脑胶质瘤靶向纳米药物及其与放疗联用的实验研究

发布时间：2018-12-29 13:25

【摘要】：颅内恶性肿瘤中脑胶质瘤以44%的发病率居于首位,具有病程短、治愈难、存活时间短和预后差等特点,脑胶质瘤病人的生存期仅有6~12个月。脑胶质瘤的治疗方案以手术、放疗和化疗为主,但手术治疗由于其解剖特点无法全部切除,化疗由于血脑屏障的存在可用的药物很少,且疗效不佳。因此,提高脑肿瘤的放化疗靶向性和疗效,延长胶质瘤患者的生存期已成为研究的热点。纳米靶向输送系统因其独特的理化性质,可通过载药高分子和靶向高分子的共组装,将药物选择性地送到病变部位或病变细胞,提高药物的生物利用度,降低毒副作用。各种肿瘤靶向的给药体系成为近年来人们关注的热点[1]。狂犬病病毒(RVG)是自然界存在的嗜神经病毒,很容易越过血脑屏障,侵袭脑组织和神经中枢。研究发现,RVG中的29个氨基酸序列构成的嗜神经性病毒衍生肽(RVG29-YTIWMPENPRPGTPCDIFTNSRGKRASNG)保留了RVG的嗜神经特性。有研究将RVG29键合到聚酰胺-胺树状聚合物(PAMAM)和聚亚乙胺(PEI)上,用以担载和输送核酸类药物,具有一定的脑靶向效果,但对细胞毒类抗癌药物的脑靶向输送应用未见报道。葡聚糖可以在肝、脾、肾和消化道被葡聚糖酶降解,毒性极低,用葡聚糖修饰的高分子胶束在体内具有类似聚乙二醇(PEG)的“隐形”效果。载药葡聚糖纳米颗粒可减少肝脏对药物的清除,提高在高度血管化的癌变组织中的药物浓度。在本研究中,我们采用水溶性的葡聚糖作为药物载体合成了靶向纳米胶束M(RVG,PTX),用立体定向技术建立原位脑胶质瘤动物模型,通过活体成像、激光共聚焦显微镜、MTT、流式细胞术、Western Blot、real-time PCR、核磁共振等方法,研究了RVG29靶向纳米药物对脑胶质瘤的抑制效果,并探讨与放疗结合对C6细胞生长抑制的增效作用。首先,通过靶向实验证实RVG29修饰后纳米胶束能通过血脑屏障达到颅内,并证实其靶向作用与RVG29有关。其次,通过MTT和核磁检测及生存期统计,证实靶向纳米胶束能够提高紫杉醇对胶质瘤的抑制率,减小颅内肿瘤的体积,延长荷瘤动物的生存期。同时,纳米靶向胶束给药与体外放疗联用可减低药物剂量,提高对胶质瘤的抑制率。并进一步证明C6细胞的凋亡伴随着Caspase家族和Bax的表达升高和Bcl-xl的表达下调,以及MDR和P-gp的相应变化。本研究结果将为脑胶质瘤等颅内病变的治疗提供新思路和实验依据。
[Abstract]:The incidence of glioma in intracranial malignant tumors was 44%, which was characterized by short course of disease, difficult to cure, short survival time and poor prognosis. The survival time of glioma patients was only 6 ~ 12 months. Surgery, radiotherapy and chemotherapy are the main treatments for glioma, but the surgical treatment can not be completely resected because of its anatomical characteristics. Because of the existence of blood-brain barrier, there are few drugs available for chemotherapy, and the curative effect is not good. Therefore, improving the radio-chemotherapeutic targeting and efficacy of brain tumors and prolonging the survival time of glioma patients has become a hot topic. Because of its unique physical and chemical properties, nano-targeted delivery system can selectively deliver drugs to diseased sites or cells through co-assembly of drug-loaded and targeted polymers, thus increasing the bioavailability of drugs and reducing side effects. Various tumor targeting drug delivery systems have become the focus of attention in recent years [1]. Rabies virus (RVG) is a neurotropic virus that exists in nature. It can easily cross the blood-brain barrier and invade the brain tissue and nerve center. It was found that the neurovirus derived peptide (RVG29-YTIWMPENPRPGTPCDIFTNSRGKRASNG) composed of 29 amino acid sequences in RVG preserved the neurotropic properties of RVG. It has been studied that RVG29 is bonded to polyamide-amine dendrimer (PAMAM) and polyethylamine (PEI) to support and transport nucleic acid drugs, and has a certain brain targeting effect. However, the brain targeted delivery of cytotoxic anticancer drugs has not been reported. Dextran can be degraded by glucanase in liver, spleen, kidney and digestive tract, and its toxicity is very low. The polymer micelles modified with dextran have the "invisible" effect similar to polyethylene glycol (PEG) in vivo. Drug loading dextran nanoparticles can reduce liver drug clearance and increase drug concentration in highly vascularized cancerous tissues. In this study, we synthesized targeted nano-micelle M (RVG,PTX) using water-soluble dextran as drug carrier, and established in situ brain glioma animal model by stereotactic technique. In vivo imaging, confocal laser microscopy, MTT, Flow cytometry (FCM) and Western Blot,real-time PCR, NMR were used to study the inhibitory effect of RVG29 targeting nanopharmaceuticals on glioma and to explore the synergistic effect of combination with radiotherapy on the growth inhibition of C6 cells. Firstly, it was proved by targeting experiment that RVG29 modified nanoparticles could reach the brain through the blood-brain barrier, and that its targeting effect was related to RVG29. Secondly, through MTT, nuclear magnetic detection and survival statistics, it is proved that targeted micelles can improve the inhibition rate of paclitaxel on glioma, reduce the volume of intracranial tumor, and prolong the survival time of tumor bearing animals. At the same time, the combination of nano-targeted micelle administration and in vitro radiotherapy can reduce the dosage of drug and increase the inhibition rate of glioma. It was further demonstrated that the apoptosis of C6 cells was accompanied by the increased expression of Caspase family and Bax, the down-regulation of Bcl-xl expression, and the corresponding changes of MDR and P-gp. The results of this study will provide new ideas and experimental evidence for the treatment of intracranial lesions such as gliomas.
【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】：R739.41

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