转铁蛋白受体结合肽与超声微泡联合介导抗肿瘤药物治疗脑胶质瘤的实验研究

发布时间：2018-05-04 08:52

本文选题：转铁蛋白受体结合肽 + 5-氟尿嘧啶　；参考：《重庆医科大学》2016年博士论文

【摘要】：目的脑胶质瘤是中枢神经系统发生率最高的恶性肿瘤,因血脑屏障(Blood brain barrier,BBB)的存在以及传统抗肿瘤药物对肿瘤细胞选择性不强,导致目前脑胶质瘤的临床疗效较差,生存期短。研究发现,超声微泡能无创、可逆开放BBB;转铁蛋白受体(Transferrin receptor,TfR)在脑胶质瘤细胞膜上高表达;转铁蛋白受体结合肽(HAIYPRH)能与脑胶质瘤细胞膜TfR特异性结合。本课题首次将超声微泡和结合肽两种不同的靶向技术联合运用,超声微泡介导使药物透过BBB,在脑部定点释放;结合肽介导使药物对脑胶质瘤细胞产生选择性,从而实现高效率的脑肿瘤靶向治疗。鉴于此,本课题以5-氟尿嘧啶(5-fluorouracil,5-FU)和表柔比星(Epirubicin,EPI)为工具药,评价HAIYPRH-5-FU和HAIYPRH-EPI两种复合物体外抗脑胶质瘤细胞活性;同时评价HAIYPRH-5-FU复合物超声微泡的体内靶向性及抗脑胶质瘤活性。方法在第一部分试验中,以EPI为工具药,谷氨酸为连接物,制备HAIYPRH-EPI复合物;以5-FU为工具药,6-氨基己酸为连接物,制备HAIYPRH-5-FU复合物。在第二部分试验中,主要明确复合物的抗肿瘤活性及其与TfR表达的相关性。首先采用流式细胞术检测U87与LN229脑胶质瘤细胞膜表面TfR1表达差异;然后以U87和LN229胶质瘤细胞为研究对象,采用mtt法考察各组药物对细胞增殖抑制作用,计算存活率评价细胞毒性,通过透射电镜和检测caspase3,8,9活性观察细胞凋亡,利用epi本身的荧光特性,借助荧光显微镜定性和流式细胞术半定量观察epi和haiyprh-epi细胞摄取,采用hplc-ms/ms法定量观察5-fu和haiyprh-5-fu细胞摄取。在第三部分试验中,我们通过正交设计优化处方,成功制备载haiyprh-5-fu超声微泡,测定包封率及载药量,并进行形态观察、稳定性观察、粒径、粒度分布与电位检测。在第四部分试验中,主要考察载haiyprh-5-fu超声微泡的体内靶向性及抗脑胶质瘤活性,采用立体定向技术建立大鼠ln229胶质瘤模型,通过mri和he染色验证模型制作成功,采用hplc-ms/ms法测定各组织和血液中的5-fu及haiyprh-5-fu的浓度,考察其靶向效率,并通过mri观察复合物超声微泡对脑胶质瘤的抑制作用,同时比较各组大鼠的生存期和体重。结果在第一部分试验中,我们成功制备haiyprh-epi和haiyprh-5-fu两种复合物,质谱鉴定分子量与预期一致,hplc测定其纯度分别为99.03%和99.07%。在第二部分试验中,流式细胞术检测脑胶质瘤细胞膜表面tfr1表达结果显示:u87细胞膜表面tfr1呈低表达,而ln229细胞膜表面tfr1呈高表达;细胞毒性与细胞凋亡结果显示:haiyprh-epi与haiyprh-5-fu对tfr呈低表达的u87细胞的细胞毒性和促凋亡活性较弱,而在tfr高表达的ln229细胞中则呈现明显的细胞毒性和促凋亡活性,且细胞毒性呈剂量依赖性,外源性加入25μmtf能明显加强复合物对ln229细胞的细胞毒性和促凋亡活性,而epi和5-fu在u87和ln229细胞中的细胞毒性和细胞凋亡活性无明显差别,tf的加入对该类效应也无明显影响;细胞摄取结果与细胞毒性、细胞凋亡结果相吻合,显示:主要依赖被动转运入细胞的epi或5-fu在u87和ln229细胞中的摄取无明显差别,但haiyprh-epi或haiyprh-5-fu在两种细胞中的摄取存在显著差异,两种复合物在ln229细胞中的摄取明显高于u87细胞,且外源性加入25μmtf能够显著加强ln229细胞对两种复合物的摄取。在第三部分实验中,以haiyprh-5-fu为试验药物,对制备影响较显著的4个因素进行正交试验,以包封率为指标进行评分,优选出最佳制备处方,即聚乳酸-羟基乙酸共聚物(plga)800mg,司盘801.5ml,初乳水相体积1ml,聚乙烯醇(pva)体积45ml,并以最佳处方成功制备载haiyprh-5-fu超声微泡。采用hplc-ms/ms法测得其包封率与载药量分别为(45.87±0.71)%和(0.86±0.19)%;在形态及稳定性观察项中,冻干粉重悬于去离子水中,显微镜下微泡分布均匀,无粘连,7d时两种微泡仍分布均匀,分散度较好;malvernzatasizernanozs激光粒度分析仪检测其平均粒径与zeta电位分别为(476.3±143.2)nm和-(12.8±5.86)mv。在第四部分试验中,mri检查和he染色证实大鼠脑胶质瘤模型建立成功。荷瘤大鼠给药并进行超声辐照,于1h、2h、4h后测定组织分布和靶向效率,结果显示,5-fu超声微泡组与haiyprh-5-fu超声微泡组均呈现明显的脑部靶向性;药物处理前后通过mri观察肿瘤体积,结果提示,与5-fu组相比,同剂量的5-fu超声微泡组第21d肿瘤体积明显减小,与haiyprh-5-fu(0.5mm/kg)组相比,低(0.1mM/kg)、中(0.5mM/kg)、高(1mM/kg)浓度的HAIYPRH-5-FU超声微泡组在第14d和第21d肿瘤体积均明显减少,并呈剂量依赖性;各试验组从建模第10天起开始记录体重,与空白微泡组、5-FU(0.5mM/kg)组和HAIYPRH-5-FU(0.5mM/kg)组相比,中(0.5mM/kg)、高浓度(1mM/kg)HAIYPRH-5-FU超声微泡组大鼠体重从第28天开始出现显著性差异,且HAIYPRH-5-FU超声微泡组浓度越大体重变化越小,呈现剂量依赖性;在生存曲线中,空白对照组、空白微泡组、5-FU(0.5mM/kg)组、HAIYPRH-5-FU(0.5mM/kg)组、5-FU超声微泡(0.5mM/kg)组、低浓度HAIYPRH-5-FU超声微泡(0.1mM/kg)组、中浓度HAIYPRH-5-FU超声微泡(0.5mM/kg)组、高浓度HAIYPRH-5-FU超声微泡(1mM/kg)组的中位生存期分别为26天、24天、34天、25天、45天、49天、52天和63天。结论HAIYPRH-抗肿瘤药物复合物对TfR呈高表达的脑胶质瘤细胞呈现出明显的选择性和抗肿瘤活性,HAIYPRH-抗肿瘤药物复合物超声微泡对接种TfR呈高表达的脑胶质瘤细胞的荷瘤大鼠呈现出明显的体内靶向性和抗肿瘤活性。本课题的实施为开发新的具有临床应用前景的抗肿瘤药物提供了新的理论和试验依据。
[Abstract]:Objective glioma is a malignant tumor of the highest incidence in the central nervous system. The existence of Blood brain barrier (BBB) and the poor selectivity of traditional antitumor drugs on tumor cells lead to the poor clinical efficacy and short survival time of brain glioma. Transferrin receptor (TfR) is highly expressed on the glioma cell membrane, and transferrin receptor binding peptide (HAIYPRH) can be specifically associated with the TfR cell membrane of glioma cells. This subject is the first time to combine the two different target techniques of ultrasonic microbubbles and binding peptides. Ultrasound microbubbles mediate the release of drugs through BBB, binding to the brain; In view of this, 5- fluorouracil (5-fluorouracil, 5-FU) and epirubicin (Epirubicin, EPI) are used as a tool to evaluate the activity of two kinds of compound objects of HAIYPRH-5-FU and HAIYPRH-EPI against glioma cells, and evaluate HAIY at the same time. In vivo targeting and anti brain glioma activity of PRH-5-FU complex ultrasound microbubbles. Methods in the first part of the experiment, EPI was used as a tool and glutamic acid was used as a connector to prepare HAIYPRH-EPI complexes; 5-FU was used as a tool and 6- amino hexanic acid was used as a connector to prepare HAIYPRH-5-FU complex. In the second part of the experiment, the main determination of the resistance of the complex was made. Tumor activity and its correlation with TfR expression. First, flow cytometry was used to detect the difference of TfR1 expression on the surface of U87 and LN229 glioma cells. Then U87 and LN229 glioma cells were used as the research object. The inhibitory effect of the drugs on cell proliferation was investigated by MTT method and the survival rate was calculated to evaluate the cytotoxicity by transmission electron microscopy and examination. Caspase3,8,9 activity was observed to observe the apoptosis of cells. Using fluorescence characteristics of EPI itself, the uptake of EPI and haiyprh-epi cells was observed by fluorescence microscopy and flow cytometry. The uptake of 5-FU and haiyprh-5-fu cells was quantitatively observed by hplc-ms/ms. In the third part, we optimized the prescription by orthogonal design and successfully prepared the prescription. Haiyprh-5-fu ultrasound microbubbles were carried out and the encapsulation efficiency and drug loading were measured. Morphological observation, stability observation, particle size, particle size distribution and potential detection were carried out. In the fourth part, the target of haiyprh-5-fu ultrasound microbubbles and the activity of anti glioma were mainly investigated. The model of rat ln229 glioma was established by using stereotactic technique, and the model was established by using stereotactic technique. MRI and he staining verification model was made successfully. The concentration of 5-FU and haiyprh-5-fu in tissues and blood was measured by hplc-ms/ms method, and the target efficiency was investigated. The inhibitory effect of ultrasonic microbubbles on brain glioma was observed by MRI, and the survival time and weight of the rats were compared. The results were successful in the first part of the experiment. Two compounds of haiyprh-epi and haiyprh-5-fu were prepared. The molecular weight of mass spectrometry was the same as expected. The purity of HPLC was 99.03% and 99.07%. in second parts. The results of TfR1 expression on the membrane surface of brain glioma cells by flow cytometry showed that the TfR1 in the membrane surface of U87 cells was low, and the TfR1 of ln229 cell membrane was highly expressed. The results of cytotoxicity and apoptosis showed that the cytotoxicity and apoptosis activity of haiyprh-epi and haiyprh-5-fu cells with low expression of TfR were weak, but in ln229 cells with high expression of TfR, the cytotoxicity and apoptosis activity were obvious, and the cytotoxicity was dose-dependent. The exogenous addition of 25 mu MTF could obviously strengthen the compound. The cytotoxicity and apoptosis activity of ln229 cells were not significantly different from that of EPI and 5-FU in U87 and ln229 cells. The addition of TF had no obvious effect on this effect, and the results of cell uptake coincided with the cytotoxicity and apoptosis results, which showed that it mainly depended on the passive transport of EPI or 5-FU into cells. There was no significant difference in the uptake of U87 and ln229 cells, but there was a significant difference in the uptake of haiyprh-epi or haiyprh-5-fu in the two cells. The uptake of the two compounds in ln229 cells was significantly higher than that of U87 cells, and the exogenous addition of 25 mu MTF could significantly enhance the uptake of the two compounds by ln229 cells. In the third experiment, Hai Yprh-5-fu was a test drug. Orthogonal test was carried out on 4 factors which had significant influence on preparation. The optimum preparation prescription was selected by the index of encapsulation efficiency, namely, poly (PLGA) 800mg, 801.5ml, 1ml of colostrum and 45ml of polyvinyl alcohol (PVA), and the preparation of haiyprh-5-fu with the best prescription. The encapsulation efficiency and drug loading of ultrasonic microbubbles were measured by hplc-ms/ms method (45.87 + 0.71)% and (0.86 + 0.19)% respectively. In the observation items of morphology and stability, the freeze-dried powder was suspended in the deionized water. The microbubbles were evenly distributed under the microscope without adhesion, and the two kinds of microbubbles were still evenly distributed, and the dispersion degree was better at 7d, and the malvernzatasizernanozs laser particle size fraction was better. The average particle size and zeta potential were measured by (476.3 + 143.2) nm and - (12.8 + 5.86) mv. respectively in fourth parts. MRI examination and he staining proved that the rat glioma model was established successfully. The tumor bearing rats were given and irradiated by ultrasound, and the tissue distribution and target efficiency were measured after 1h, 2h, 4h, and the results showed that 5-FU ultrasound microbubbles and haiy were found. The prh-5-fu ultrasound microbubble group showed obvious brain targeting, and the tumor volume was observed by MRI before and after the drug treatment. The results suggested that the volume of 21d tumor in the same dose 5-FU ultrasound microbubble group decreased significantly, and compared with the haiyprh-5-fu (0.5mm/kg) group, the low (0.1mM/kg), middle (0.5mM/kg), and high (1mM/kg) concentration of HAIYPRH-5-FU ultrasound microscale was compared with the haiyprh-5-fu (0.5mm/kg) group. The volume of the tumor in the group 14d and 21d decreased significantly and was dose-dependent. The body weight of the experimental groups began to be recorded from the tenth day of modeling, compared with the blank microbubble group, the 5-FU (0.5mM/kg) group and the HAIYPRH-5-FU (0.5mM/kg) group, and the weight of the rats in the high concentration (1mM/kg) HAIYPRH-5-FU ultrasonic microbubble group began to appear from twenty-eighth days. Difference, and the greater the concentration of HAIYPRH-5-FU ultrasound microbubbles, the smaller the weight change, the more dose dependence; in the survival curve, the blank control group, the blank microbubble group, the 5-FU (0.5mM/kg) group, the HAIYPRH-5-FU (0.5mM/kg) group, the 5-FU ultrasound microbubble (0.5mM/kg) group, the low concentration HAIYPRH-5-FU ultrasonic microbubble group (0.1mM/kg), and the middle concentration HAIYPRH-5-FU ultrasonic microbubbles (0.5 MM/kg group, the median survival time of the high concentration HAIYPRH-5-FU ultrasound microbubbles (1mM/kg) group was 26 days, 24 days, 34 days, 25 days, 45 days, 49 days, 52 days and 63 days. Conclusion the HAIYPRH- antitumor drug complex showed obvious selectivity and antitumor activity to the high expression of TfR glioma cells, HAIYPRH- antitumor drug complex ultrasound microbubbles. The tumor bearing rats inoculated with high expression of TfR glioma cells showed significant in vivo targeting and antitumor activity. The implementation of this topic provides a new theoretical and experimental basis for the development of new antitumor drugs with clinical applications.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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