基于巯基化壳聚糖季铵盐纳米粒及胶原支架的基因递释研究

发布时间：2018-08-12 18:15

【摘要】：1巯基化壳聚糖季铵盐(TMC-Cys)用于pDNA的体内外转染以及机理研究我们结合了壳聚糖季铵盐(TMC)和巯基化壳聚糖的优点,设计合成了巯基化壳聚糖季铵盐(TMC-Cys)作为新型的非病毒基因给药载体。不同分子量(30,100 and 200kDa)和季铵化程度(15% and 30%)的TMC-Cys通过聚电解质法与表达绿色荧光蛋白的pEGFP质粒形成自组装纳米复合物(nanocomplex,NC)。毒性实验的数据显示在浓度小于2mg/ml时聚合物均未显示出细胞毒性。EB排阻实验和凝胶阻滞实验显示TMC-Cys可以有效的缩合pDNA,在适合的N/P比条件下,形成平均粒径在120nm-200nm、zeta电势为+15mV-+20mV的纳米粒。TMC-Cys/pEGFP NC表现出很好的物理稳定性并且可以保护pEGFP免受核酶的降解。TMC-Cys/pEGFPNC的红细胞粘附率比TMC/pEGFP NC高2.7倍,粘蛋白粘附率TMC/pEGFP NC高1.5倍,HEK293细胞的摄取率比TMC/pEGFPNC高2.6倍,比Lipofectamine2000高3倍。将培养温度从37℃降至4℃可以使TMC-Cys/pEGFP NC的HEK293细胞摄取率降低3/4,而叠氮化钠的预处理也可以使TMC-Cys/pEGFP NC的HEK293细胞摄取率降低1/3,表明TMC-Cys/pEGFP NC的HEK293细胞摄取是一个依赖代谢热量的过程。用氯丙嗪预处理可以使TMC-Cys/pEGFP NC的HEK293细胞摄取率降低70%,暗示了TMC-Cys/pEGFP NC是通过网格蛋白介导的内吞方式入胞。胞内高浓度的还原性谷胱甘肽使得TMC-Cys/pEGFP NC以比胞外快3.5倍的速度释放pEGFP,使得核质内的pEGFP浓度迅速增加。共聚焦激光显微镜的定量定性实验都表明TMC-Cys/pEGFP NC释放pEGFP入核的浓度比TMC/pEGFP NC高3..7倍。正因为上述这些理由,TMC-Cys/pEGFP NC在HEK293细胞中的转染效率比TMC/pEGFP NC高1.4-3.2倍,其中适合的TMC-Cys (100,30)/pEGFP NC的转染效率比Lipofectamine2000高1.5倍。在小鼠后腿腓肠肌中注射纳米复合物的实验也证明TMC-Cys (100,30)/pEGFP NC的体内转染效率比TMC/pEGFP NC高2.3倍,比Lipofectamine 2000高4.1倍。 2载有TMC-Cys/pDNA NC的3D胶原支架用于皮肤增生性瘢痕治疗的初步研究增生性瘢痕通常是皮肤受损后创口愈合异常产生的结果,通常表现为皮肤和皮下组织中细胞外基质尤其是Ⅰ型胶原和Ⅲ型胶原的过度沉积。转化生长因子β1(TGFβ1)在皮肤纤维化病变中扮演着重要的角色。Smad蛋白是细胞内TGFβ1通路的重要信号传导分子,参与调节胶原的合成。因此我们用TMC-Cys和表达smad2 siRNA的pSUPER质粒组装成纳米复合物,载入冻干法制备的多孔胶原支架中,用于抑制TGFβ1信号通路的活性。多孔胶原支架提供了三维的支持空间,包封率在60%以上并受载药量的控制,在体外表现出持续的释放特性,第三天累积释放量达到70%左右。阿尔玛兰细胞增殖实验的数据显示皮肤成纤维细胞可以在其中良好的生长繁殖,7天内细胞数量可以扩增一倍。RT-PCR的结果显示载有TMC-Cys/pSUPER-smad2 NC的胶原支架可以有效的抑制生长于其中的皮肤成纤维细胞中smad2、I型胶原和Ⅲ型胶原的1nRNA表达,抑制效率达到80-87%。酶联免疫实验也在蛋白水平上证明了皮肤成纤维细胞中Ⅰ型、Ⅲ型胶原合成量下降。
[Abstract]:1 mercapto chitosan quaternary ammonium salt (TMC-Cys) was used to transfect pDNA in vivo and in vitro and its mechanism.
Combining the advantages of chitosan quaternary ammonium salt (TMC) and thiol-chitosan, we designed and synthesized thiol-chitosan quaternary ammonium salt (TMC-Cys) as a novel non-viral gene delivery vector. TMC-Cys with different molecular weight (30,100 and 200 kDa) and quaternary ammonium degree (15% and 30%) expressed green fluorescent protein pEGFP by polyelectrolyte method. EB exclusion test and gel blockade test showed that TMC-Cys could effectively condense pDNA, forming an average particle size of 120 nm-200 nm under suitable N/P ratio, zeta potential of + 15 mV-+ 20 mV. TMC-Cys/pEGFPNC showed good physical stability and protected pEGFP from ribozyme degradation. The adherence rate of erythrocytes to TMC-Cys/pEGFPNC was 2.7 times higher than that of TMC/pEGFPNC, the adherence rate of mucin to TMC/pEGFPNC was 1.5 times higher, and the uptake rate of HEK293 cells was 2.6 times higher than that of TMC/pEGFPNC and 3 times higher than that of Lipofectamine 2000. The uptake of HEK293 cells from TMC-Cys/pEGFP NC decreased by 3/4 from 37 to 4, while pretreatment with sodium azide also reduced the uptake of HEK293 cells from TMC-Cys/pEGFP NC by 1/3, indicating that the uptake of HEK293 cells from TMC-Cys/pEGFP NC was a heat-dependent process. Pretreatment with chlorpromazine could reduce the uptake of HEK2 from TMC-Cys/pEGFP NC by 1/3. The uptake of 93 cells decreased by 70%, suggesting that TMC-Cys/pEGFP NC was endocytosis mediated by reticulin. The high concentration of reduced glutathione in TMC-Cys/pEGFP NC led to the release of pEGFP at a rate of 3.5 times faster than that outside the cell, resulting in a rapid increase in the concentration of pEGFP in the nucleus. For these reasons, the transfection efficiency of TMC-Cys/pEGFP NC in HEK293 cells was 1.4-3.2 times higher than that of TMC/pEGFP NC, and the transfection efficiency of suitable TMC-Cys(100,30)/pEGFP NC was 1.5 times higher than that of Lipofectamine 2000. The in vivo transfection efficiency of TMC-Cys(100,30)/pEGFP NC was 2.3 times higher than that of TMC/pEGFP NC and 4.1 times higher than that of Lipofectamine 2000.
2 3D collagen scaffold containing TMC-Cys/pDNA NC for the treatment of hypertrophic scar of skin
Hypertrophic scars are usually the result of abnormal wound healing after skin injury, usually manifested by excessive deposition of extracellular matrix (ECM) in skin and subcutaneous tissues, especially type I and type III collagen. Transforming growth factor beta 1 (TGF beta 1) plays an important role in skin fibrosis. Smad protein is the intracellular TGF beta 1 pathway. Important signal transduction molecules are involved in regulating collagen synthesis. Therefore, we assembled nanocomposites from TMC-Cys and pSUPER plasmids expressing Smad2 siRNA into porous collagen scaffolds prepared by freeze-drying method to inhibit the activity of TGF-beta 1 signaling pathway. The cumulative release of TMC-Cys/pSUPER-smad2 NC-loaded collagen was about 70% on the third day. The data of Almaran cell proliferation experiment showed that the skin fibroblasts could grow and reproduce well and the number of cells could be doubled within 7 days. The results of RT-PCR showed that the collagen was loaded with TMC-Cys/pSUPER-smad2 NC. The scaffolds could effectively inhibit the expression of smad2, collagen I and collagen I I I in the fibroblasts, and the inhibition efficiency was 80-87%. Enzyme-linked immunosorbent assay (ELISA) showed that the synthesis of collagen I and collagen I I I in the fibroblasts decreased at the protein level.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

【相似文献】