二硫键紫杉醇聚合物胶束抑制乳腺肿瘤的体内外评价

发布时间：2019-04-22 20:30

【摘要】：紫杉醇(PTX)被广泛用于乳腺癌、非小细胞肺癌等实体肿瘤的一线治疗。但紫杉醇水溶性差,须将无水乙醇和聚氧乙基代蓖麻油作为溶媒,但易引发严重的过敏反应,限制了其临床使用。为克服紫杉醇的水溶性差及实现肿瘤靶向控释,诸多紫杉醇的新型制剂应运而生。其中,氧化还原型纳米紫杉醇胶束制剂利用肿瘤细胞内外及肿瘤细胞与正常细胞之间的氧化还原电位差,可实现高效的肿瘤细胞内药物触发释放。减少血液循环或正常细胞内的药物泄漏,降低相关的药物毒副作用。目的:本研究以一种选择性氧化还原型紫杉醇纳米制剂[P(PEGMEA)-co-P(PDPHEMA)-g-PTX]作为研究对象,通过体内外实验评价其作用于乳腺癌细胞株和动物模型的效果,为该新型紫杉醇的临床应用提供实验依据。方法:(1)以生理盐水稀释成不同浓度梯度的二硫键紫杉醇处理乳腺癌MCF-7细胞株,24h后,MTT法测定MCF-7细胞生长抑制情况,并绘制浓度抑制率曲线,摸索其IC50。将MCF-7、MDA-MB-231,MCF-10A及U937共4种细胞系分别按照空白对照组:共聚物,阳性对照组:紫杉醇(PTX),给药组:二硫键紫杉醇纳米制剂。24h后用MTT法检测细胞生长的抑制情况。另用受试药物培养一组MDA-MB-231细胞,48h后,流式细胞仪检测细胞凋亡情况。(2)制作MCF-7裸鼠乳腺癌动物模型。选择荷瘤裸鼠18只,随机分为3组(空白基质对照组:共聚物,16mg/kg。阳性对照组:PTX,5mg/kg。给药组:二硫键紫杉醇纳米制剂,16mg/kg),并为裸鼠称重、测量瘤体大小。给药结束后检测血常规,计算肿瘤抑制率;同时,留取裸鼠心、肝、脾、瘤等脏器进行HE染色。另用同样方法培养三组裸鼠,但投药浓度为(共聚物,5mg/kg,PTX,5mg/kg;二硫键紫杉醇,5mg/kg)。(3)实验结果以均数±标准差(x±SD)表示,组间比较采用ANOVA方差分析,两组间比较用t-检验,用SPSS 18.0对组间进行统计分析,以P0.05为统计学差异具有显著性。结果:(1)MTT试验结果显示二硫键紫杉醇纳米制剂随着浓度增加,对MCF-7细胞活力抑制作用逐渐加强,存在非常显著的剂量与效应关系,IC50=6.13μg/ml。以10μg/ml进行投药,与共聚物相比,对MCF-7细胞、MDA-MB-231细胞以及人正常乳腺细胞MCF-10A的抑制率分别为:52.4%,56.0%,22.4%(p0.05),对U937细胞活力具有促进作用(24.3%),与10μg/ml紫杉醇组相比,对不同细胞的抑制率差异无统计学意义。二硫键紫杉醇与紫杉醇具有诱导MDA-MB-231细胞凋亡的作用。(2)体内试验中,共聚物对照组和二硫键紫杉醇组裸鼠状态较好。紫杉醇组裸鼠在给药2周后精神状态开始萎靡,活动量减少并出现2只裸鼠死亡。(1)表观药物浓度相同时,紫杉醇对照组和二硫键紫杉醇给药组肿瘤生长被抑制,分别为:3590±877、4125±792mm3;瘤重分别为:3.62±1.0、4.58±0.9g。(2)实际紫杉醇含量相同,PTX组为:1936±399mm3,二硫键紫杉醇组为:1637±371 mm3。紫杉醇及二硫键紫杉醇组瘤体重量均为2.00±0.18g,抑瘤率为33.7%。二硫键紫杉醇无明显骨髓抑制。HE染色组织切片显示:肿瘤组织可见明显坏死区域,心肺肾无明显改变,肝、脾见少量炎细胞浸润,二硫键紫杉醇组与紫杉醇组对肿瘤组织杀伤方面在病理学上未见明显差别。结论:二硫键紫杉醇为水溶性药物,无需聚氧乙基代蓖麻油和乙醇作为溶媒,消除了过敏反应。二硫键紫杉醇纳米制剂对乳腺癌MCF-7细胞活力具有非常显著的抑制作用,其抑制作用具有浓度依赖性。二硫键紫杉醇与PTX均明显抑制乳腺癌细胞增殖,对巨噬细胞U937具有免疫促进作用,可诱导MDA-MB-231细胞凋亡。在体内实验中,在取得与紫杉醇相同的抑瘤效果时,二硫键紫杉醇组的紫杉醇含量更低。在达到最佳有效浓度后,再加大二硫键紫杉醇的浓度并不能明显提高抑瘤效果。其抑制性作用主要集中于肿瘤组织,毒副作用低。
[Abstract]:Paclitaxel (PTX) is widely used for the first-line treatment of solid tumors such as breast cancer and non-small cell lung cancer. However, the water-solubility of paclitaxel is poor, and absolute ethyl alcohol and polyoxyethylene ether can be used as the solvent, but it is easy to cause serious allergic reaction and limit its clinical use. In order to overcome the water-solubility difference of the paclitaxel and to realize the targeted controlled release of the tumor, the novel preparation of the taxol has emerged. In which, the oxidation-reduction type nano-taxol micelle preparation can realize the drug-triggered release of the high-efficiency tumor cell by utilizing the redox potential difference between the inside of the tumor cells and the tumor cells and the normal cells. And the drug leakage in the blood circulation or normal cells is reduced, and the related medicine toxic and side effect is reduced. Objective: In this study, a selective oxidation-reduction type paclitaxel nano-preparation[P (PEGMEA)-co-P (PDPHEMA)-g-PTX] was used as the research object, and the effect of it on the breast cancer cell line and animal model was evaluated by out-of-vivo experiment, and the experimental basis for clinical application of the novel paclitaxel was provided. Methods: (1) The MCF-7 cell line of breast cancer was treated with the disulfide-bond paclitaxel which was diluted with normal saline to different concentration gradient. After 24 h, the growth inhibition of MCF-7 cells was determined by MTT method, and the concentration-inhibition curve was plotted and the IC50 of the MCF-7 cell line was determined. MCF-7, MDA-MB-231, MCF-10A and U937 cell lines were divided into two groups according to the control group: the copolymer, the positive control group, the paclitaxel (PTX) and the treated group: the disulfide-bond paclitaxel nano-preparation. The inhibition of cell growth was detected by the MTT method after 24 h. After a group of MDA-MB-231 cells were cultured with the test drug, the cell apoptosis was detected by flow cytometry after 48 h. And (2) preparing the MCF-7 nude mouse mammary cancer animal model. 18 tumor-bearing nude mice were randomly divided into 3 groups (blank matrix control group: copolymer,16 mg/ kg). Positive control group: PTX,5 mg/ kg. Treated group: disulfide-bond paclitaxel nano-preparation,16 mg/ kg) and weighed in nude mice to measure the size of the tumor. After the administration is over, the blood routine is detected and the tumor inhibition rate is calculated; at the same time, the viscera of the naked mouse heart, the liver, the spleen, the tumor and the like are left to carry out HE staining. Three groups of nude mice were cultured in the same manner, but the administration concentration was (copolymer,5 mg/ kg, PTX,5 mg/ kg; disulfide bond paclitaxel,5 mg/ kg). (3) The results of the experiment were represented by mean square standard deviation (x-SD). The ANOVA and variance analysis were used for the comparison between the two groups. The statistical analysis between the two groups was performed with SPSS 18.0, and the difference was significant between the two groups. Results: (1) The results of the MTT assay showed that the concentration of the disulfide-linked paclitaxel nano-preparation increased with the increase of the concentration, and the inhibitory effect on the activity of the MCF-7 cells was gradually enhanced, and there was a very significant dose-and-effect relationship, with the IC50 of 6.13. m The inhibition rate of MCF-7 cells, MDA-MB-231 cells and human normal breast cells (MCF-10A) was 52.4%, 56.0% and 22.4% (p0.05), respectively. The inhibition rate of different cells was not statistically significant. Disulfide and paclitaxel play a role in inducing the apoptosis of MDA-MB-231 cells. (2) In vivo test, the state of the nude mice of the copolymer control group and the disulfide bond paclitaxel group was better. After 2 weeks of administration, the state of mental state of the nude mice of the Paclitaxel group began to fall, and the amount of activity decreased and two nude mice died. (1) When the apparent drug concentration was the same, the tumor growth of the paclitaxel-treated group and the paclitaxel-treated group was inhibited, namely,3590-877,4125-792 mm3, and the tumor weight: 3.62-1.0, 4.58-0.9g, respectively. (2) The actual taxol content was the same, the PTX group was:1936-399 mm3, and the disulfide-bond paclitaxel group was 1637-371mm3. The weight of the paclitaxel and the disulfide-bonded paclitaxel group was 2.00-0.18 g, and the tumor-inhibiting rate was 33.7%. Disulfide-bound paclitaxel had no significant bone marrow inhibition. The histological sections of HE staining showed that the tumor tissue had obvious necrotic area, no obvious change in the heart and lung, and small amount of inflammatory cell infiltration in the liver and spleen, and no significant difference was seen in the pathology of the anti-killing of the tumor by the combination of the paclitaxel group and the paclitaxel group. Conclusion: The disulfide-bond paclitaxel is a water-soluble drug, and it is not necessary to use the polyoxyethylene ether and the ethanol as the solvent to eliminate the allergic reaction. The disulfide-linked taxol nano-preparation has a very significant inhibitory effect on the activity of the breast cancer MCF-7 cell, and the inhibitory effect of the disulfide-linked taxol nano-preparation has the concentration dependence. Both the disulfide bond and PTX inhibit the proliferation of breast cancer cells, and it has an immune function to the macrophage U937 and can induce the apoptosis of the MDA-MB-231 cells. In vivo experiments, the paclitaxel content of the disulfide-bond paclitaxel group was lower when the same tumor-inhibiting effect as paclitaxel was obtained. After the optimal effective concentration is reached, the concentration of the disulfide-bound paclitaxel can not be increased obviously, and the tumor-inhibiting effect can not be obviously improved. The inhibitory effect is mainly concentrated on the tumor tissues, and the toxic and side effects are low.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

【参考文献】