基于金纳米粒的新型基因载体构建与其经皮治疗皮肤黑色素瘤的体内外研究

发布时间：2018-02-09 17:22

本文关键词： 经皮给药金纳米粒细胞穿膜肽 microRNA-221 黑色素瘤　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的：采用还原法制备聚乙烯亚胺(PEI)和修饰的金纳米粒,并进一步用穿膜多肽对纳米粒进行修饰。探究其作为多功能基因载体,携载编码有microRNA-221抑制基因的质粒DNA (Mi221),对黑色素瘤细胞B16F10的基因调控和体外抗肿瘤效果,同时考察了其作为透皮载体,输送基因治疗皮肤黑色素瘤的体内疗效。方法：1、采用NaBH4还原法制备连接有聚乙烯亚胺的金纳米粒,随后使用细胞穿膜肽对其表面进行修饰,最终制得多功能金纳米粒(AuPT);2、对AuPT的形态、粒径、电位和表面PEI与TAT的接枝率进行表征；3、制备不同质量比的AuPT/质粒DNA复合物,考察不同复合物的粒径、包裹质粒DNA(pDNA)的效率与细胞转染效率三项指标,并据此筛选AuPT/pDNA的最佳质量比；4、考察AuPT携载报告基因的入胞效率与入胞机制；5、考察不同载体携载Mi221转染黑色素瘤细胞后,对microRNA-221以及其下游两种基因的调控作用,并评价体外基因治疗后,肿瘤细胞的迁移、增殖和周期的变化。6、使用FITC-pDNA作为荧光标记药物,用不同载体携载FITC-pDNA进行体外透皮实验,定量考察透皮效率,并使用激光共聚焦显微镜观察载体与基因复合物在不同皮肤层的分布以及透皮机制。7、使用B16F10细胞建立小鼠皮肤黑色瘤模型,使用不同载体携载Mi221进行透皮基因治疗,考察Mi221的抑瘤效果和体内抗肿瘤机制,并用透射电镜(TEM)观察瘤内金纳米粒的分布。结果：AuPT在水溶液中分布均匀,粒径大约为10-20 nm且能够有效地携载pDNA并保护其不被降解。当TAT与载体孵育的浓度为25 μg/mL, AuPT:pDNA=1:8时,复合物粒径最小,并对黑色素瘤细胞B16F10转染效率最高。基因调控结果显示,AuPT/Mi221可以显著降低肿瘤细胞内microRNA-221的表达水平,上调p27基因和c-Kit基因的表达。这种基因调控作用明显降低了B16F10细胞的迁移能力,将更多的细胞阻滞在细胞周期的G1期,从而成功抑制了肿瘤细胞的增殖。体外透皮实验表明,细胞穿膜肽可以增强载体携载基因类药物的透皮效率。使用激光共聚焦显微镜(CLSM)观察皮肤内复合物的分布情况,可以初步判断,AuPT/FITC-pDNA复合物主要通过皮肤毛囊、汗腺等附属器官穿透皮肤。最终体内实验证明,AuPT/Mi221经皮给药成功抑制了小鼠黑色素瘤的生长,小鼠未产生明显的毒副反应。在电子透射显微镜(TEM)下,能够在肿瘤内部观察到黑色的金纳米粒,进一步证明了AuPT的体内透皮效果。通过肿瘤切片染色发现,经皮给药后,小鼠体内肿瘤细胞的分裂受到抑制,同时肿瘤细胞出现了大量的凋亡和坏死等现象。结论：AuPT不仅可以携载并保护基因类药物,高效转染肿瘤细胞,调控肿瘤细胞内基因的表达。同时还具有很强的透皮能力,在经皮治疗皮肤黑色素瘤方面具有良好的潜力。
[Abstract]:Objective: to prepare polyethylene imine (PEI) and modified Jinnanmi granules by reduction method, and further modify the nanoparticles with transmembrane polypeptide. Carrying the plasmid DNA Mi221 encoding microRNA-221 suppressor gene, the gene regulation of melanoma cell line B16F10 and its antitumor effect in vitro were investigated. In vivo efficacy of gene delivery in the treatment of skin melanoma. Methods NaBH4 reduction method was used to prepare Jinnanmi granules with polyethyleneimine, and then the surface was modified with cell membrane penetrating peptide. The morphology, particle size, potential and grafted ratio of AuPT with TAT were characterized by AuPTT / plasmid DNA complex with different mass ratio, and the particle size of the complex was investigated. The efficiency of encapsulating plasmid DNA and the efficiency of cell transfection were determined. According to the best mass ratio of AuPT/pDNA, the entry efficiency of AuPT carrying reporter gene and its mechanism were investigated. After transfection of Mi221 with different vectors, melanoma cells were transfected into melanoma cells. The regulation of microRNA-221 and its downstream genes was evaluated. After gene therapy in vitro, the migration, proliferation and cycle changes of tumor cells were evaluated. FITC-pDNA was used as fluorescent labeling drug and FITC-pDNA was carried on transdermal experiments in vitro. The transdermal efficiency was quantitatively investigated. The distribution of carrier and gene complex in different skin layers and the transdermal mechanism were observed by laser confocal microscope. B16F10 cells were used to establish mouse skin melanoma model. Transdermal gene therapy with Mi221 was carried out with different vectors. The inhibitory effect and anti-tumor mechanism of Mi221 in vivo were investigated. The distribution of Jinnanmi granules in the tumor was observed by transmission electron microscopy (TEM). The particle size is about 10-20 nm and can effectively carry pDNA and protect it from degradation. When TAT incubates with the carrier at a concentration of 25 渭 g / mL, AuPT:pDNA=1:8 has the smallest particle size. The results of gene regulation showed that AuPTP / Mi221 could significantly reduce the expression of microRNA-221 in melanoma cells. The expression of p27 gene and c-Kit gene was upregulated. This gene regulation significantly reduced the migration ability of B16F10 cells and blocked more cells in G1 phase of cell cycle, thus successfully inhibiting the proliferation of tumor cells. The transmembrane peptide can enhance the transdermal efficiency of the carrier carrying gene drugs. Using the laser confocal microscope (CLSM) to observe the distribution of the complex in the skin, we can preliminarily judge that the AuPTR / FITC-pDNA complex mainly passes through the skin hair follicles. The final in vivo experiments showed that AuPTR / Mi221 successfully inhibited the growth of melanoma in mice, and the mice did not produce any obvious side effects under the electron transmission microscope (TEM). Black Jinnanmi granules could be observed inside the tumor, which further proved the transdermal effect of AuPT in vivo. By the staining of tumor sections, it was found that the division of tumor cells in mice was inhibited after percutaneous administration. At the same time, a large number of apoptosis and necrosis occurred in tumor cells. Conclusion: AuPT can not only carry and protect gene drugs, transfect tumor cells efficiently, regulate gene expression in tumor cells, but also have strong transdermal ability. It has good potential in percutaneous treatment of skin melanoma.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450;R739.5

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