【摘要】：1巰基化殼聚糖季銨鹽(TMC-Cys)用于pDNA的體內(nèi)外轉(zhuǎn)染以及機理研究 我們結(jié)合了殼聚糖季銨鹽(TMC)和巰基化殼聚糖的優(yōu)點,設(shè)計合成了巰基化殼聚糖季銨鹽(TMC-Cys)作為新型的非病毒基因給藥載體。不同分子量(30,100 and 200kDa)和季銨化程度(15% and 30%)的TMC-Cys通過聚電解質(zhì)法與表達綠色熒光蛋白的pEGFP質(zhì)粒形成自組裝納米復(fù)合物(nanocomplex,NC)。毒性實驗的數(shù)據(jù)顯示在濃度小于2mg/ml時聚合物均未顯示出細胞毒性。EB排阻實驗和凝膠阻滯實驗顯示TMC-Cys可以有效的縮合pDNA,在適合的N/P比條件下,形成平均粒徑在120nm-200nm、zeta電勢為+15mV-+20mV的納米粒。TMC-Cys/pEGFP NC表現(xiàn)出很好的物理穩(wěn)定性并且可以保護pEGFP免受核酶的降解。TMC-Cys/pEGFPNC的紅細胞粘附率比TMC/pEGFP NC高2.7倍,粘蛋白粘附率TMC/pEGFP NC高1.5倍,HEK293細胞的攝取率比TMC/pEGFPNC高2.6倍,比Lipofectamine2000高3倍。將培養(yǎng)溫度從37℃降至4℃可以使TMC-Cys/pEGFP NC的HEK293細胞攝取率降低3/4,而疊氮化鈉的預(yù)處理也可以使TMC-Cys/pEGFP NC的HEK293細胞攝取率降低1/3,表明TMC-Cys/pEGFP NC的HEK293細胞攝取是一個依賴代謝熱量的過程。用氯丙嗪預(yù)處理可以使TMC-Cys/pEGFP NC的HEK293細胞攝取率降低70%,暗示了TMC-Cys/pEGFP NC是通過網(wǎng)格蛋白介導(dǎo)的內(nèi)吞方式入胞。胞內(nèi)高濃度的還原性谷胱甘肽使得TMC-Cys/pEGFP NC以比胞外快3.5倍的速度釋放pEGFP,使得核質(zhì)內(nèi)的pEGFP濃度迅速增加。共聚焦激光顯微鏡的定量定性實驗都表明TMC-Cys/pEGFP NC釋放pEGFP入核的濃度比TMC/pEGFP NC高3..7倍。正因為上述這些理由,TMC-Cys/pEGFP NC在HEK293細胞中的轉(zhuǎn)染效率比TMC/pEGFP NC高1.4-3.2倍,其中適合的TMC-Cys (100,30)/pEGFP NC的轉(zhuǎn)染效率比Lipofectamine2000高1.5倍。在小鼠后腿腓腸肌中注射納米復(fù)合物的實驗也證明TMC-Cys (100,30)/pEGFP NC的體內(nèi)轉(zhuǎn)染效率比TMC/pEGFP NC高2.3倍,比Lipofectamine 2000高4.1倍。 2載有TMC-Cys/pDNA NC的3D膠原支架用于皮膚增生性瘢痕治療的初步研究 增生性瘢痕通常是皮膚受損后創(chuàng)口愈合異常產(chǎn)生的結(jié)果,通常表現(xiàn)為皮膚和皮下組織中細胞外基質(zhì)尤其是Ⅰ型膠原和Ⅲ型膠原的過度沉積。轉(zhuǎn)化生長因子β1(TGFβ1)在皮膚纖維化病變中扮演著重要的角色。Smad蛋白是細胞內(nèi)TGFβ1通路的重要信號傳導(dǎo)分子,參與調(diào)節(jié)膠原的合成。因此我們用TMC-Cys和表達smad2 siRNA的pSUPER質(zhì)粒組裝成納米復(fù)合物,載入凍干法制備的多孔膠原支架中,用于抑制TGFβ1信號通路的活性。多孔膠原支架提供了三維的支持空間,包封率在60%以上并受載藥量的控制,在體外表現(xiàn)出持續(xù)的釋放特性,第三天累積釋放量達到70%左右。阿爾瑪蘭細胞增殖實驗的數(shù)據(jù)顯示皮膚成纖維細胞可以在其中良好的生長繁殖,7天內(nèi)細胞數(shù)量可以擴增一倍。RT-PCR的結(jié)果顯示載有TMC-Cys/pSUPER-smad2 NC的膠原支架可以有效的抑制生長于其中的皮膚成纖維細胞中smad2、I型膠原和Ⅲ型膠原的1nRNA表達,抑制效率達到80-87%。酶聯(lián)免疫實驗也在蛋白水平上證明了皮膚成纖維細胞中Ⅰ型、Ⅲ型膠原合成量下降。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346

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