携带奥沙利铂、赫赛汀的复合金属纳米粒子对HER2阳性胃癌的分子靶向作用

发布时间：2018-06-07 03:15

本文选题：人类表皮生长因子2阳性 + 胃癌　；参考：《浙江大学》2014年硕士论文

【摘要】：背景：胃癌是严重危害人类生命健康的重大疾病,世界卫生组织癌控项目数据显示,全球每年胃癌新发病例约93万,死亡人数约70万,占所有癌症死亡率的1/10;我国每年新发胃癌患者达40万人,死亡人数达30万,患病率和死亡率均是世界平均水平的2倍多。目前在我国胃癌早期检出率不到10%。超过90%以上的胃癌患者于就诊时已是进展期以上,或在确诊时已经发生了转移或播散,从而失去了手术的机会。因此,胃癌的化学治疗是国内绝大多数胃癌患者的主要治疗方法,在我国有着重要的研究意义。但是,当前临床应用的常规化疗药物全身毒副作用较大,患者耐受性差；肿瘤细胞对化疗药物产生耐药等,都容易导致化疗的失败。针对胃癌常规化疗药物的全身用量大,毒副作用大的研究,具有极大地学术价值、社会效应及应用前景。近年来,由于各种纳米粒子能理想地在体内传递各种物质并具有可调的表面化学靶向结构,使得它在肿瘤治疗中凸显出独特的优势。目的：本研究在胃癌细胞人类表皮生长因子2受体为靶向的基础上,以贵金属Au纳米颗粒通过交换偶合与Fe304形成纳米复合物作为载体,化学结合单克隆抗体赫赛汀及一线化疗药物奥沙利铂,从而构建以人类表皮生长因子2为靶向的磁共振成像示踪控释奥沙利铂的新型纳米复合物Oxaliplatin-Au-Fe3O4-Herceptin,并且评价其在人类表皮生长因子2阳性胃癌中的价值。方法： 1.应用交换偶合法及热降解技术在金表面降解乙酰丙酮铁完成Au-Fe3O4金属纳米复合物的构建。通过表面修饰剂的加入对Au-Fe3O4纳米颗粒的形貌进行调控。透射电镜表征纳米粒子。 2.液相Oxaliplatin-Au-Fe3O4-Herceptin与1-(3-二甲氨基丙基)-3-乙基二亚胺及N-羟基硫代琥珀酰混合形成连接键,再与赫赛汀溶液混合培养,形成Au-Fe3O4-Herceptin。快速层析法纯化得到奥沙利铂连接配体。混合奥沙利铂混悬液、Au-Fe3O4-Herceptin及连接配体,优化比例,培养纯化得到Oxaliplatin-Au-Fe3O4-Herceptin。MALDI-MS验证产物,体外透析袋不同pH值下检测奥沙利铂结合效率。 3. MTS法分别验证Au-Fe3O4、Au-Fe3O4-Herceptin Oxaliplatin-Au-Fe3O4-Herceptin细胞毒性。 4.常规培养HER2阳性胃癌细胞系SGC-7901。分别与Au-Fe3O4、 Oxaliplatin-Au-Fe3O4-Herceptin孵育2小时,透射电镜观察纳米复合物在胃癌细胞表面胞吞作用的程度。 5.胃癌原位移植瘤模型的建立及分组：研究不同时限注射Oxaliplatin-Au-Fe3O4-Herceptin的胃癌裸鼠模型,调整建立磁共振扫描参数,观察小鼠肿瘤组织变化。结果 1.透射电镜下,见纳米粒子大小约25nm,铁粒子与金粒子1:1恒定； 2.成功构建了Oxaliplatin-Au-Fe3O4-Herceptin纳米复合物,并通过质谱、红外及透析袋缓释奥沙利铂证实了各分子间通过化学连接键相连。细胞毒性实验显示：当铁离子浓度在30ug/ml以内时,Oxaliplatin-Au-Fe3O4-Herceptin纳米复合物对细胞基本没有毒性； 3.体外胞吞实验：将Oxaliplatin-Au-Fe3O4-Herceptin纳米复合物、Au-Fe3O4纳米粒子以相同铁粒子浓度与SGC-7901细胞共孵育2h后透射电镜可见,Oxaliplatin-Au-Fe3O4-Herceptin纳米复合物被大量吞入细胞,而Au-Fe3O4纳米粒子未见被吞噬入细胞内； 4.小鼠尾静脉注射纳米粒子复合物一段时间后,磁共振扫描老鼠皮下移植瘤,见肿瘤组织内T2相低密度信号灶。肿瘤组织普鲁士染色后可见肿瘤血管旁大量铁粒子聚集。结论：本课题通过学科交叉研究,研制成功针对人类表皮生长因子2受体金属纳米化合物Oxaliplatin-Au-Fe3O4-Herceptin,能结合奥沙利铂靶向胃癌,并实现磁共振示踪控释,获得拥有自主知识产权。研究明确了该种化合物的体内外作用作用机制和生物安全性,为进一步的临床应用和人类表皮生长因子2阳性胃癌患者的个性化治疗,以及新药的开发提供坚实的实验基础。同时,亦为开发新型靶向治疗药物和靶向治疗提供新的思路和技术支持。
[Abstract]:Background :

Gastric cancer is a major disease which seriously endangers the health of human life . The data of World Health Organization Cancer Control Project shows that the number of new cases of gastric cancer in the world is about 9.3 million , the number of deaths is about 700,000 , accounting for 1 / 10 of all cancer mortality . In our country , more than 90 % of patients with gastric cancer have progressed more than 10 % each year .
In recent years , various kinds of nano - particles can transfer various substances in vivo and have adjustable surface chemical targeting structure , so that it has unique advantages in tumor treatment .

Purpose :

Based on the targeting of human epidermal growth factor - 2 receptor in gastric cancer cells , a novel nano - complex Oxhide - Au - Fe _ 3O _ ( 4 - -Fe _ 3O _ 3O _ 4 - ) was prepared by exchange coupling with Fe304 as a carrier , and it was used as a carrier to construct a novel nano - composite Oxhide - Au - Fe3O4 - Hercee with magnetic resonance imaging and release oxaliplatin targeting human epidermal growth factor 2 , and its value in human epidermal growth factor 2 positive gastric cancer was evaluated .

Method :

1 . The Au - Fe _ 3O _ 4 metal nanoparticles were prepared by using exchange coupling method and thermal degradation technique on gold surface . The morphology of Au - Fe3O4 nanoparticles was controlled by the addition of surface modifier . The nano - particles were characterized by TEM .

2 . The preparation of Oxhide - Au - Fe _ 3O _ 3 - ethyldiimine with 1 - ( 3 - dimethylaminopropyl ) -3 - ethyldiimine and N - hydroxythiosuccinyl was carried out to form the bond , and then mixed with Hercystatin solution to form Au - Fe _ 3O _ 3O _ 4 - HerceII . Rapid chromatographic method was used to purify the oxaliplatin - linked ligand .

3 . The toxicity of Au - Fe _ 3O _ 4 , Au - Fe _ 3O _ 3O _ 3O _ 3 - 2 - Fe _ 3O _ 3O _ 3O _ 3 - 4 - Fe _ 3O _ 3O _ 4 - 2 was verified by MTS method .

4 . The HER2 - positive gastric cancer cell line SGC - 790was incubated with Au - Fe _ 3O _ 3O _ 4 and Oxhide - Au - Fe _ 3O _ 3O _ 3O _ 3O _ 4 for 2 hours , and the extent of endocytosis of the nano - complexes on the surface of gastric cancer cells was observed by transmission electron microscope .

5 . Establishment and grouping of in - situ transplanted tumor model of gastric cancer : The nude mouse model of gastric cancer with different time - frame injection of Oxhide - Au - Fe3O4 - HerceII was studied , and the parameters of magnetic resonance scan were adjusted to observe the changes of tumor tissues in mice .

1 . Under the transmission electron microscope , the size of the nanoparticles is about 25nm , and the iron particles are constant with the gold particles 1 : 1 ;

2 . Oxhide - Au - Fe _ 3O _ 4 - Hertin nanocomposites were successfully constructed . Through mass spectrometry , IR and slow release of oxaliplatin , it was confirmed that there was no toxicity to the cells .

3 . In vitro endocytosis experiment : Oxhide - Au - Fe _ 3O _ 3O _ 4 - Hertin nanocomposites were incubated for 2 hours at the same concentration of Fe particles and SGC - 790cells for 2 hours . The Oxhide - Au - Fe _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 4 nanoparticles were absorbed into the cells and the Au - Fe _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 3O _ 4 nanoparticles were swallowed into the cells .

4 . After intravenous injection of nano - particle complex for a period of time , magnetic resonance scanning the mouse subcutaneous transplantation tumor to see the T2 - phase low - density signal focus in the tumor tissue . After the tumor tissue , the tumor tissue was stained and the large amount of iron particles near the tumor vessel were collected . Conclusion :

The study on the mechanism and biological safety of human epidermal growth factor - 2 receptor metal nano - compound Oxhide - Au - Fe _ 3O _ 3O _ 3O _ 4 - Her2 combined with oxaliplatin targeted gastric cancer , and achieved independent intellectual property rights . It also provides a solid experimental basis for further clinical application and personalized treatment of human epidermal growth factor 2 positive gastric cancer patients , and provides new ideas and technical support for the development of new targeted therapy drugs and targeted therapy .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：TB383.1

【共引文献】