本文選題：聚乙二醇(PEG) + 聚乳酸(PLA)��； 參考：《中南大學》2007年碩士論文

【摘要】： 近年來，隨著基因轉移技術的日趨成熟，基因治療成為生物科學和臨床醫(yī)學的研究熱點之一�；蛑委煹妮d體問題，以及載體相關的免疫反應、細胞毒性與安全性問題是限制基因治療發(fā)展和臨床應用的瓶頸。目前基因治療的研究和臨床應用中常用的載體分為病毒載體和非病毒載體，各個載體均有各自的優(yōu)缺點，病毒載體主要的潛在缺點包括：①致病可能性；②免疫原性；③大規(guī)模生產的困難性，而非病毒導入系統(tǒng)正受到更多的關注，它的主要的優(yōu)點包括安全，容易大規(guī)模生產等，主要缺點是不能獲得較高水平的基因導入和表達。納米�；蜣D運體是近年發(fā)展起來的一種新型的非病毒基因轉運載體。它是將DNA、RNA等基因治療分子包裹在納米顆粒之中或吸附在其表面，同時也在顆粒表面耦聯(lián)特異性的靶向分子，如特異性配體、單克隆抗體等，通過靶向分子與細胞表面特異性受體結合，在細胞攝取作用下進入胞內，實現(xiàn)安全有效的靶向性基因治療。 納米粒材料的選擇是成功進行納米基因轉運和治療的關鍵。所選擇的材料必須是生物可降解型或者易于從體內排泄，而不產生有害的降解產物，且無免疫原性，不引起機體的免疫排斥反應。高分子生物降解材料制備的納米顆粒具有穩(wěn)定、無毒、無抗原性、生物相容性好、對所轉運基因的表達有緩釋作用及對基因有保護作用等優(yōu)點，是良好的納米基因轉運載體材料。研究證實，納米粒與DNA的連接多通過靜電吸引作用來完成。DNA的磷酸骨架所帶的負電荷只能與表面帶正電性的載體結合。因此，需要利用生物活性分子對納米顆粒的表面進行改性，使其表面攜帶陽離子物質，起到防止顆粒團聚，并有利于結合DNA分子的作用。 目的：構建一種理想的納米基因載體轉運系統(tǒng)，使其能有效的保護轉運基因不被核酶降解，并具備較高的基因轉染效率、有效的輸送基因至靶位點以及良好的生物相容性。 方法：在本研究中，基于我們的前期工作，首次創(chuàng)新性的選擇單甲氧基聚乙二醇-聚乳酸(MePEG-PLA)和殼聚糖(chitosan，CS)通過透析過濾法來制備MePEG-PLA-CS納米粒作為基因轉運載體，并用正交實驗設計方法安排實驗，優(yōu)選制備工藝條件。使用AFM(原子力顯微鏡)和Zetasizer(Marlven，粒徑分析儀)來檢測MePEG-PLA-CS納米粒和MePEG-PLA-CS／DNA復合物的Zeta電位以及粒徑分布。我們通過靜電吸附作用將MePEG-PLA-CS納米粒和帶正電荷的DNA的結合在一起形成MePEG-PLA-CS／DNA復合物，通過分光光度計法來檢測MePEG-PLA-CS／DNA復合物中DNA的濃度，從而計算DNA的結合效率。通過凝膠阻滯實驗和DNaseI消化實驗來檢測該基因載體轉運系統(tǒng)對DNA的保護能力。采用MTT法研究MePEG-PLA-CS納米粒對人肝癌細胞(HepG2)及正常肝細胞(L-02)的影響。使用MePEG-PLA-CS攜帶pEGFP-C1(綠色熒光蛋白質粒)pDNA作為報導基因轉染COS7細胞來評價體外轉染效率，其中商業(yè)化脂質體(Lipofectamine2000，Invitrogen)作為陽性對照而裸DNA作為陰性對照。進一步的，構建BALB／C瘤鼠模型，采用MePEG-PLA-CS納米粒載報告質粒綠色熒光蛋白(EGFP)研究體內基因轉染的能力。 結果：通過正交實驗，我們得到了制備MePEG-PLA-CS NP的最優(yōu)條件，AFM和粒徑分析儀證明該納米粒表面平滑完整，分散良好，無粘附團聚現(xiàn)，，其表面電位為正，該納米基因載體轉運系統(tǒng)DNA結合效率(DNA loading efficiency)為91％±4.5％。MePEG-PLA-CS納米粒由于表面帶正電荷而獲得結合帶負電DNA的能力。同時，實驗也證實，MePEG-PLA-CS納米顆粒／DNA復合物能保護所攜帶的DNA免受核酸酶的降解。本研究發(fā)現(xiàn)，MePEG-PLA-CS納米顆粒對正常的肝細胞(L-02)在一定的劑量范圍內無細胞毒性，只在高濃度下才會表現(xiàn)出一定的細胞毒性作用。在體外基因轉運中，該納米顆�？捎行мD運EGFP(綠色熒光蛋白)報道基因表達質粒進入COS7細胞，其轉運效率達40％，強于相同條件下脂質體的轉染效率。在體內基因轉染實驗中，由MePEG-PLA-CS納米粒處理過的裸鼠一組，在腫瘤組織中EGFP基因表達最高，這證實了MePEG-PLA-CS納米粒借助PEG能夠在循環(huán)系統(tǒng)中長期滯留而不被網狀內皮系統(tǒng)捕獲，通過EPR(選擇性滯留效應)效應富集到腫瘤組織中，從而具有靶向治療腫瘤的能力。 結論：采用透析過濾法，優(yōu)化工藝條件，成功制備粒徑較小、分布均勻的聚MePEG-PLA-CS納米顆粒。使該納米顆粒表面帶正電荷從而結合質粒DNA，組裝成MePEG-PLA-CS納米顆�；蜣D運體系。通過體內外基因轉染及保護DNA等試驗，證實該轉運體系可保護所攜帶DNA免受核酸酶的降解，并且是一種低毒高效的納米基因載體，我們的研究為應用納米粒來實現(xiàn)腫瘤的基因治療奠定了良好的科學基礎。
[Abstract]:In recent years, with the growing maturity of gene transfer technology, gene therapy has become one of the hotspots in biological science and clinical medicine. The problem of gene therapy carrier, carrier related immune response, cytotoxicity and safety are bottlenecks restricting the development and clinical application of gene therapy. The research and clinical study of gene therapy at present The commonly used carriers are divided into virus carriers and non viral vectors. Each carrier has its own advantages and disadvantages. The main potential shortcomings of the virus carrier include: (1) the possibility of disease; (2) immunogenicity; (3) the difficulty of large-scale production; and the non virus introduction system is being paid more attention, and its main advantages include safety and ease. Large scale production, such as mass production, is the main disadvantage of not obtaining high level of gene introduction and expression. The nanoparticle gene transporter is a new type of non viral gene transport carrier developed in recent years. It is DNA, RNA and other gene therapy molecules wrapped in nanoparticles or adsorbed on its surface, but also on the surface of particles. Targeted molecules, such as specific ligands, monoclonal antibodies, are combined with specific receptors on the cell surface by targeting molecules and enter the cell under cell uptake to achieve a safe and effective targeted gene therapy.
The selection of nanoparticles is the key to the successful transport and treatment of nanoscale genes. The selected materials must be biodegradable or excreted easily from the body without producing harmful degradation products, without immunogenicity, and no immune rejection. The nanoparticles prepared by polymer biodegradable materials are stable. It is a good nanoscale transport carrier material, which has the advantages of non-toxic, antigenicity and biocompatibility. It is a good nanoscale transport carrier material for the expression of the transporter gene and the protective effect on the gene. It is confirmed that the connection of the nanoparticles and DNA is mostly through the electrostatic attraction to complete the negative charge of the phosphoric acid skeleton of.DNA only with the positive surface of the surface. Therefore, it is necessary to use bioactive molecules to modify the surface of the nanoparticles to carry the cationic substance on the surface, which prevents the agglomeration of particles and is beneficial to the interaction of DNA molecules.
Objective: to construct an ideal nanoscale carrier transport system, which can effectively protect the transport genes from ribozyme degradation, and have high gene transfection efficiency, effective gene delivery to target loci and good biocompatibility.
Methods: in this study, based on our previous work, the first innovative selection of mono methoxy poly (MePEG-PLA) and chitosan (chitosan, CS) was used to prepare MePEG-PLA-CS nanoparticles as a gene transport carrier by dialysis filtration method, and the experiment was arranged by orthogonal test design method, and the preparation conditions were optimized. AF M (atomic force microscopy) and Zetasizer (Marlven, particle size analyzer) are used to detect the Zeta potential and particle size distribution of MePEG-PLA-CS nanoparticles and MePEG-PLA-CS / DNA complexes. By electrostatic adsorption, we combine MePEG-PLA-CS nanoparticles with a positive charge DNA together to form a MePEG-PLA-CS / DNA complex, through a spectrophotometer. The method was used to detect the concentration of DNA in the MePEG-PLA-CS / DNA complex and calculate the binding efficiency of DNA. The protective ability of the gene carrier transport system to DNA was detected by gel block experiment and DNaseI digestion experiment. The effect of MePEG-PLA-CS nanoparticles on human liver cancer cells (HepG2) and normal liver cells (L-02) was studied by MTT method. PLA-CS carrying pEGFP-C1 (green fluorescent protein particle) pDNA as the reporter gene transfected COS7 cells to evaluate the transfection efficiency in vitro, in which commercial liposomes (Lipofectamine2000, Invitrogen) were used as positive controls and naked DNA as negative control. Further, the BALB / C tumor mouse model was constructed with MePEG-PLA-CS nanoparticles carrying the report plasmid green. Color fluorescence protein (EGFP) is used to study the ability of gene transfection in vivo.
Results: through the orthogonal experiment, we obtained the optimal conditions for the preparation of MePEG-PLA-CS NP. AFM and particle size analyzer proved that the nanoparticles were smooth and complete, dispersed well, and the surface potential was positive. The DNA binding efficiency (DNA loading efficiency) of the nanoscale carrier transport system (DNA loading efficiency) was 91% + 4.5%.MePEG-PLA-CS nanometers. It is also proved that the MePEG-PLA-CS nanoparticles / DNA complexes can protect the carrier DNA from nuclease degradation. This study found that MePEG-PLA-CS nanoparticles were not cytotoxic to normal liver cells (L-02) in a certain dose range, only in high concentration. The results showed that MePEG-PLA-CS nanoparticles were not cytotoxic to normal liver cells (L-02) in a certain dose range. In vitro gene transport, the nanoparticles can effectively transport EGFP (green fluorescent protein) to report that the gene expression plasmid entered COS7 cells, and its transport efficiency is 40%, stronger than the transfection efficiency of liposomes under the same condition. In the gene transfection experiment, the MePEG-PLA-CS nanoparticles are located at the nanoparticles. A group of nude mice has the highest expression of EGFP gene in tumor tissue, which confirms that MePEG-PLA-CS nanoparticles can be retained in the circulatory system for a long time without being captured by the reticuloendothelial system with the help of PEG, and are enriched in the tumor tissue through the effect of EPR (selective retention effect), and have the ability to target the tumor targeting the tumor.
Conclusion: using the dialysis filtration method and optimizing the technological conditions, the particles with small size and uniform distribution of MePEG-PLA-CS nanoparticles were successfully prepared. The nanoparticles were charged with positive charge and then combined with plasmid DNA to assemble the MePEG-PLA-CS nanoparticle gene transport system. The transfer system was confirmed through the transfection of the body and the body and the protection of DNA. The DNA is protected from the degradation of nuclease, and is a low toxic and efficient nanoscale gene carrier. Our research has laid a good scientific basis for the application of nanoparticles in the treatment of tumor gene therapy.

【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346

【引證文獻】

相關博士學位論文 前1條

1 宋遠見;腦靶向高分子聚合物納米藥物載體的制備、表征和應用[D];中國礦業(yè)大學;2012年

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