多柔比星—五味子乙素共载脂质体克服肿瘤多药耐药的研究

发布时间：2018-03-21 11:29

本文选题：多药耐药　切入点：多柔比星　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:肿瘤细胞对抗肿瘤药物产生多药耐药性(multidrug resistance,MDR)是肿瘤化疗失败的主要原因,也是困扰肿瘤治疗的一大难题。药物载体作为一种新型的MDR逆转策略,具有多种优势而成为研究热点,前期多柔比星(Doxorubicin,DOX)脂质体已被证明可部分克服肿瘤多药耐药。而从植物五味子提取的五味子乙素(Schisandrin B,Sch B)已被证明具有抑制P-gp和促进凋亡作用。目的:本论文试制多柔比星-五味子乙素共载脂质体(Doxorubicin-Schisandrin B co-liposomes,DSCL),探讨五味子乙素与脂质体对克服肿瘤MDR的协同作用。方法:探讨其制剂的优化、对多柔比星的细胞积聚、细胞摄取及细胞外排的影响,并分析该制剂克服肿瘤细胞MDR的可能机制,同时建立荷瘤耐药小鼠以评价其药效学。结果:首先采用硫酸铵梯度法制备DSCL,建立HPLC药物浓度测定方法,分别通过药物磷脂比和磷脂胆固醇比优化制备方法,最终获得多柔比星包封率为93.96±3.30%,五味子乙素包封率为87.81±1.97%,粒径为163±102nm,Zeta电位为-23±4mv,体外体外释放实验和体外稳定性实验说明DSCL符合后续研究。然后,以人白血细胞系K562/DOX耐药细胞为模型细胞,以多柔比星溶液(F-DOX)、五味子乙素+多柔比星混合液(SchB+DOX)、多柔比星脂质体(DOX-L)、多柔比星脂质体与五味子乙素混合液(DOX-L+Sch B)、维拉帕米+多柔比星混合液(Ver+DOX)为对照,分别比较DSCL组和对照组的细胞毒性不同,并测定不同时间点进入耐药细胞的DOX药物量差别,绘制药物浓度随时间变化曲线。结果显示DSCL逆转倍数分别是SchB+DOX组、DOX-L组和DOX-L+Sch B组的1.68、14.52和1.42倍。DSCL使DOX进入耐药细胞K562/DOX的药物量是DOX-L、Sch B+DOX的1.30和1.21倍,且具有延缓外排作用。说明DSCL具备良好的克服肿瘤多药耐药作用,脂质体和五味子乙素显示了良好的协同作用。为了进一步了解DSCL克服肿瘤MDR机制,我们设计了内吞抑制剂和低温摄取实验,结果显示K562/DOX细胞摄取DSCL主要通过耗能的内吞途径。而P-gp表达和凋亡实验证实了 DSCL克服肿瘤MDR可能是通过抑制P-gp表达和促进凋亡双通道途径。最后,运用K562/DOX细胞嫁接法建立了荷瘤耐药小鼠模型,测定其肿瘤体积和瘤重的结果表明,其抑制率高达84.14%,说明DSCL具备有效克服多药耐药特性,五味子乙素和脂质体在克服MDR是具有良好的协同作用。结论:DSCL具备良好的克服MDR作用,脂质体和五味子乙素显示了良好的协同作用。
[Abstract]:Background: multidrug resistance (MDR) produced by tumor cells against tumor drugs is the main reason for the failure of chemotherapy and a difficult problem in tumor therapy. Drug vector is a new strategy of MDR reversal. Has a variety of advantages and has become a research hotspot, Doxorubicindox) liposomes have been shown to overcome multidrug resistance in some cases. Schisandrin B Sch B) extracted from Schisandrin B has been proved to be able to inhibit P-gp and promote apoptosis. The cocarrier liposome Doxorubicin-Schisandrin B co-liposome DSCL was used to study the synergistic effect of Schisandin-B and liposome on overcoming tumor MDR. The effects of doxorubicin on cell accumulation, cell uptake and extracellular efflux of doxorubicin were analyzed. The possible mechanism of the preparation in overcoming MDR in tumor cells was analyzed. Results: first, the ammonium sulfate gradient method was used to prepare DSCLs, and the method for the determination of drug concentration was established. The preparation methods were optimized by the ratio of phospholipid to phospholipid and the ratio of phospholipid to cholesterol, respectively. Finally, the entrapment efficiency of doxorubicin was 93.96 卤3.30, the entrapment efficiency of Schisandrin was 87.81 卤1.97, and the particle size was 163 卤102 nm 路m ~ (-1) Zeta potential was -23 卤4 MV. The results of in vitro release test and in vitro stability test showed that DSCL was suitable for further study. Human white blood cell line K562% DOX resistant cells were used as model cells. Compared with doxorubicin solution (F-DOXN), schisandrin doxorubicin (SchB DOXX), doxorubicin liposome (DOX-LX), doxorubicin liposome and schisandrin B (SchB), verapamitoxin (Verapamil) was used as control. The cytotoxicity of DSCL group was compared with that of control group, and the amount of DOX drug entering drug-resistant cells at different time points was measured. The results showed that the reversal times of DSCL were 1.68 ~ 14.52 and 1.42 times of those of SchB DOX group and DOX-L SchB group, respectively. The amount of drugs that DOX entered into resistant cell line K562% DOX was 1.30 and 1.21 times as much as that of DOX-LX Sch B DOX. DSCL has good effect of overcoming multidrug resistance, liposome and schisandrin showed good synergism. In order to further understand the mechanism of DSCL to overcome tumor MDR, DSCL has the function of delaying efflux. We designed endocytosis inhibitors and hypothermic uptake tests, The results showed that the uptake of DSCL by K562 / DOX cells was mainly through the energy-consuming endocytosis pathway. The results of P-gp expression and apoptosis experiments confirmed that DSCL could overcome tumor MDR by inhibiting P-gp expression and promoting apoptosis. A mouse model of drug resistance was established by K562% DOX cell grafting. The results of tumor volume and tumor weight test showed that the inhibition rate of DSCL was as high as 84.14, which indicated that DSCL had the ability to overcome multidrug resistance. Conclusion: Schisandrin and liposome have a good synergistic effect on overcoming MDR, and liposome and Schisandrin B have good synergistic effect on overcoming MDR.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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