【作者】 孙自玲；

【导师】 白伦；

【作者基本信息】 苏州大学 ， 纺织工程， 2010， 博士

【摘要】 多孔丝素蛋白膜(PSFFs)是具有良好的机械性能和理化性质,如柔韧性和抗拉伸强度、降解性、有效的血管化能力等,所以在医学上广泛应用于人造血管、皮肤、骨来代替、修补、辅助修复人体组织器官。利用多孔丝素蛋白膜辅助修复真皮时,必然要求能够快速有效地血管化,而多孔丝素蛋白膜血管化过程是多孔丝素蛋白膜材料、细胞因子、细胞和细胞外基质等因素相互协调作用和适应的过程。但是生物材料中血管新生的细节及至材料与机体组织协同作用的的创伤环境条件下血管化的详细过程相关的研究报告也比较少。为了探讨多孔丝素蛋白膜材料应用于创伤修复中的血管化机制,需要对材料植入体内时血管化相关的组织细胞与微环境在组织学和分子生物学水平上探讨与研究。本研究以多孔丝素蛋白膜材料在创伤修复时血管化进程的动态观察作为研究研究目标。通过对多孔丝素蛋白膜材料血管化过程的超微观察分析、血管化有关分子如HIF、VEGF、整合素αvβ3和血管内皮细胞CD34等免疫组化分析,反转录聚合酶链式扩增结合技术(RT-PCR)等手段对丝蛋白生物材料植入大鼠真皮层以后的血管化相关因子HIF、VEGF及受体、Ang-1的行为趋势等进行观察与分析,以期探究材料与组织共存情况下的血管新生过程的真实面貌,从而在分子水平和免疫组化水平对多孔丝素蛋白膜材料血管化过程进行系统研究。研究的内容主要分为以下三个部分:第一部分为毛细血管形态学变化过程的实验跟踪研究及结果。这部分的工作采用苏木素伊红(HE)染色从组织形态学上对血管形成过程和模式进行分析,并用透射电镜的方法从超微结构分析内皮细胞形成的毛细血管成熟过程进行观察,并对血管新生模式进行分析。结果说明了多孔多孔丝素蛋白膜材料内的血管化过程是血管网从无到有,再从有经调整到优化、合理的血管网的过程以及内皮细胞从少到多,形成新生毛细血管成熟的生长阶段。手术7天后多孔多孔丝素蛋白膜材料周围血管主要以出芽方式侵入材料,内皮细胞周围没有完整的基底膜; 25天后主要是血管网形成,以套叠方式优化血管,内皮细胞和周细胞连接紧密、基底膜完整。在利用多孔丝素蛋白膜材料进行真皮创伤修复过程中,植入早期血管主要以出芽方式新生,而随着修复过程的进行,血管新生方式中出芽方式逐渐减少,而套叠方式逐渐增多,创伤修复后期毛细血管主要以套叠方式新生。第二部分为毛细血管新生过程的免疫组化实验跟踪研究及结果。这部分的工作通过制备创伤动物模型,以免疫组化的方法研究HIF、VEGF、血管内皮细胞标记物CD34、整合素avβ3和纤维连接蛋白(FN)、层连接蛋白(LN)在PSFFs和PVA创伤修复中表达的时相性变化,对他们定位、定性和定量研究,并对他们的变化趋势分析,从而进一步探讨多孔丝素蛋白膜材料中的创伤修复和血管化的分子机制。按照统计标准,结果表明手术1天时,多孔丝素蛋白膜和PVA材料里的内皮细胞CD34表达阴性,材料里充满成纤维细胞和炎症细胞;手术7天,15天,25天时PSFFs中微血管密度(MVD)值分别是3.54,11.6,24.5,与PVA中的MVD值比较,统计分析二者具有显著差异(P﹤0.05)。PSFFs中的VEGF在手术1天、7天、15天和25天分值分别是3.4,1.9, 2.5, 2.6。和PVA相比在手术7天时具有显著差异(P﹤0.05),其他时间段差异不显著(P﹥0.05);手术1天、7天、15天和25天PSFFs中HIF表达分别是12,4.8,8.6,9.0,和PVA相比在1天、15天和25天二者之间没有显著差异(P﹥0.05),在手术7天时具有显著差异(P﹤0.05)。另外本章还研究了血管出芽的标记物整合素avβ3、细胞外基质的主要成分FN和基底膜LN的变化趋势。得出PSFFs的血管化过程遵循创伤修复血管形成的一般特点,但也体现了自己的优越性,加快了修复过程。PSFFs比PVA具有良好的血管化能力,并且有利于细胞入侵、血管新生、细胞外基质的重建,从而使组织修复达到良好的效果。另外此结果对血管在多孔丝素蛋白材料中的生长模型进行了完善。第三部分为以分子生物学的方法对毛细血管新生过程相关分子,生长因子进行实验跟踪研究及结果。这部分的工作通过逆转录-聚合酶链式反应(RT-PCR)方法对VEGF及受体、Ang-1、整合素、HIF进行扩增,观察他们在4个时间段的变化趋势。结果显示VEGF、HIF和整合素的变化趋势和免疫组化结果一致。PSFFs植入活体后血管形成相关分子为适应环境发生了相关的改变。通过以上三部分内容的研究,对多孔丝素蛋白膜材料植入实验中的血管形成过程进行了系统观察分析与研究,通过与对照材料PVA实验结果比较,确认了丝素蛋白膜材料在创伤修复中提供有利于毛细血管新生的正常生理微环境;在毛细血管形成中各种相关因子的动态过程检测或是毛细血管形态数量变化实验结果,都显示了与PVA材料有显著性差异。研究观察到在多孔丝素蛋白膜植入体内的血管新生过程中,修复进程前期主要以出芽方式新生、后期主要以套叠新生。在丝素蛋白材料中看到这一现象,为这一现象在新的条件下的再现提供实验证据。对多孔丝素膜蛋白材料血管化过程,首先通过对创伤修复进程进行组织学和透射电镜的显微追踪观察;其次通过免疫组化实验对血管化重要因子及要素,如HIF、VEGF、微血管密度等研究分析了他们在材料中的时空性表达趋势,并研究了细胞外基质、血管基底膜的变化过程;最后通过在mRNA分子水平上对多孔丝素蛋白材料膜中的HIF、VEGF和Ang-1、整合素的表达分析。在多方面的实验观察研究的基础上,提出了在丝素蛋白材料的血管化进程的初步模式。为丝蛋白作为医用生物材料的设计制作及其应用的进一步优化等提供了的基础的研究结果。更多还原

【Abstract】 Silk fibroin has been widely applied in dermis, bone, vessel regeneration due to its biocompatibility, degradability and remarkable mechanical properties. Angiogenesis is a key step during wound healing in most tissues, so biomaterials aiding wound healing requires rapid and effective neovascularization of biomaterials after implantation. Process of neovascularization involved in interactions between cell-cell, cell-growth factors and silk fibroin–extracellular matrix (ECM). Although numbers of researches have focused on the design and preparation of porous biomaterials for excellent neovascularization, the analysis about processes and model on angiogenesis of biomaterial in vivo has been little reported so far.It is necessary to explore and analyze the characters and process of porous silk fibroin films( PSFFs )neovascularization. So this paper aims to study the dynamic process involved in the vascularization of PSFFs for dermis regeneration using immunochemical method, transmission electron microscopy (TEM), Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. Angiogenic factors VEGF and its receptor, hypoxia inducible factor-1(HIF),integrinαvβ3, angiopoietin-1 and so on were observed for exploring a real and systemic process of PSFFs neovascularization.This paper includes three parts:The first part is to observe process and mode of angiogenesis in PSFFs by hematoxylin-eosin staining (HE) in histology and TEM in ultrastructure. The result showed capillaries formation in PSFFs and polyvinyl alcohol (PVA) sponges went through form avascular to vascular, and matured through remodeling into a complex vascular net. At early stage of wound healing capillaries were formed in PSFFs by sprouting, and intussusceptive angiogenesis were mainly present at late stage. The second part is to study vascular endothelial growth factor (VEGF), hypoxia inducible factor-1(HIF), integrinαvβ3, CD34, fibronectin (FN) and laminin(LN) expression in PSFFs and PVA through immunochemical method. The results show that mean value of MVD in PSFFs is 0, 3.54, 11.6, 24.5 respectively after 1, 7, 15, 25 days implantation. It is significant difference compared with that of PVA.VEGF expression in PSFFs is 3.4,1.9, 2.5, 2.6 respectively by day 1,7,15,25. Mean value of HIF expression in PSFFs was 12,4.8,8.6,9.0 by day1,7,15,25. Mean value of HIF and VEGF expression in PVA compared with those of PSFFs are significant difference only by day 7. Trends of HIF expression is consistent with that of VEGF in PSFFs, otherwise integrinαvβ3, FN and LN expression were discussed too. PSFFs have better neovascularization than that of PVA due to excellent characters in angiogenesis, degradation.The third part is to study the mRNA expression about VEGF and its receptor, angiopoietin-1(Ang-1), integrinαvβ3, HIF at different times. More proliferating cells in PSFFs were in the absence of oxygen after implanting to dermis defects. The potent angiogenic factors VEGF and its receptor,integrinαvβ3 and Ang-1 may be activated in response to hypoxia under the control of the transcription factor HIF-1.The results of VEGF, HIF -1 and integrinαvβ3 were consistent with that of immunochemical level.In all, Processes and model of capillaries formed in PSFFs were studies in immunochemical and mRNA level, PSFFs have better neovascularization than that of PVA due to excellent characters in degradation, angiogenesis. PSFFs as scaffolds can promote angiogenesis by sprouting at early stage and intussusception at late stage in wound healing; HIF, VEGF and integrinαvβ3, FN and LN expression in PSFFs and PVA were in a dynamic process in mRNA and immuochemical level in order to adapt environment.General regulations in angiogenesis were followed by neovascularization in PSFFs. Primary model for processes of neovascularization were put forward.更多还原