载基因微球的制备及其在促进内皮细胞增殖方面的初步应用

发布时间：2018-10-13 16:43

【摘要】：本文制备了可降解载基因微球(Microparticles，MPs)，利用MPs/pEGFP-ZNF580复合物转染人脐静脉内皮细胞（HUVECs），达到了促进HUVECs增殖和迁移目的。针对高分子量聚乙烯亚胺(Polyethyleneimine，PEI)对细胞毒性高的问题，本文利用低分子量PEI制备了可降解型基因载体，并进行了评价。 1.为了克服高分子量PEI作为基因载体引起的细胞毒性问题，以聚乙二醇单甲醚(mPEG)、3(S)-甲基-吗啉-2,5-二酮(MMD)、乙交酯(GA)和低分子量PEI为原料，制备了甲氧基-聚乙二醇-嵌段-聚(3(S)-甲基-吗啉-2,5-二酮-共聚-乙交酯)-接枝-聚乙烯亚胺三嵌段共聚物(mPEG-b-P(MMD-co-GA)-g-PEI)，经自组装形成“核-壳”结构MPs。其中，MPs的疏水“核”由P(MMD-co-GA)组成，以它作为交联点，连接着众多的PEI和PEG链段，从而形成具有亲水性且带正电荷的“壳”。 MPs吸附并压缩ZNF580基因，形成MPs/pEGFP-ZNF580复合物。与高分子量PEI相比，MPs和MPs/pEGFP-ZNF580复合物的细胞毒性大大降低。MPs/pEGFP-ZNF580复合物的转染效果与LipofectamineTM2000的转染效果相似，经转染60小时后，HUVECs的增殖和迁移得到提高。本文提供了一种制备低毒性非病毒基因载体有效方法。 2.为了达到pEGFP-ZNF580从MPs/pEGFP-ZNF580复合物中持续释放的目的，合成了具有不同降解速率的三嵌段共聚物，进而制备了具备不同降解速率疏水核心的MPs。首先，合成了甲氧基-聚乙二醇-嵌段-聚(3(S)-甲基-吗啉-2,5-二酮)-接枝-聚乙烯亚胺(mPEG-b-PMMD-g-PEI)、甲氧基-聚乙二醇-嵌段-聚(3(S)-甲基-吗啉-2,5-二酮-共聚-丙交酯)-接枝-聚乙烯亚胺(mPEG-b-P(MMD-co-LA)-g-PEI)和甲氧基-聚乙二醇-嵌段-聚(3(S)-甲基-吗啉-2,5-二酮-共聚-丙交酯-共聚-乙交酯)-接枝-聚乙烯亚胺(mPEG-b-P(MMD-co-LA-co-GA)-g-PEI)三嵌段共聚物。之后，，经自组装形成了MPs。 pEGFP-ZNF580能够通过该复合物转染进入HUVECs，western blot分析表明ZNF580关键蛋白表达水平升高至35.74%-46.11%，pEGFP-ZNF580能够持续释放25天。这种MPs/pEGFP-ZNF580复合物在实现组织工程支架和人工血管的快速内皮化方面，具有潜在的应用价值。 3.为了实现MPs对HUVECs的选择性转染，通过接枝Arg-Glu-Asp-Val(REDV)多肽，制备了靶向性三嵌段共聚物，即REDV-接枝-聚乙烯亚胺-接枝-聚(丙交酯-共聚-乙交酯)-接枝-聚乙烯亚胺-接枝-REDV (REDV-g-PEI-g-P(LA-co-GA)-g-PEI-g-REDV)和REDV-接枝-聚乙烯亚胺-接枝-聚(丙交酯-共聚-乙交酯-共聚-3(S)-甲基-吗啉-2,5-二酮)-接枝-聚乙烯亚胺-接枝-REDV (REDV-g-PEI-g-P(LA-co-GA-co-MMD)-g-PEI-g-REDV)。以此为基材制备了HUVECs靶向性MPs。在HUVECs和人脐动脉平滑肌(HUASMCs)的共培养体系中，MPs/pEGFP-ZNF580复合物能够特异性地选择转染HUVECs，促进内皮细胞增殖，并抑制HUASMCs增殖。结果表明，这种MPs/ZNF580复合物具有特异性内皮细胞选择功能。
[Abstract]:Biodegradable gene carrying microspheres (Microparticles,MPs) were prepared and transfected into human umbilical vein endothelial cells (HUVECs),) by MPs/pEGFP-ZNF580 complex to promote the proliferation and migration of HUVECs. Aiming at the high toxicity of high molecular weight polyethylene imine (Polyethyleneimine,PEI), a biodegradable gene vector with low molecular weight PEI was prepared and evaluated. In order to overcome the cytotoxicity caused by high molecular weight PEI as a gene vector, polyethylene glycol monomethyl ether (mPEG), 3 (S)-methyl-morpholine-2o-5-dione (MMD), glycolide (GA) and low molecular weight PEI) were used as raw materials. The mPEG-b-P (MMD-co-GA)-g-PEI triblock copolymers (mPEG-b-P (MMD-co-GA)-g-PEI) with methoxy-polyethylene glycol-block-poly (3 (S)-methyl-morpholine-2-diketone-5-diketone-ethylene lactide) were prepared. The MPs. with "core-shell" structure was formed by self-assembly. Among them, the hydrophobic "nucleus" of MPs is composed of P (MMD-co-GA), which acts as the crosslinking point and connects a large number of PEI and PEG segments to form a hydrophilic and positively charged "shell". MPs adsorbs and compresses ZNF580 gene to form MPs/pEGFP-ZNF580 complex. Compared with high molecular weight PEI, the cytotoxicity of MPs and MPs/pEGFP-ZNF580 complex was much lower. The transfection effect of MPs/pEGFP-ZNF580 complex was similar to that of LipofectamineTM2000. After 60 hours of transfection, the proliferation and migration of HUVECs were improved. This paper provides an effective method for preparation of low toxicity nonviral gene vector. 2. In order to continuously release pEGFP-ZNF580 from MPs/pEGFP-ZNF580 complexes, triblock copolymers with different degradation rates were synthesized, and MPs. with different degradation rate hydrophobic cores were prepared. First Methoxy-polyethylene glycol-block-poly (3 (S)-methyl-morpholine-2o-5-dione)-grafted polyimide (mPEG-b-PMMD-g-PEI), methoxy-polyethylene glycol-block-poly (3 (S)-methyl-morpholine-2o-5-diketone-copolymerization-lactide)-graft copolymerization-poly (ethylglycolide) were synthesized MPEG-b-P (MMD-co-LA)-g-PEI and mPEG-b-P (MMD-co-LA-co-GA)-g-PEI triblock copolymers of mPEG-b-P (MMD-co-LA)-g-PEI and methoxy-polyethylene glycol-block-poly (3 (S)-methyl-morpholine-2o-5-diketone-copolymerized-lactide-ethylene-lactide)-graft-polyethylene imide (mPEG-b-P (MMD-co-LA-co-GA)-g-PEI). After self-assembly, MPs. pEGFP-ZNF580 could be transfected into HUVECs,western blot by the complex. The expression level of ZNF580 key protein was increased to 35.74-46.11%, and pEGFP-ZNF580 could be released continuously for 25 days. This MPs/pEGFP-ZNF580 complex has potential application value in realizing rapid endothelialization of tissue engineering stents and artificial blood vessels. In order to realize the selective transfection of HUVECs by MPs, the targeted triblock copolymers were prepared by grafting Arg-Glu-Asp-Val (REDV) polypeptides. That is, REDV- grafted polyethyleneimine grafted poly (lactide copolymerizate-glycolide) grafted polyethyleneimide-grafted REDV (REDV-g-PEI-g-P (LA-co-GA) -g-PEI-g-REDV) and REDV- grafted polyvinyleneimide-grafted poly (lactide-co-ethyllactide 3 (S)- Methyl-morpholine-2o-5-diketone)-graft-polyimide-graft-REDV (REDV-g-PEI-g-P (LA-co-GA-co-MMD)-g-PEI-g-REDV). HUVECs targeting MPs. was prepared by using this material as substrate. In the co-culture system of HUVECs and human umbilical artery smooth muscle (HUASMCs), MPs/pEGFP-ZNF580 complex can selectively transfect HUVECs, to promote endothelial cell proliferation and inhibit HUASMCs proliferation. The results showed that the MPs/ZNF580 complex had specific endothelial cell selection function.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943;O631.11

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