【作者】 吴迪；

【导师】 范志民；

【作者基本信息】 吉林大学 ， 外科学， 2016， 博士

【摘要】 背景:乳腺癌已经成为中国女性最常见的恶性肿瘤之一,在世界范围内也是如此。紫杉醇(Paclitaxel,PTX)是乳腺癌的一线化疗药物。但其水溶性差,且会引起较严重的不良反应,如过敏反应等,使其临床应用受到了一定限制[4]。易溶于水、毒性更低的新型紫杉醇药物传送系统就成为研究的热点。中国科学院应用化学研究所合成了一系列功能性聚乙二醇-聚(乳酸-碳酸酯)[poly(ethylene glycol)-b-poly(L-lactide-co-carbonate)]及其与紫杉醇和靶向基团的键合物。但这种药物传递系统对乳腺癌在体内及体外的作用情况仍未知晓。本研究中,我们选择叶酸作为靶向基团,并制成紫杉醇胶束(PTX micelles,M(PTX))和叶酸靶向紫杉醇胶束(folic acid-bearing polymer-PTX micelles,(M(FA/PTX))。我们期待这些胶束能拥有多种功能,包括水溶性、缓释作用、生物降解能力、叶酸受体靶向能力等,与目前临床应用的药物剂型相比较,疗效相近或更优,但副作用更低。目的:探索紫杉醇胶束和叶酸靶向紫杉醇胶束作用于EMT-6小鼠乳腺癌细胞株和动物模型的效果,检测两种胶束的肿瘤抑制率、肿瘤细胞的凋亡效果和对生存率的影响。材料与方法:制备含25%PTX的(M(PTX))和含22.5%PTX的(M(FA/PTX))两种PTX键合物胶束。(1)对EMT-6小鼠乳腺癌细胞进行常规培养、传代。应用MTT法检测PTX、M(PTX)和(M(FA/PTX))以等量PTX的不同浓度梯度(10ug/ml、1ug/ml、0.1ug/ml、0.01ug/ml、1ng/ml)作用EMT-6细胞24、48、72小时的细胞抑制率。应用流式细胞仪检测含等量PTX浓度为1ug/ml时,PTX、M(PTX)和(M(FA/PTX))作用于EMT-6细胞24、48、72小时的细胞凋亡率。应用倒置显微镜观察含等量PTX 1μg/ml浓度的PTX、M(PTX)、M(PTX)作用于EMT-6细胞24和72小时的细胞形态变化。应用Hoechst 33258荧光染色检测含等量PTX 1μg/ml浓度的PTX、M(PTX)、M(PTX)作用于EMT-6细胞72小时的凋亡指数。(2)制作EMT-6小鼠乳腺癌动物模型。在抑瘤实验中,分为空白对照、PTX、M(PTX)和M(FA/PTX)4个组,每组12只小鼠,给药总剂量为20mg/kg为标准(以等量PTX计算),第5天处死动物。测量瘤重,计算肿瘤抑制率。瘤组织一部分做流式细胞检测,其余的迅速用4%多聚甲醛固定,石蜡包埋,进行H&E染色、Bax和Bcl-2的免疫组化检测。生存实验中,分为空白对照、PTX、M(PTX)和M(FA/PTX)4个组,每组18只小鼠,给药总剂量为26.7mg/kg为标准(以等量PTX计算),每日观察记录小鼠生存状态。(3)应用SPSS18.0统计软件,计量资料用均数±标准差描述,组间差异应用单项方差分析比较,以p<0.05为有统计学意义。计数资料应用χ2检验进行样本率间比较,以p<0.05为有统计学意义。应用Kaplan-Meier法计算生存率,绘制生存曲线。各组间生存差异比较采用Log-rank检验。结果:(1)在体外实验中,MTT试验结果显示在72h时,在1ug/ml的浓度下M(PTX)和M(FA/PTX)的抑制效果达到最佳。随着作用时间的延长,各组药物对EMT-6乳腺癌细胞生长的抑制率也随之增加。流式细胞结果显示在药物作用72h时,各用药组的细胞凋亡率均高于对照组,对照组、PTX组、M(PTX)组和M(FA/PTX)组的细胞凋亡率分别为14.4%、20.7%、19.7%和25.7%,M(FA/PTX)组的细胞凋亡率最高,与其他两用药组比较其差异有统计学意义(p<0.05)。倒置显微镜下,PTX、M(PTX)和M(FA/PTX)三种药物作用于EMT-6小鼠乳腺癌细胞后可以观察到细胞形态较对照组细胞发生明显的改变。Hoechst 33258染色结果显示对照组、PTX组、M(PTX)组和M(FA/PTX)组的细胞凋亡率分别为5.8%、15.8%、15.2%和21%。(2)在体内抑瘤实验中,PTX组的动物在通过尾静脉给药时,均烦躁不安,持续尖叫,而M(PTX)和M(FA/PTX)两组的小鼠在给药时均无明显的异常反应出现。在给药5天后,PTX、M(PT X)和M(FA/PTX)组的瘤重抑制率分别为29.0%、46.5%和54.9%。在组织学观察中,三个用药组均可观察到坏死区域,M(FA/PTX)组的坏死区域的面积要比PTX和M(PTX)组明显增大。对照组、PTX、M(PTX)和M(FA/PTX)组的Bax蛋白阳性表达率分别为8.3%、50%、60%和75%,Bcl-2蛋白阳性表达率分别为91.7%%、40%、60%和50%。流式细胞检测结果显示,对照组、PTX、M(PTX)和M(FA/PTX)组的细胞凋亡率分别为1.0%、36.6%、55.9%和66.1%。生存时间结果显示,对照组、PTX组、M(PTX)组和M(FA/PTX)组在用药30天时的生存率分别为50%、55.6%、72.2%和77.8%,中位生存时间分别为33天、31天、34天和42天(log-rank test:c2=9.789,P=0.02)。结论:在体外细胞试验中,M(PTX)和M(FA/PTX)两种药物可以获得与PTX近似的对EMT-6小鼠乳腺癌细胞的肿瘤抑制效果,且M(FA/PTX)表现出比M(PTX)更好的肿瘤抑制效果。在体内动物实验中,与PTX和M(PTX)相比,M(FA/PTX)显示出更好地对小鼠皮下移植瘤的抑制效果,能够更有效诱导肿瘤细胞凋亡。M(FA/PTX)能更好的延长荷瘤小鼠的生存时间。M(FA/PTX)在人乳腺癌中治疗效果值得期待。更多还原

【Abstract】 Introduction: Breast cancer is the most common solid malignant tumor for women in China, in the United States and in other countries Paclitaxel(PTX) is a first line chemotherapy drug for breast cancer. However, because of its low water solubility and strong adverse reactions such as allergies, low blood pressure, etc., its clinical use has been greatly limited. Great efforts have been made to develop new delivery systems of PTX,including liposomes, polymeric micelles, protein–PTX conjugates, etc. Recently, our group synthesized a series of functionalized poly(ethylene glycol)-b-poly(L-lactide-co-carbonate)and their conjugates with PTX and targeting moieties. In this paper, therefore, we chose FA as the targeting moiety, and attached it to a carrier polymer as its side-chain groups rather than a terminal group. PTX, a real anti-tumor drug, is used instead of a model drug. These micelles(PTX micelles, M(PTX) and folic acid-bearing polymer-PTX micelles,(M(FA/PTX)) are expected to have multiple functions, including water solubility, prolonged circulation, biodegradability, folatereceptor targeting and rapid cell-uptake.Objective: To study the therapeutic efficacy of M(PTX) and(M(FA/PTX)) in EMT-6breast cancer in vitro and in vivo, including anti-tumor activity, tumor cell apotosis and survival experiment.Methods: Two kinds of PTX conjugate micelles, one of which(M(PTX)) contained 25 wt.% of PTX and the other(M(FA/PTX)) contained 22.5 wt.% of PTX and 1.4 wt.% of folate(FA), were prepared for cell apoptosis and antitumor activity evaluation on EMT-6mice breast cancer cell line and models in comparison with 0.9 wt.% saline(control) and equivalent PTX.(1) The cytotoxic effect of PTX, M(PTX) and(M(FA/PTX) on EMT-6 breast cancer cell was evaluated by MTT at different time in different concentration. Cell apoptosis was analyzed by flow cytometry with 1ug/ml equivalent PTX. Inverted microscope was used to observe to the morphologic change of tumor cell between before and after drug administration. Hoechst 33258 fluorescence staining was used to detect the apotosis index with 1ug/ml equivalent PTX at 72 hours after drug administration.(2) EMT-6 breast cancer model was prepared. On day 5 after drug administration, at an equivalent paclitaxel dose of 20 mg/kg, mice were killed. Tumor inhibition rate was calculated. Cell apoptosis was analyzed by flow cytometry. Breast tumors were examined histologically with H&E staining and immunohistochemically by examining Bax and Bcl-2expression. And at an equivalent paclitaxel dose of 26.7mg/kg survival status of tumor-bearing mice with different treatments was also examined.(3) The results were analyzed using statistical software SPSS 18.0, each set of data was given as mean ± SD. Difference comparison between groups was carried out by one-way ANOVA. The χ2 test was used to test for associations between factors and the odds ratio.Life-tables were calculated according to the Kaplan–Meier method. Survival curves were compared with the log-rank test. The level of statistical significance was set as p< 0.05.Results:(1) In vitro experiment, MTT results showed that the inhibition effect of M(PTX) and M(FA/PTX) was best at 72 hours with1ug/ml equivalent PTX after drug administration.With the prolonged administration time, the inhibition raqte of each drug on EMT-6 breast cancer cell line was also increased. Flow cytometry results showed that the cell apotosis rate of control group, PTX, M(PTX) and M(FA/PTX) group was 14.4%, 20.7%, 19.7%, 25.7%.respectively. the cell apotosis rate of M(FA/PTX) group was the highest(p<0.05).With inverted microscope the tumor cell morphologic change of PTX, M(PTX) and M(FA/PTX)group was significant. Hoechst 33258 fluorescence staining showed that the cell apotosis rate of control group, PTX, M(PTX) and M(FA/PTX) group was 5.8%,15.8%, 15.2% and21%, respectively.(2) In vivo, The PTX group mice were dysphoric and screamed from time to time during drug injection. Mice of other groups had no obvious adverse reactions. On day 5 after drug administration, the average tumor masses were 0.49, 0.33, 0.22, and 0.18 g for the control, PTX, M(PTX) and M(FA/PTX) groups, respectively. The inhibition rates of tumor growth calculated for the three drug groups were 32.6%, 51.6% and 62.3%, respectively.H&E staining results showed that the necrosis area in M(FA/PTX) micelle group is significantly increased compared with other groups. Bcl-2 expression difference is statistically significant between PTX and M(FA/PTX) groups and control group(p<0.05).Bax expression difference is statistically significant between drug groups and control group(p<0.05). The percentage of cell apoptosis based on flow cytometry was 1.0%, 36.6%,55.9% and 66.1%, respectively, which showed statistically significant differences(P<0.05)between three drug groups and the control group. At an equivalent paclitaxel dose of 26.7mg/kg, the average survival time was 33 days, 31 days, 34 days and 42 days, respectively,and the survival rate at 30 days was 50%, 55.6%, 72.2% and 77.8%, respectively(p<0.05).Conclusion: In vitro, M(PTX) and M(FA/PTX) demonstrated the similar anti-tumor activity as PTX on EMT-6 breast cancer cell line,and the effect of M(FA/PTX) was more better. In vivo, the M(FA/PTX) have better anti-tumor activity on EMT-6 breast cancer model and can prolong the survival time of tumor-bearing mice. the M(FA/PTX) are promising in treatment of human breast cancers.更多还原