本文關(guān)鍵詞： 內(nèi)皮細(xì)胞 轉(zhuǎn)染效率 兩親性基因載體 靶向識別 自組裝　出處：《天津大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目前,心腦血管疾病手術(shù)治療存在的主要問題是移植物中新生內(nèi)膜增生以及再狹窄等,這些都嚴(yán)重危及患者生命。鑒于內(nèi)皮細(xì)胞的一些天然優(yōu)勢,植入材料表面快速內(nèi)皮化是材料在體內(nèi)移植成功的關(guān)鍵�；蛑委熀筒牧媳砻娴墓δ芑揎検菍崿F(xiàn)人工血管植入材料內(nèi)皮化的兩種有效方法。本論文系統(tǒng)地綜述了基因治療和多肽修飾的生物材料在促進(jìn)內(nèi)皮化方面取得的成果。由于內(nèi)皮細(xì)胞固有的轉(zhuǎn)染效率低下,使植入材料在體內(nèi)的內(nèi)皮化效果大受影響。因此,有效提高內(nèi)皮細(xì)胞的轉(zhuǎn)染效率對于促進(jìn)人工血管材料的內(nèi)皮化有著最為直接的意義。鑒于陽離子基因載體的分子量和靶向活性多肽對基因的傳遞有重要影響,本論文首先探討了陽離子基因載體的分子量對內(nèi)皮細(xì)胞轉(zhuǎn)染效率的影響,進(jìn)而又設(shè)計了多肽修飾的新型基因載體,并用于內(nèi)皮細(xì)胞的體外轉(zhuǎn)染研究。第二章.為了考察陽離子基因載體的分子量對內(nèi)皮細(xì)胞轉(zhuǎn)染效率的影響,本章以聚甲基丙烯酸羥乙酯(PHEMA)為載體主鏈,通過引發(fā)L-丙交酯(LA)開環(huán)聚合,得到PHEMA-PLA嵌段共聚物。不同分子量的親水鏈聚乙二醇單甲醚(PEG)和聚乙烯亞胺(PEI)通過酯化和酰胺化反應(yīng)連接到羧基化的PHEMA-PLA嵌段共聚物上,制備得到四種不同分子量的陽離子基因載體:PHEMA-PLA-PEG 2kDa-PEI 1.8 kDa、PHEMA-PLA-PEG 5 kDa-PEI 1.8 kDa、PHEMA-PLA-PEG 2kDa-PEI 10 kDa和PHEMA-PLA-PEG 5 kDa-PEI 10 kDa。進(jìn)而通過溶液自組裝的方法分別構(gòu)建得到四種對應(yīng)的納米粒:G2I1.8、G5I1.8、G2I10和G5I10。采用動態(tài)光散射技術(shù)考察了這四種納米粒載體及其與pEGFP-ZNF580基因形成的復(fù)合物的流體力學(xué)直徑和表面zeta電位的大小。并選取EA.hy926為模型內(nèi)皮細(xì)胞,考察這四種基因載體及其復(fù)合物對內(nèi)皮細(xì)胞的生物學(xué)毒性及轉(zhuǎn)染活性的影響。結(jié)果表明適當(dāng)?shù)靥岣哧栯x子基因載體的分子量和在載體中引入PEG有助于提高載體復(fù)合物在內(nèi)皮細(xì)胞中的轉(zhuǎn)染效率。第三章.本章制備了一種REDV(Arg-Glu-Asp-Val)多肽修飾的基因載體,用于負(fù)載pEGFP-ZNF580基因,考察了靶向活性多肽修飾的載體復(fù)合物在內(nèi)皮細(xì)胞中的轉(zhuǎn)染效率。通過開環(huán)聚合和接枝聚合的方法制備得到聚乙二醇單甲醚-聚(丙交酯-co-己內(nèi)酯)-聚乙烯亞胺(mPEG-P(LA-co-CL)-PEI)兩親性嵌段共聚物。通過CREDVW多肽與mPEG-P(LA-co-CL)-PEI的共價鍵合反應(yīng),制備得到REDV多肽修飾的基因載體mPEG-P(LA-co-CL)-PEI-REDV。并通過自組裝的方法制備得到聚合物mPEG-P(LA-co-CL)-PEI-REDV和mPEG-P(LA-co-CL)-PEI對應(yīng)的納米粒載體REDV-NP和NP。通過凝膠電泳的方法考察了兩種載體納米粒對pEGFP-ZNF580基因的包覆能力。以EA.hy926為模型內(nèi)皮細(xì)胞,借助MTT實驗考察了載體基因復(fù)合物對內(nèi)皮細(xì)胞生物毒性的影響,并考察了載體復(fù)合物在內(nèi)皮細(xì)胞中的轉(zhuǎn)染效率。實驗結(jié)果表明,靶向活性多肽REDV修飾的基因載體對pEGFP-ZNF580基因包覆能力強(qiáng),對內(nèi)皮細(xì)胞生物相容性較好,體外轉(zhuǎn)染效率比PEI 25 kDa復(fù)合物高,且能有效促進(jìn)內(nèi)皮細(xì)胞的遷移。第四章.在本章研究中,為了制備多位點連接REDV多肽修飾的基因載體,考察其在內(nèi)皮細(xì)胞中遞送基因的能力,我們以PHEMA主鏈為大分子引發(fā)劑,采用開環(huán)聚合的方法將己內(nèi)酯引入到PHEMA的多條臂肢中,進(jìn)而通過酯化反應(yīng)和酰胺化反應(yīng)將PEG和小分子量的PEI與羧基化后的PCL鏈端連接,制備得到兩親性的梳型基因載體PHEMA-PCL-PEG-PEI。借助一種異雙官能團(tuán)連接劑,將REDV活性多肽引入到PEI鏈端,制備得到REDV多肽功能化的兩親性基因載體(PHEMA-PCL-PEG-PEI-REDV)。再通過自組裝的方法構(gòu)筑了這種聚合物納米粒,用于對pEGFP-ZNF580基因的包載。通過動態(tài)光散射技術(shù)和凝膠成像電泳分析的方法考察了靶向載體對pEGFP-ZNF580基因包覆能力的大小。以EA.hy926細(xì)胞為模型細(xì)胞,考察了靶向基因載體復(fù)合物對內(nèi)皮細(xì)胞的生物毒性及在內(nèi)皮細(xì)胞中的轉(zhuǎn)染效果,并對細(xì)胞中的蛋白表達(dá)含量進(jìn)行測定。結(jié)果表明該REDV多肽修飾的雙親梳型基因載體可有效包載pEGFP-ZNF580基因,且不會對內(nèi)皮細(xì)胞造成嚴(yán)重的細(xì)胞毒性,在內(nèi)皮細(xì)胞中轉(zhuǎn)染效率好,相應(yīng)的蛋白表達(dá)含量高。實驗結(jié)果表明陽離子基因載體的分子量對內(nèi)皮細(xì)胞的轉(zhuǎn)染效率有著很大的影響,且生物活性多肽修飾的載體及基因復(fù)合物可有效提高內(nèi)皮細(xì)胞的體外轉(zhuǎn)染效率。本文的研究工作對提高生物材料的內(nèi)皮化有著潛在的指導(dǎo)意義,有望應(yīng)用于心腦血管疾病的介入治療中。
[Abstract]:At present, the main problems of surgical treatment of cardiovascular and cerebrovascular diseases is the graft neointimal hyperplasia and restenosis, which seriously endanger the lives of patients. In view of some natural advantages of endothelial cells, implant materials quick endothelialization of materials is the key to success in the transplantation in vivo. Functional modification of the gene therapy and the surface of the material is two effective methods of artificial vascular endothelial implants. This thesis systematically summarizes the gene therapy and the peptide modified biological material made in promoting endothelialisation achievements. Due to the low transfection efficiency of endothelial cell intrinsic, make implant materials in vivo endothelialization effect greatly. Therefore, effectively improve the transfection of endothelial cells to promote the efficiency of endothelialization of artificial blood vessel materials have the most direct significance. In view of the molecular weight and cationic gene vector targeting activity Peptides have important influence on gene transfer, this paper discusses the influence of molecular weight of cationic gene vector transfection efficiency, and then design a new gene carrier peptide modified, and used for transfection of endothelial cells in vitro. In the second chapter. In order to investigate the effect of molecular weight of cationic gene vector transfection the efficiency of this chapter to poly 2-hydroxyethyl methacrylate (PHEMA) as the carrier backbone, by triggering L- lactide ring opening polymerization (LA), PHEMA-PLA block copolymer. The hydrophilic polyethylene glycol with different molecular weight (PEG) and polyethyleneimine (PEI) through esterification and amidation of carboxyl attached to the PHEMA-PLA block copolymer, prepared four kinds of different molecular weight cationic gene vector: PHEMA-PLA-PEG 2kDa-PEI 1.8 kDa, PHEMA-PLA-PEG 5 kDa-PEI 1.8 kDa, PHEMA-PLA-PEG 2kDa-PEI 10 KDa and PHEMA-PLA-PEG 5 kDa-PEI 10 kDa. respectively and then construct four corresponding nanoparticles by solution self-assembly: G2I1.8, G5I1.8, fluid mechanics, composite diameter and surface zeta potential of G2I10 and G5I10. on these four kinds of nanoparticles and pEGFP-ZNF580 gene formed by dynamic light scattering technology and select EA.hy926 size. As a model of endothelial cells, biological toxicity and transfection activity to examine these four gene vector and its compounds on endothelial cells. The results show that properly increasing the molecular weight and cationic gene vector PEG was introduced into the carrier is helpful to improve the carrier complex in endothelial cells transfection efficiency. In the third chapter, a kind of REDV. Were prepared in this chapter (Arg-Glu-Asp-Val) gene carrier peptide modified, used to load pEGFP-ZNF580 gene, investigated the carrier complex targeting peptide modified The efficiency of transfection in endothelial cells. Methods through ring opening polymerization and graft polymerization to prepare polyethylene glycol monomethyl ether poly (-co- lactide caprolactone) - polyethyleneimine (mPEG-P (LA-co-CL) -PEI) two amphiphilic copolymers by CREDVW peptide and mPEG-P (LA-co-CL) -PEI covalent bond reaction. Preparation of mPEG-P peptide modified REDV gene vector (LA-co-CL) and -PEI-REDV. by self-assembly method prepared by polymer mPEG-P (LA-co-CL) -PEI-REDV and mPEG-P (LA-co-CL) -PEI corresponding to REDV-NP and NP. nanoparticles were investigated with two carrier nanoparticles on pEGFP-ZNF580 gene by gel electrophoresis method with EA.hy926 as a model. With the help of MTT endothelial cells, the effects of the gene complex on endothelial cell toxicity, and the effects of the carrier complex in endothelial cells in transfection efficiency. The experimental results Show that the gene vector targeting peptide modified REDV of pEGFP-ZNF580 gene with strong ability of endothelial cells have good biocompatibility, in vitro transfection efficiency than the PEI 25 kDa complex, and can effectively promote the migration of endothelial cells. In the fourth chapter. In this chapter the research, in order to gene carrier preparation of multilocus REDV connection peptide modified gene delivery, investigate its ability in endothelial cells, we used PHEMA as the backbone of macroinitiator, using the method of ring opening polymerization of multiple limbs caprolactone into PHEMA, then the PEG and PEI of small molecular weight and carboxylated PCL after the chain end connected by esterification the reaction and amidation reaction, preparation of two amphiphilic comb type gene vector PHEMA-PCL-PEG-PEI. with the help of a hetero bifunctional linker, REDV active polypeptide into the PEI chain end, preparation of two amphiphilic functionalized REDV polypeptide gene carrier Body (PHEMA-PCL-PEG-PEI-REDV). Through the self-assembly method to build the polymer nanoparticles for the encapsulation of pEGFP-ZNF580 gene. Methods by dynamic light scattering and gel electrophoresis analysis examines the imaging ability of pEGFP-ZNF580 gene targeting vector coated size. Using EA.hy926 cells as model cells, effects of targeting gene carrier complex biological toxicity of endothelial cells and in endothelial cells and the cell transfection effect, the protein expression levels were determined. The results showed that the REDV peptide modified amphiphilic comb gene carrier can be effectively loaded pEGFP-ZNF580 gene, and will not cause severe cytotoxicity to endothelial cells, endothelial cell transfection efficiency well, the corresponding protein content is high. The experimental results show that the transfection efficiency of cationic gene vector on endothelial cells has a great influence The carrier, and biological activity of peptide modified and gene complexes can effectively improve transfection efficiency in vitro endothelial cells. This research work has potential significance for improving endothelialization of biomaterials, interventional therapy is expected to be applied to cardiovascular and cerebrovascular diseases.

【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R318.08;R54

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