叶酸靶向紫杉醇胶束对EMT-6乳腺癌细胞株及动物模型的抗肿瘤作用

发布时间：2018-05-17 22:17

本文选题：乳腺癌 + 药物靶向　；参考：《吉林大学》2016年博士论文

【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HE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p0.05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p0.05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(p0.05)。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。
[Abstract]:Background: breast cancer has become one of the most common malignant tumors in Chinese women, and is also the case in the world. Paclitaxel (PTX) is a first-line chemotherapy drug for breast cancer. But its water solubility is poor, and it can cause serious adverse reactions, such as allergic reaction, so that its clinical application is limited to [4]. soluble in water and toxicity. A lower new taxol drug delivery system has become a hot spot of research. A series of functional polyethylene glycol poly (lactic acid carbonate) [poly (ethylene glycol) -b-poly (L-lactide-co-carbonate)) and the bonds with taxol and target groups have been synthesized by the Institute of Applied Chemistry of the Chinese Academy of Sciences. In this study, we chose folic acid as a target group and made paclitaxel micelles (PTX micelles, M (PTX)) and folic acid targeted paclitaxel micelles (folic acid-bearing polymer-PTX micelles, (M (FA/PTX)) in this study. We expect these micelles to have a variety of functions, including water solubility and sustained release. The effect, biodegradability, and the targeting ability of folic acid receptor, compared with the current clinical drug formulations, are similar or better, but have lower side effects. Objective: To explore the effect of paclitaxel micelles and folic acid targeted paclitaxel micelles on EMT-6 mouse breast cancer cell lines and animal models, and to detect the tumor inhibition rate of two kinds of micelles. The effect of apoptosis and the survival rate of tumor cells. Materials and methods: two kinds of PTX binding micelles containing 25%PTX (M (PTX)) and 22.5%PTX (M (FA/PTX)) were prepared. (1) conventional culture of EMT-6 mice breast cancer cells was carried out. MTT method was used to detect PTX, M (PTX) and (0.1) concentration gradient (0.1) Ug/ml, 0.01ug/ml, 1ng/ml) acts on the cell inhibition rate of EMT-6 cells at 24,48,72 hours. Using flow cytometry to detect the cell apoptosis rate of PTX, M (PTX) and (M (FA/PTX)) when the concentration of PTX is 1ug/ml. Cell morphology changes at 24 and 72 hours. Hoechst 33258 fluorescence staining was used to detect PTX, M (PTX), M (PTX), M (PTX), and M (PTX) for 72 hours of apoptosis index of EMT-6 cells. (2) the mammary carcinoma animal model of EMT-6 mice was made. In the tumor suppressor experiment, 4 groups, 12 mice in each group, were divided into 4 groups, 12 mice in each group. The total dose was 20mg/kg as the standard (calculated by PTX), the animals were killed fifth days. The tumor weight was measured and the tumor inhibition rate was calculated. A part of the tumor tissue was detected by flow cytometry. The rest were fixed with 4% polyformaldehyde, paraffin embedded, HE staining, Bax and Bcl-2 immunohistochemistry. The survival experiments were divided into blank control, PTX, M (PTX) and M (FA/PT). X) 4 groups, each group of 18 mice, the total dose of 26.7mg/kg as the standard (with equal amount of PTX), the daily observation of the survival state of mice. (3) the application of SPSS18.0 statistical software, the measurement data were described with mean standard deviation, the difference between groups was compared with the single variance analysis, and P0.05 was statistically significant. The count data were carried out by the x 2 test. The sample rate was compared with P0.05. The survival rate was calculated by Kaplan-Meier method and the survival curve was drawn. The survival difference between each group was compared with the Log-rank test. (1) in the experiment in vitro, the results of MTT test showed that the inhibition effect of M (PTX) and M (FA/PTX) was best at the concentration of 1ug/ml at the concentration of 72h. With the time of action. The inhibition rate of EMT-6 breast cancer cell growth was also increased in each group. Flow cytometry showed that the apoptosis rate of each drug group was higher than that of the control group when the drug action was 72h. The apoptosis rate of the control group, the PTX group, the M (PTX) group and the M (FA/PTX) group were 14.4%, 20.7%, 19.7% and 25.7%, and the apoptosis rate of M (FA/PTX) group. The difference was statistically significant (P0.05). Under the inverted microscope, three drugs of PTX, M (PTX) and M (FA/PTX) were used to observe the cell morphology of the breast cancer cells in EMT-6 mice. The results showed that the cell morphology of the cells in the control group was obviously changed by.Hoechst 33258 staining, and the control group, PTX, M (PTX) and M. The rate of apoptosis was 5.8%, 15.8%, 15.2% and 21%. (2) in the tumor suppressor experiment in vivo. The animals in the PTX group were restless and screaming, while the mice in the M (PTX) and M (FA/PTX) two groups had no obvious abnormal response to the drug delivery. After 5 days of administration, the tumor repressor rate of PTX, M (PT X) and M (FA/PTX) groups was respectively For 29%, 46.5%, and 54.9%. in histological observation, the necrotic area was observed in three groups, and the area of the necrotic region in the M (FA/PTX) group was significantly higher than that in the PTX and M (PTX) group. The positive rates of the Bax protein expression in the PTX, M (PTX) and M (FA/PTX) groups were 8.3%, 50%, 60% and 75% in the control group, respectively, and 40%, 60% respectively. The results of 50%. flow cytometry showed that the apoptosis rates of PTX, M (PTX) and M (FA/PTX) groups were 1%, 36.6%, 55.9% and 66.1%., respectively, and the survival time of control group, PTX group, M (PTX) group and M (FA/PTX) group were 50%, 55.6%, 72.2% and 77.8%, and the median survival time was 33 days, 31 days, 34 days, respectively. And 42 days (log-rank test:c2=9.789, P=0.02). Conclusion: in vitro cell test, M (PTX) and M (FA/PTX) drugs can obtain the tumor inhibition effect on EMT-6 mouse breast cancer cells similar to PTX, and M (FA/PTX) shows a better tumor inhibition effect than M. It is better to induce tumor cell apoptosis.M (FA/PTX) more effectively to prolong the survival time of tumor bearing mice (.M (FA/PTX) in human breast cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9;R-332

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