,and vorinostat,a ferroptosis inducer,were co-encapsulated in the water-soluble lumen and lipid soluble membrane of liposomes.Simvastatin inhibits Myeloid leukemia 1(MCL-1)in SKOV3 cells,a protein in the B-cell lymphoma 2(BCL-2)family,induces apoptosis and inhibits drug resistance of ovarian cancer cells.Vorinostat,on one hand,as a histone deacetylase inhibitor(HDACi),sensitizes the inhibitory effect of simvastatin on MCL-1 by stabilizing the structure of MCL-1.On the other hand,vorinostat can induce ferroptosis in ovarian cancer cells through the Recombinant Solute Carrier Family 7,Member 11(SLC7A11)pathway,realizing the synergistic antitumor effect of simvastatin and vorinostat.The liposomes were characterized and analyzed by dynamic light scattering(DLS)and transmission electron microscopy(TEM).The prepared liposomes possessed suitable particle size distribution,high encapsulation efficiency and good stability.Cytotoxicity test was used to verify the synergistic effect of simvastatin and vorinostat.The results showed that the combined index values of the proportions of the three drugs were all less than 1,indicating that the drugs had synergistic effect.The results of protein western blot analysis and various intracellular factors analysis showed that vorinostat could increase the inhibitory effect of simvastatin on MCL-1 and promote the apoptosis of ovarian cancer cells.In addition,quantitative analysis of intracellular markers of ferroptosis,including glutathione(GSH),reduced nicotinamide adenine dinucleotide phosphate(NADPH),peroxide lipids and degradation products(malondialdehyde,MDA),prostaglandin E2(PGE-2),and western blot analysis of SLC7A11 showed that vorinostat could also promote ferroptosis by SLC7A11 inhibition.The combined anticancer effect of apoptosis and ferroptosis was realized.In summary,we prepared a liposome delivery system capable of carrying both SIM and SAHA at the same time,and verified in vitro that the liposome had good safety and significantly enhanced in vitro tumor suppressive activity,providing various possibilities for combined treatment of OC.更多还原