紫杉醇纳米粒时辰给药对肺癌的抑制作用研究

发布时间：2018-05-14 08:55

本文选题：肺癌 + 紫杉醇　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:本实验的研究目的是为了探究载紫杉醇(PTX)的聚己内酯-聚乙二醇-聚己内酯(poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone)(PCL-PEG-PCL,PCEC))聚合物纳米颗粒(PTX-NPs)时辰给药对肺癌的抗肿瘤作用及其相关机制,并初步筛选出一天中最适合给药的时辰点。方法:本实验具体分为体外实验和体内实验两部分完成。在体外研究中,首先以聚乙二醇(poly(ethylene glycol),PEG)和己内酯(ε-caprolactone)为原料,成功制备出PCEC聚合物,并将其作为载体;然后,通过薄膜分散法成功制备出PTX-NPs,用透射电子显微镜考察其形貌,通过动态光散射扫描仪评估其粒径,采用高效液相色谱仪(High performance liquid chromatography,HPLC)测得其百分包封率、载药率及在体外的药物释放特性。采用噻唑蓝(methylthiazoletetrazolium,MTT)试验研究PTX-NPs和空载体PCEC聚合物纳米颗粒的细胞毒性。通过荧光显微镜(Fluorescence microscope)观察PCEC聚合物纳米颗粒的体外细胞摄取功能。在体内研究中,先用常规方法培养A549肺癌细胞。然后,在相同的光照环境下饲养裸小鼠3周,建立相同的生物节律,光照时间(07:00-19:00),黑暗时间(19:00-07:00)。构建A549肺癌皮下异种移植瘤裸小鼠动物模型,待肿瘤体积长大至100mm3左右,随机分成三组,每组按0、5、10、15HALO(Hours After Light Onset设光照后小时)(即分别对应一天中的07:00、12:00、17:00、22:00四个时辰点)再分成四个亚组。其中两组作为治疗组,严格按照昼夜24小时的四个不同时辰点尾静脉注射ptx注射液和ptx-nps溶液。第三组作为对照组,尾静脉注射体积相同的生理盐水(ns)。ptx注射液和ptx-nps溶液均按ptx10mg/kg给药,每隔3天一次,共给药3次。每隔一天测量裸鼠体重、肿瘤长短径,计算肿瘤体积,绘制出肿瘤生长曲线、计算出肿瘤抑制率,并用小动物18f-fdg(fluorine-18-deoxyglucose)pet/ct(positronemissiontomography/computedtomography)显像观察与分析。治疗12天后处死裸小鼠,剥离瘤块,固定,采用免疫组织化学技术(immunohistochemistry,ihc)检测各组肿瘤标本中cd-31和ki-67的表达情况,并采用末端标记法(tdt-mediateddutpnickendlabeling,tunel)检测各组肿瘤标本中细胞凋亡的情况。结果:本研究制备的ptx-nps的外观呈球形结构,平均粒径约168nm,粒径分布均匀,具有初始快速释放而随后缓慢而持续释放药物的特点。mtt试验研究结果表明,空载体pcec聚合物纳米颗粒没有明显的细胞毒性作用;ptx-nps的体外细胞毒性随着药物浓度的增大而增大,并且在四个不同的时辰点给药ptx-nps溶液(ptx浓度相同),细胞活性呈现出逐渐递增的趋势:22:0017:0012:0007:00。体内抗肿瘤作用研究表明,ptx-nps溶液组和ptx注射液组的肿瘤抑制率均明显高于对照组(p0.01),并且两者均在22:00给药时的肿瘤抑制率最大,其数值分别为84.36%和68.84%(p0.05)。此外,治疗组的cd-31和ki-67的表达均呈现出递减的趋势:07:0012:0017:0022:00;治疗组的细胞凋亡数呈现出递增的趋势:07:0012:0017:0022:00。并且,在22:00给药时,PTX-NPs溶液组的CD-31和Ki-67的表达均比PTX注射液组低(P0.05),细胞凋亡数比PTX注射液组高(P0.05)。小动物18F-FDG PET/CT检查结果显示,在四个不同时辰点(07:00、12:00、17:00和22:00)给药,对照组的SUVmax值分别为:2.15±0.23,2.10±0.20,2.5±0.25和2.12±0.18。PTX注射液组的SUVmax值分别为:1.44±0.44,1.26±0.11,1.15±0.06和0.83±0.06。PTX-NPs溶液组的SUVmax值分别为:1.14±0.09,0.97±0.07,0.86±0.04和0.58±0.03。结论:1.本实验通过开环聚合法成功制备出PCEC聚合物,并将其制备成纳米颗粒,其毒性相对较低,且该纳米颗粒能比较容易地被肿瘤细胞摄取,是一种比较理想的、安全的药物载体。2.通过薄膜分散法成功制备出PTX-NPs,外观呈均匀球形,具有良好的载药率、包封率和药物释放特性;其粒径较小,且呈单分散分布,非常适合静脉给药。3.PTX-NPs的体外细胞毒性随着药物浓度的增大而增大,并具有时辰依赖性。4.在体内抗肿瘤作用研究中发现,PTX-NPs时辰给药对肺癌表现出显著的抑制作用,具有协同效应,并在15HALO给药时其抗肿瘤作用达到最佳,其机制与抑制肿瘤血管的生成、肿瘤细胞的增殖和促进细胞凋亡有关。
[Abstract]:Objective: the purpose of this study was to explore the antitumor effect and mechanism of the drug on lung cancer by PTX (poly (ethylene glycol) -poly (ethylene glycol) -poly (PCEC) -poly (PCEC) -poly (PCL-PEG-PCL, PCEC)). Methods: the most suitable time point for drug delivery. Methods: this experiment is divided into two parts in vitro and in vivo. In the study in vitro, first of all, poly (ethylene glycol), PEG) and caprolactone (epsilon -caprolactone) are used as raw materials to prepare PCEC polymers successfully and take them as carriers; and then, the membrane dispersion method is successfully made. PTX-NPs was prepared by transmission electron microscopy, and its particle size was evaluated by dynamic light scattering scanner (High performance liquid chromatography, HPLC). The percentage of encapsulation, drug loading rate and drug release in vitro were measured by high performance liquid chromatography (chromatography, HPLC). P (methylthiazoletetrazolium, MTT) was used to study P Cytotoxicity of TX-NPs and empty carrier PCEC polymer nanoparticles. The cell uptake of PCEC polymer nanoparticles in vitro was observed by fluorescence microscopy (Fluorescence microscope). In vivo, A549 lung cancer cells were cultured in a conventional method. Then, 3 weeks of nude mice were raised in the same light environment, and the same organisms were established. Rhythm, light time (07:00-19:00), dark time (19:00-07:00). A nude mouse model of subcutaneous xenograft tumor of A549 lung cancer was constructed, and the tumor volume grew up to about 100mm3, and was randomly divided into three groups, each group was treated with 0,5,10,15HALO (Hours After Light Onset set light after light) (that is, four times of 07:00,12:00,17:00,22:00 in one day, respectively. " The two groups were divided into four subgroups, of which two groups were treated with PTX injection and ptx-nps solution strictly according to the caudal vein at 24 hours of day and night. The third group was used as the control group. The same volume of saline (NS).Ptx injection and ptx-nps solution were given by ptx10mg/kg, once every 3 days. 3 times. Every day, the body weight of the nude mice was measured, the tumor size was measured, the tumor growth curve was drawn, the tumor inhibition rate was calculated, and the 18F-FDG (fluorine-18-deoxyglucose) pet/ct (positronemissiontomography/computedtomography) imaging of the small animals was observed and analyzed. The nude mice were killed, the lump of the tumor was stripped and fixed after 12 days. Immunohistochemistry (IHC) was used to detect the expression of cd-31 and Ki-67 in all the tumor specimens, and the apoptosis of the tumor specimens was detected by tdt-mediateddutpnickendlabeling (tdt-mediateddutpnickendlabeling, TUNEL). Results: the appearance of ptx-nps in this study was spherical, with an average size of about 16 8nm, the particle size distribution is uniform, with the initial rapid release and the subsequent slow and sustained release of the drug.Mtt test results show that the no-load pCEC polymer nanoparticles have no obvious cytotoxicity, and the cytotoxicity of ptx-nps in vitro increases with the increase of drug concentration, and is given to ptx-np at four different hour points. The s solution (PTX concentration was the same), the cell activity showed a gradual increasing trend: the tumor inhibition rate in the ptx-nps solution group and PTX injection group was significantly higher than that of the control group (P0.01), and the tumor inhibition rates were the highest at 22:00, and the values were 84.36% and 68.84% respectively (P 0.05). In addition, the expression of cd-31 and Ki-67 in the treatment group showed a decreasing trend: 07:0012:0017:0022:00; the number of apoptotic cells in the treatment group showed an increasing trend: 07:0012:0017:0022:00. and the expression of CD-31 and Ki-67 in PTX-NPs solution group was lower than that of PTX injection group at 22:00 (P0.05), and the number of apoptotic cells was higher than that of PTX injection. Group high (P0.05). The results of 18F-FDG PET/CT examination of small animals showed that at four different hour points (07:00,12:00,17:00 and 22:00), the SUVmax values of the control group were respectively: 2.15 + 0.23,2.10 + 0.20,2.5 + 0.25 and 2.12 + 0.18.PTX injection groups, respectively: 1.44 + 0.44,1.26 +, 0.11,1.15 + 0.06 and 0.83 + 0.06.PTX-NPs solution groups The values are as follows: 1.14 + 0.09,0.97 + 0.07,0.86 + 0.04 and 0.58 + 0.03. conclusion: 1. this experiment successfully prepared PCEC polymer by open ring polymerization, and prepared it into nano particles, its toxicity is relatively low, and the nanoparticles can be easily absorbed by tumor cells. It is an ideal, safe drug carrier.2. through thin film. The dispersion method successfully prepared PTX-NPs with a uniform appearance and spherical appearance, with good drug loading rate, encapsulation efficiency and drug release characteristics. Its particle size is small, and it is monodisperse distribution. The cytotoxicity of.3.PTX-NPs in vitro is very suitable for the increase of drug concentration, and the antitumor effect of time dependent.4. in the body is studied. It is found that PTX-NPs time administration has a significant inhibitory effect on lung cancer and has a synergistic effect, and its anti-tumor effect is best when 15HALO is administered. The mechanism is related to inhibiting the formation of tumor vessels, proliferation of tumor cells and promoting cell apoptosis.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

【参考文献】