【作者】 闻正顺；

【导师】 潘晓亮； 许梓荣；

【作者基本信息】 石河子大学 ， 动物营养与饲料科学， 2008， 硕士

【摘要】 多聚糖是一类数量巨大的海洋资源。由于其良好的生物相容性、可降解性、无毒以及独特的理化性质和生理活性,最近几十年在世界范围内掀起了研究热潮,在医药、生物、化工、轻工和农业等各方面都展示了巨大的应用潜力。其相关的理论研究和应用探索将会对糖生物学、糖工程以及海洋药物的发展和产业化产生深刻的影响。随着纳米技术的兴起,多聚糖纳米微粒在药物控释、靶向制剂与基因转导等医药方面的研究将会为海洋糖工程创新药物的开发提供新的思路。本课题以多糖(CS)为主要原料,应用纳米技术,构建了纳米多聚糖微粒(CNP),通过对CNP结构与性质的表征,裸鼠肝癌移植瘤病理试验,荧光定量PCR检测移植瘤组织血管内皮生长因子(VEGF)基因mRNA丰度,探讨了多聚糖纳米微粒对裸鼠肝癌移植瘤及其血管内皮生长因子(VEGF)表达的影响。主要研究结果如下:1、CNP的结构与性质表征AFM观察显示,CNP纳米粒子呈现为分散的规则微球形式;纳米粒度及Zeta电位分析仪测定显示,平均粒径为45.19nm.粒径范围为36.49～51.68nm;CNP的平均表面电荷为40.05mV,电荷分布范围25.03～56.18mV。2、肝癌移植瘤裸鼠试验及肿瘤病理变化观察选择健康、体重相近、遗传基础相似的6周龄雌性无胸腺裸鼠12只,按随机原则分为4组,3个剂量梯度组M1、M2、M3和对照组M4。在裸鼠背部皮下接种2×10~6个/0.5毫升人肝癌细胞BEL7402,当移植瘤直径约为0.5cm时(裸鼠背部皮下移植瘤直径为0.4～0.5cm为成瘤标准),开始每天灌胃给药一次,其中M1组为30mg/kg、M2组为60mg/kg、M3组为90mg/kg、M4组为等体积生理盐水。由肝癌移植瘤肿瘤生长曲线图可以看出,实验组(M1、M2、M3)曲线的斜率依次减小,均低于对照组(M4),分别为:15.223、11.983、6.1851。这表明随着药物CNP的剂量的增加,肿瘤的生长具有减慢的趋势。CNP动物给药21天后,肿瘤组织透射电镜观察发现对照组肿瘤细胞形态结构完整,细胞微绒毛丰富;剂量组肿瘤组织呈坏死性病理变化,肿瘤细胞多为坏死性与变性细胞,核固缩、核均质化溶解及核碎裂,胞浆内有大量空泡,细胞膜破裂形成细胞碎片,线粒体肿胀空化,内质网扩张囊泡化,并出现纤维化病变。3、荧光定量PCR试验荧光定量PCR检测各组瘤组织血管内皮生长因子(VEGF)mRNA水平相对表达量,M1、M2、M3、M4分别为0.1052、0.06731、0.02933、0.1465。由此可见VEGF基因mRNA水平随着CNP剂量增加而呈现表达量降低的趋势。综上所述,CNP主要通过破坏肿瘤细胞膜与亚细胞结构,使癌细胞呈现坏死性变化;在分子水平抑制血管内皮生长因子的表达,抑制肿瘤血管的生成来抑制肿瘤的生长与发展的。更多还原

【Abstract】 Chitin/Chitosan is one of the most important marine polysaccharides.Chitin and chitosan have attracted more attention in the world due to their good biocompatibility,degradability,low toxicity and special physicochemical properities and biological activities.They have prospective applications in many fields such as biomedicine,chemical industry,light industry and agriculture.The research on application and action mechanism of chitin/chitosan could contribute to the development of glycobiology, glycotechnology and marine drug.With the development of nanotechnology,nanoparticles and nanomaterials have been broadly applied in biomedicine and pharmacy.Reseach on the application of chitosan nanoparticles in drug controlled delivery,targeted drugs and gene carder will provide novel orientation for development of marine glycotechnology drug.In this study,polysaccharide nanoparticles(CNP)were constructed by nanotechnology using polysaccharide(CS)as the raw material.The structure and character of CNP was analyzed,the pathology assay of hepatocellular carcinoma xenografts in nude mice was demonstrated,as well as the vascular endothelial growth factor(VEGF)mRNA levels of hepatocellular carcinoma xenografts in nude mice were detected by fluorescence quantitative PCR.Therefore,the effects of CNP on growth of hepatocellular carcinoma xenografts and expression of VEGF in nude mice were discussed.The main results were as follows:1、Structure and characterization of polysaccharide nanoparticles(CNP):AFM observation indicated that the morphology of CNP was well-proportioned solid sphere and its size ranged from 36.49 to 51.68 nm with a size of 45.19 nm.The average potential was 40.05 mV,it ranged from 25.03 to 56.18 mV with 100%by Zetasizer Nano-ZS90.2、Test of liver carcinoma xenografts in nude mice and tumor pathology:Twelve female athymismus nude mice(6-week-old)were used in this study.After the period of acclimation,animals were randomly divided into four groups(M1、M2、M3、control(M4)).The nude mice were vaccinated with 2×10~6/0.5 ml human liver cancer cell line BEL7402 containing matrigel in the back skin for 7 to 10 days.As tumor diameter was about 0.5 cm(the standard is tumor diameter of 0.4 cm to 0.5cm),M1 was fed a 30mg/kg·Only CNP solution,M2 was fed a 60mg/kg·Only CNP solution,M3 was fed a 90mg/kg·Only CNP solution,Control(M4)was fed a physiological saline by intragastric administration.The result of liver carcinoma xenografts experiment in nude mice showed that the curve slope of the experimental groups of M1,M2 and M3 were 15.223,11.983 and 6.1851,respectively.In this experiment,it was showed that the more dose of CNP,the slower growth speed of tumor.Therefore,CNP could inhibit the growth of tumor.Tumor pathological test showed that after 21-day CNP administration,electron microscopy showed that the tumor cell morphology was complete and cell microvilli was rich in the control group.However, necrosis pathology changes of the tumor tissue with necrosis cells and degenerative cells,pyknosis,nuclear homogenization dissolved and nuclear fragmentation could be observed by transmission electron microscopy in the trial group.In the cytoplasm,there were a large number of vacuolus.Moreover,cells debris,swelling empty mitochondrial,dilated endoplasmic reticulum vesicles and fibrosis lesions could be investigated.Thus,CNP can promote tumor necrosis lesions.3、Fluorescence quantitative PCR experimentThrough the administration of different doses CNP for 21 days,it showed that the relative level of VEGF mRNA expression(M3,M2,M1,Control)were 0.02933,0.06731,0.1052,0.1465,respectively,by fluorescence quantitative PCR detection.It was demonstrated that when the dose of CNP increased,the levels of VEGF mRNA expression were reduced,indicating that there were some dose-effect relationship. Therefore,it was revealed that CNP could inhibit vascular endothelial growth factor(VEGF)gene mRNA expression.In conclusion,CNP could destroy the cell membrane and subcellular structure of tumor,resulting in the necrosis lesions of hepatocellular carcinoma cells.The expression mRNA level of VEGF was inhibited by CNP.Therefore,the growth and development of tumor was suppressed via angiogenesis blocking.更多还原