發(fā)布時間：2018-08-12 18:15

【摘要】：1巰基化殼聚糖季銨鹽(TMC-Cys)用于pDNA的體內外轉染以及機理研究 我們結合了殼聚糖季銨鹽(TMC)和巰基化殼聚糖的優(yōu)點,設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通過聚電解質法與表達綠色熒光蛋白的pEGFP質粒形成自組裝納米復合物(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是通過網格蛋白介導的內吞方式入胞。胞內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,使得核質內的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的轉染效率比Lipofectamine2000高1.5倍。在小鼠后腿腓腸肌中注射納米復合物的實驗也證明TMC-Cys (100,30)/pEGFP NC的體內轉染效率比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)口愈合異常產生的結果,通常表現(xiàn)為皮膚和皮下組織中細胞外基質尤其是Ⅰ型膠原和Ⅲ型膠原的過度沉積。轉化生長因子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TGFβ1通路的重要信號傳導分子,參與調節(jié)膠原的合成。因此我們用TMC-Cys和表達smad2 siRNA的pSUPER質粒組裝成納米復合物,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天內細胞數(shù)量可以擴增一倍。RT-PCR的結果顯示載有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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R346

【相似文獻】

相關期刊論文 前10條

1 孫文娟;陸景華;蔡霞;唐勝建;;應用膠原蛋白構建組織工程皮膚的實驗研究[J];菏澤醫(yī)學�？茖W校學報;2007年03期

2 陳大福;田偉;行勇剛;周一新;曹晶晶;王猛;;膠原支架增強自固化磷酸鈣骨水泥的力學及成骨性能研究[J];中國矯形外科雜志;2006年18期

3 李靜;顧漢卿;;組織工程化尿路細胞-支架復合體的制備及其體外培養(yǎng)的初步研究[J];透析與人工器官;2009年01期

4 蘇青和,楊敏杰,周紅梅;用含活性細胞的膠原支架復合皮修復小鼠全層皮膚缺損的研究[J];中華燒傷雜志;2003年06期

5 劉玲蓉,張立海,馬東瑞,任百智,張其清;碳化二亞胺交聯(lián)的膠原-硫酸軟骨素支架材料構建人工真皮的研究[J];中國修復重建外科雜志;2003年02期

6 趙東鍔,汪鋼,俞世強,周更須,金振曉,劉維永;應用膠原支架構建組織工程心臟瓣膜的初步研究[J];中華胸心血管外科雜志;2002年05期

7 黃漢萍,牟善松,馬安德;膠原支架材料復合膜的制備及其組織培養(yǎng)性能觀察[J];第一軍醫(yī)大學學報;2004年07期

8 丁曉飛;組織工程技術修復軟骨缺損的進展[J];廣西醫(yī)學;2003年04期

9 鄭小飛;骨和軟骨組織工程中細胞種植基質材料的研究進展[J];國外醫(yī)學.骨科學分冊;1999年03期

10 熊猛,艾玉峰,王潘勇;采用組織工程方法體外構建血管模型的初步實驗研究[J];中國修復重建外科雜志;2001年02期

相關會議論文 前5條

1 張炎;劉太華;劉波;李玉泉;張秀花;姜宗來;;動脈脫細胞三維膠原支架的制備和血管平滑肌細胞種植[A];第七屆全國生物力學學術會議論文集[C];2003年

2 劉太華;張炎;李玉泉;劉波;張秀花;姜宗來;;動脈脫細胞三維膠原支架主要組織相容復合體I(MHC I)抗原的免疫組化觀察[A];第七屆全國生物力學學術會議論文集[C];2003年

3 張穎;楊柳;王富友;譚洪波;陳光興;郭林;段小軍;;Ⅰ/Ⅲ型膠原雙層支架復合自體MSCs移植修復關節(jié)軟骨缺損研究[A];第六屆西部骨科論壇暨貴州省骨科年會論文匯編[C];2010年

4 劉興茂;陳昭烈;劉紅;熊福銀;;心肌細胞體外三維培養(yǎng)的初步研究[A];中國生物工程學會第三次全國會員代表大會暨學術討論會論文摘要集[C];2001年

5 馬列;高長有;周杰;施躍文;沈家驄;;豬皮膠原構建人工真皮支架的研究[A];第九屆全國生物材料學術會議（CBMS-9）論文集[C];2002年

相關碩士學位論文 前6條

1 齊義營;關節(jié)軟骨組織工程研究：自體生長因子或分化后的胚胎干細胞復合雙層膠原支架修復關節(jié)軟骨缺損[D];浙江大學;2008年

2 王奇;神經干細胞體外三維培養(yǎng)及其模型研究[D];大連理工大學;2009年

3 劉振寧;GDF-5誘導脂肪干細胞復合膠原支架成軟骨細胞分化修復椎間盤退變的前期實驗性研究[D];中國醫(yī)科大學;2008年

4 夏文森;組織工程血管模型體外分層構建的實驗研究[D];第四軍醫(yī)大學;2002年

5 徐俊華;納米陶瓷仿生人工骨的研制及體內外活性研究[D];浙江大學;2004年

6 謝青靖;痰瘀相關理論及治療高血壓左心室肥厚的臨床研究[D];廣州中醫(yī)藥大學;2000年

，

本文編號：2179890