【作者】 邓诚；

【导师】 董念国；

【作者基本信息】 华中科技大学 ， 外科学， 2012， 博士

【摘要】 第一部分：基质金属蛋白酶酶解控释血管内皮生长因子聚乙二醇水凝胶制备与鉴定第一节四枝化状丙烯酰化聚乙二醇的合成与鉴定目的：运用接枝聚合方法制备四枝化状丙烯酰化聚乙二醇(4branched-PEG-DA),并鉴定其化学结构及属性是否符合实验设计要求。方法：以分子量20,000Da线性聚乙二醇(PEG)单体为原料,通过四步合成方法,在单体末端引入四个丙烯酰基双键官能团。经红外光谱、1H核磁波谱、高效液相色谱、基质辅助激光解吸电离及凝胶过滤色谱检测聚合物产品在分子结构、目标官能团置换度、终产物产率、分子量及产物纯度方面是否符合实验设计、满足后续实验要求。结果：自制4branched-PEG-DA产物经红外光谱、1H核磁共振检测证实分子结构与设计相符、末端双键官能团置换度96.3%；高效液相色谱检测显示产物纯度为98.4%；基质辅助激光解吸电离检测提示产物实际平均分子量与设计结构分子量相符；凝胶过滤色谱检测结果显示产物多分散性数值为1.02,产物分子量均一,纯度较高。结论：通过接枝聚合方法制备4branched-PEG-DA在分子结构、目标官能团置换度、终产物产率、分子量及产物纯度方面均符合实验设计、满足后续实验要求。第二节基质金属蛋白酶酶解控释血管内皮生长因子聚乙二醇水凝胶制备及释药性能研究目的：制备基质金属蛋白酶酶解控释血管内皮生长因子聚乙二醇水凝胶(VEGF-MMP多肽PEG gel),观察其形貌结构并研究酶解控释效应,为后续实验奠定基础。方法：运用9-芴甲氧羰基(Fmoc)多肽固相法合成含有巯基(-SH)的基质金属蛋白酶(matrix metalloproteinase, MMP)底物多肽。利用4branched-PEG-DA与血管内皮生长因子165(vascular endothelial growth factor 165, VEGF-165)共价结合后,根据Michael加成反应原理,与含有巯基的MMP底物多肽结合生成VEGF载药聚乙二醇水凝胶(VEGF-MMP多肽PEG gel)。通过大体形态及扫描电镜观察VEGF-MMP多肽-PEG gel形貌及微观结构。运用酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA),研究基质金属蛋白酶2(MMP-2)与VEGF-MMP多肽PEG gel之间酶解释药的关系。结果：室温下VEGF-MMP多肽PEG gel大体形态为透明果冻状。经扫描电镜观察,水凝胶表面微观形态呈网状。其中对MMP敏感的VEGF-MMP多肽PEG gel (VEGF-MMP (W) X-PEG gel)表面孔隙平均直径为0.251±0.10lum,孔隙率为10.1±1.21%。ELISA检测结果显示,VEGF-MMP多肽-PEG gel对VEGF-165包封率为84.33±1.18%。凝胶形态崩解时相及ELISA检测结果显示,在不同浓度MMP-2作用下,VEGF-MMP (W) X-PEG gel具有稳定的VEGF释放速率。结论：通过Michael加成反应制备的VEGF-MMP (W) X-PEG gel与MMP-2具有稳定的控释关系,满足后续实验要求。第二部分：VEGF控释水凝胶-去细胞瓣复合支架制备及体外生物相容性研究第一节VEGF控释水凝胶-去细胞瓣复合支架的制备与鉴定目的：制备VEGF控释水凝胶-去细胞瓣复合支架,并通过形态学观察和免疫荧光染色鉴定是否成功构建复合支架方法：运用生物素(Sulfo-NHS-SS-Biotin)修饰去细胞瓣支架,行免疫荧光染色鉴定。根据亲和素-生物素结合原理,将含有生物素的VEGF控释水凝胶(VEGF-bio-MMP(W)X-PEG gel)与去细胞瓣支架复合,行HE染色和扫描电镜观察。结果：结合生物素的去细胞瓣支架,经携带Cy3荧光亲和素染色,荧光显微镜显示瓣膜基质呈红色。形态学观察显示,水凝胶-去细胞瓣复合支架具有去细胞瓣三层基质结构和凝胶规则网状孔隙微观结构。结论：按实验设计成功制备VEGF控释水凝胶-去细胞瓣复合支架。第二节VEGF控释水凝胶-去细胞瓣复合支架体外生物相容性研究目的：参照GB/T1688国家标准,体外研究VEGF控释水凝胶-去细胞瓣复合支架生物相容性。方法：参照GB/T1688国家标准,对复合支架进行血液相互作用实验,研究其对血小板激活、凝血时间、溶血的影响；对复合支架及其浸提液进行细胞毒性实验,研究其对细胞增殖与凋亡的影响。结果：经扫描电镜,流式细胞仪检测,证实复合支架可减少去细胞瓣对血小板的粘附与激活；经PT/APTT/INR检测,证实复合支架对凝血时间无明显影响；经间接、直接溶血实验,证实复合支架溶血率低于GB/T16886国家标准；经细胞增殖、凋亡检测,证实复合支架及其浸提液对MRC-5人胚肺细胞无明显影响。结论：参照GB/T1688国家标准,VEGF控释水凝胶-去细胞瓣复合支架具有良好的生物相容性。第三部分：智能水凝胶-去细胞瓣复合支架制备及体内生物学性能研究第一节智能水凝胶-去细胞瓣复合支架的制备与鉴定目的：制备抗CD34抗体-VEGF控释水凝胶-去细胞瓣复合支架(智能水凝胶-去细胞瓣复合支架),并通过免疫荧光染色鉴定是否成功构建复合支架方法：运用生物素(Sulfo-NHS-SS-Biotin)修饰去细胞瓣支架。根据亲和素-生物素结合原理,将VEGF-b i o-MMP(W)X-PEG gel与去细胞瓣支架复合。同样原理,将经生物素化抗CD34抗体与水凝胶-去细胞瓣支架复合,行免疫荧光染色鉴定。结果：结合抗CD34抗体水凝胶-去细胞瓣复合支架,经FITC标记抗IgG抗体染色,荧光显微镜显示瓣膜基质呈绿色。结论：按实验设计成功制备智能水凝胶-去细胞瓣复合支架。第二节智能水凝胶-去细胞瓣复合支架体内生物学性能研究目的：建立兔颈动脉瓣膜管道移植模型,评估智能水凝胶-去细胞瓣复合支架体内生物学性能方法：运用改良兔颈动脉"Cuff"套管技术,建立兔颈动脉瓣膜管道移植模型。分别于术后24小时和7天,通过多普勒彩色超声检测管道通畅与否,判断动物模型建立是否成功。术后24小时,取出移植物,根据组织形态学检测,评估移植管道对CD34+细胞的粘附能力。术后7天,取出移植物,根据组织形态学检测及eNOS mRNA定量分析,评估移植管道内皮化程度。结果：超声结果显示移植管道通畅,兔颈动脉瓣膜管道移植模型建立成功。术后24小时,相应检测结果显示,本实验制备复合支架对CD34+细胞有明显的粘附能力。术后7天,相应检测结果显示,本实验制备复合支架内皮化程度优于对照组支架。结论：智能水凝胶-去细胞瓣复合支架具有优良的生物学性能。更多还原

【Abstract】 PartⅡThe preparation of poly (ethylene glycol) hydrogeland the research of its matrix metalloproteinase enzymatic release of vascular endothelial growth factorChapter one The preparation and identification of four branched propylene acylation poly (ethylene glycol) (4branched-PEG-DA)Objective:To prepare four branched propylene acylation poly (ethylene glycol) (4branched-PEG-DA) by grafted polymerization method, and identify the chemical structure and properties are in accordance with the experiment design and requirementsMethods:The linear polyethylene glycol (PEG) monomers (Molecular weight 20, OOODa) as raw materials, through the four-step synthesis method, four acryloyl functional groups were introduced at the monomer ends. By detections of infrared spectrometry (IR),1H-nuclear magnetic resonance (1H-NMR), high performance liquid chromatography(HPLC), Matrix-assisted laser desorption/ionization(MALDI) and gel filtration chromatography (GFC), the polymer products are in accordance with the experimental design to meet the follow-up requirements, in molecular structure, substitution of functional groups, final production rate, molecular weight and molecular weight uniformity. Results:By IR and 1HNMR detection, our 4branched-PEG-DA was confirmed the molecular structure matched the design and the substitution of functional groups was 96.3%; HPLC showed that the purity of the product was 98.4%; MALDI indicated the actual average molecular weight of the product matched the design; GFC results showed that the product Poly-dispersity value was 1.02, proved molecular weight was uniform.Conclusion:4branched-PEG-DA prepared by the grafted polymerization method is in accordance with the design of experiment and meets the requirements of follow-up, in aspects of molecular structure, functional groups substitution, final production rate, molecular weight and molecular weight uniformity.Chapter two The research of matrix metalloproteinase enzymatic release of vascular endothelial growth factor in poly (ethylene glycol) hydrogelObjective:To prepare poly (ethylene glycol) hydrogel released vascular endothelial growth factor by matrixmetalloproteinase (VEGF-MMP peptide-PEG gel),and observe the morphology and their enzymatic controlled release effect for the subsequent experiments.Methods:Using 9-fluorenyl methoxy carbonyl (Fmoc) solid phase peptide synthesis, we synthesized matrix metalloproteinase (MMP) substrate peptide containing thiol groups (-SH). After 4branched-PEG-DA and vascular endothelial growth factor 165 (VEGF-165) covalently bounded, according to Michael addition reaction, the combination of MMP substrate peptide containing-SH generated the poly (ethylene glycol) hydrogel loading VEGF (VEGF-MMP peptide-PEG gel) Observation of morphology and microstructure of VEGF-MMP peptide-PEG gel were fulfilled by camera and scanning electron microscopy. Use of enzyme-linked immunosorbent assay (ELISA), to reveal the relationship between matrix metalloproteinase 2 (MMP-2) and VEGF-MMP peptide-PEG gel.Results:At room temperature, the VEGF-MMP peptide-PEG gel was jelly-like. The scanning electron microscopy showed the microstructure of the hydrogel was reticular. The VEGF-MMP peptide-PEG gel sensitive to MMP (VEGF-MMP (W) X-PEG gel) was 0.251±0.101um in surface pore average diameter and 10.1±1.21% of porosity. ELISA showed that VEGF-165 encapsulation efficiency of VEGF-MMP peptide-PEG gel was 84.33±1.18%. The collapsed phase and ELISA results showed that under different concentrations of MMP-2, VEGF-MMP (W) X-PEG gel possessed a stable VEGF release rates.Conclusion:VEGF-MMP (W) X-PEG gel prepared by Michael addition reaction possessed a stable enzymatic controlled release with MMP-2, which to meet the further experiment requirements.PartⅡThe preparation of VEGF controlled release hydrogel-decellularized scaffold and the research of its biocompatibility in vitroChapter one The preparation and identification of VEGF controlled release hydrogel-decellularized scaffoldObjective:To prepare VEGF controlled release hydrogel- decellularized scaffold, and identify the structure by the morphology and immunofluorescence staining.Methods:Decellularized valve modified with Sulfo-NHS-SS-Biotin was identified by immunofluorescence staining. According to avidin-biotin combination, biotinylated VEGF controlled release hydrogel (VEGF-bio-MMP (W) X-PEG gel) and decellularized scaffold were compound, and identified by HE staining and scanning electron microscopy.Results:The decellularized scaffold modified with biotin was stained by avidin-Cy3, while fluorescence microscopy showed the valve matrix was red. Morphological observation showed that the hydrogel-decellularized scaffold possessed decellularized matrix structure andreticular microstructure of the hydrogel.Conclusion:VEGF controlled release hydrogel-decellularized scaffold was successfully prepared in accordance with the design of experiment.Chapter two The research of biocompatibility of VEGF controlled release hydrogel-decellularized scaffold in vitroObjective:To study the biocompatibility of VEGF controlled release hydrogel-decellularized scaffold in vitro, according to GB/T1688 national standards.Methods:Refer to GB/T1688 national standards, the hybrid scaffold, for blood interaction experiments, was studied on the influence to platelet activation, clotting time, hemolysis; the hybrid scaffold and its extract, for cytotoxic experiment, were studied on the influence to cell proliferation and apoptosis.Results:The scanning electron microscopy and flow cytometry testing, confirmed that the hybrid scaffold can reduce the decellularized influence to platelet adhesion and activation; PT/APTT/INR test showed that the hybrid scaffold had no significant effect on the clotting time; indirect and direct hemolytic experiments confirmed that the hybrid scaffold hemolysis rate was lower than the the GB/T16886 national standards; by cell proliferation and apoptosis detection, the hybrid scaffold and its extract were proved that had no significant effect on MRC-5 human embryonic lung cells.Conclusion:Refer to GB/T1688 national standards, VEGF controlled release hydrogel-decellularized scaffold possessed fine biocompatibility.PARTⅢThe preparation of Anti CD34-VEGF controlled release hydrogel-decellularized scaffold and the research of its biological properties in vivoChapter one The preparation and identification of Anti CD34-VEGF controlled release hydrogel-decellularized scaffoldObjective:To prepare Anti CD34-VEGF controlled release hydrogel-decellularized scaffold (smart hydrogel-decellularized scaffold), and identify the structure by the immunofluorescence staining.Methods:decellularized valve was modified with Sulfo-NHS-SS- Biotin. According to avidin-biotin combination, biotinylated VEGF controlled release hydrogel (VEGF-bio-MMP (W) X-PEG gel) and decellularized scaffold were compound. In the same way, the biotinylated Anti CD34 antibody and hydrogel-decellularized scaffold were compound, and identified by immunofluorescence staining.Results:Under fluorescence microscopy, hydrogel-decellularized scaffold combined with Anti CD34 antibody showed that the valve matrix was green while stained by anti-IgG-FITC.Conclusion:The smart hydrogel-decellularized scaffold was successfully prepared in accordance with the design of experiment.Chapter two The research of biological properties of smart hydrogel-decellularized scaffold in vivoObjective:To establish a rabbit carotid artery valve pipeline transplantation model, and evaluate the biological properties of smart hydrogel-decellularized scaffold in vivo.Methods:Using a modified "Cuff" method, to establish rabbit carotid artery valve pipeline transplantation model. To determine the animal model is successful by the Color Doppler Ultrasound detecting the pipes are open or not in postoperative 24 hours and 7 days. After 24 hours, take out the pipes to evaluate the adhesion to CD34+ cells by histomorphology analysis. After 7 days, take out the pipes to assess the degree of endothelialization by histomorphology and eNOS mRNA quantitative analysis.Results:The rabbit carotid artery valve pipeline transplantationmodel were successful, by the ultrasound results showing that the transplanted pipes were open. After 24 hours, the corresponding test results showed that our scaffold had an obvious adhesion to the CD34+ cells. After 7 days, the corresponding test results showed that the degree of endothelialization of our scaffold were superior to the controls.Conclusion:smart hydrogel-decellularized scaffold possessed excellent biological properties.更多还原