多孔丝素膜材料中血管新生模式机制的探索
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摘要
丝素材料由于具有良好的生物学性能，近年来在生物医学材料的研究领域受到了研究人员的关注。在生物材料修复组织过程中，为了使得生物支架材料形成适合新的组织及细胞生长的环境，就要求生物材料能够快速有效地血管化。因此生物材料的血管化问题又是生物材料修复组织过程中的关键问题。但是在生物医学材料领域，生物材料在体外或植入体内后的血管化问题并没有完全得到解决，目前仍然是组织工程和生物材料应用中的一个瓶颈问题。血管新生作为实现血管化过程的重要环节，包括出芽式血管新生和套叠式血管新生为主的各种方式，迄今已在不同的生理和病理条件下发现了这些血管新生方式，但是关于血管新生模式的机制问题并没有完全得到阐明。毛细血管新生方式的机制问题是血管新生的前沿课题，相关研究工作近二十年以来备受关注。生物材料中的血管新生模式问题，过去的研究报告较少。基于这样的现状，本课题研究以探索多孔丝素蛋白材料中血管新生模式的机制为目标，对机体生长过程及多孔丝素材料修复肌肉组织两种情况下血管新生的进程进行调查和研究。
本文采用HE染色和免疫组化实验方法观察与分析比较机体生长过程及多孔丝素材料修复肌肉组织两种情况下的血管新生过程，并对新生血管进行数量化分析，同时结合透射电镜技术分析毛细血管的新生方式，并通过半定量计分法计算机体生长过程及多孔丝素材料修复肌肉组织各阶段中缺氧诱导因子(HIF-1α)和内皮细胞生长因子(VEGF)的表达程度。研究结果显示，在大鼠肌肉生长过程中的初期既有出芽新生也有套叠新生，但出芽的现象较少；随着大鼠年龄的增长，中后期肌肉组织中的毛细血管主要以套叠新生为主。同时发现在生长过程的初期，HIF-1α及VEGF表达均比较强烈，而中后期HIF-1α及VEGF表达明显减弱。通过丝素材料植入创伤肌肉组织中的实验发现，在植入材料的早期即13天以前材料周边主要以出芽方式进行血管新生来满足材料中组织细胞的供氧需求；而后期即13天以后则主要以套叠式血管新生来重塑与调整已有血管网。并且在植入股肌肉的早期，多孔丝素材料内HIF-1α及VEGF强烈表达，随着时间的推移，中后期时HIF-1α及VEGF表达渐渐变弱。由此推测HIF-1α及VEGF的表达与血管新生方式之间有密切的联系，当HIF-1α及VEGF在血管新生过程中的表达程度由强变弱时，标志着血管新生从初期的出芽为主逐渐转变为以套叠为主。
此外，结合文献报道及实验结果，从血管新生过程中的缺氧程度差异、生长因子的表达以及内皮细胞对血管内皮生长因子的响应以及新生血管的构建等几个环节出发，根据组织中新生血管生长前期与后期的差异以及出芽新生与套叠新生的差异，对大鼠肌肉组织生长过程及多孔丝素材料中血管新生模式的发生机制作出了分析及探索，并对材料植入大鼠腿部肌肉后的血管新生过程以及新生血管网络构建模式进行了思考与讨论。
通过对多孔丝素材料中血管新生模式及其机制的研究，为阐明材料中血管化的进程及其生物学机制提供基础的工作，同时也为适应性更宽泛的医用生物材料的开发提供理论依据及实验基础。
Because of excellent biological properties, silk materials receive much attention fromresearchers in the research area of biomedical materials in recent years. During the processof biomaterials repairing tissues, in order to make biomaterials scaffold form acircumstance that is suitable for tissue regeneration and cell survival, it requires rapid andeffective neovascularization of biomaterials after implantation, thus neovascularization ofbiomaterials is also the key subject of study on the biomaterials repairing tissues. However,in the field of biomedical materials, the problem about neovascularization of biomaterialsin vitro or in vivo is not fully settled, which is still a bottleneck problem of tissueengineering and biomaterial application. As an important step of realizing theneovascularization process, angiogenesis includes various modes that are predominantlysprouting angiogenesis and intussusceptive angiogenesis, and so far these kinds of modeshave been found under different physiological and pathological conditions, but problemson the mechanism of angiogenesis mode have not been completely clarified. The researchon the capillary angiogenesis mode is the front topic of angiogenesis, and related researchwork has received much concern from researchers in the last twenty years. With regard tothe angiogenesis mode in biomaterials, the past research reports are few. Based on thepresent situation, this paper aims at exploring the mechanism of angiogenesis modes inporous silk fibroin materials, and the angiogenesis courses under these two conditions ofbody growth process and porous silk fibroin materials repairing muscle were respectivelyinvestigated and studied.
In this paper, the angiogenesis processes under these two conditions of both bodygrowth process and porous silk fibroin materials repairing muscle were observed andanalyzed by methods of HE staining and immunohistochemical assay. In addition, thequantity of the newly formed capillaries in the material was analyzed. Meanwhile, theangiogenesis mode was analyzed by TEM technology. Moreover, the expression degrees ofhypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) inthe each stage of body growth process and porous silk fibroin materials repairing musclewere calculated by semi-quantitive scoring method. The results showed that both sprouting and intussusception occurred at the early stage of tissue growth, but less sproutingphenomena were found; and with the increase of the age of the SD rat, intussusception isthe main angiogenesis mode at the middle and late stage of tissue growth. Correspondingly,there were strong expressions of both HIF-1α and VEGF at the early stage of growthprocess, while the expressions of both HIF-1α and VEGF weakened markedly at themiddle and late stage. On the other hand, according to the experiment that silk fibroinmaterials were implanted into the wound muscle of SD rats, it was found that capillarieswere mainly newly formed by sprouting before day13to supply oxygen for the cells,while by intussusception after day13to remodel and adjust the existing vascular network.Accordingly, at the early stage of implantation, there were strong expressions of bothHIF-1α and VEGF in the materials, while at the middle or latter stage of implantation, theexpressions of HIF-1α and VEGF gradually weakened. Therefore, it was speculated thatthere are close relations between the expressions of HIF-1α, VEGF and angiogenesismodes in materials. That is, when the expression levels of HIF-1α and VEGF during theangiogenesis process change from strong to weak, the main mode of capillariesangiogenesis also turn from sprouting to intussusception.
In additon, by combining the literature reports and experimental results, this paperstretched from several aspects such as differences of hypoxia degree, growth factorexpression and endothelial cells in response to VEGF, and in accordance with thedifferences between the former stage and the late stage of the growth of newly formedcapillaries as well as the differences between sprouting angiogenesis and intussusceptiveangiogenesis, the mechanism of the angiogenesis modes was analyzed and explored underthe conditions of body growth process and porous silk fibroin materials repairing tissue,moreover, the angiogenesis process and capillary network construction mode in thematerials were thought and discussed after the implantation.
This paper provided the basis for clarifying the neovascularization process andbiological mechanism of the materials as well as the theoretical basis and experimentalfoundation for the exploitation of biomedical materials with wider adaptability throughstudying on the angiogenesis mode and its mechanism in the porous silk fibroin materials.
本文采用HE染色和免疫组化实验方法观察与分析比较机体生长过程及多孔丝素材料修复肌肉组织两种情况下的血管新生过程，并对新生血管进行数量化分析，同时结合透射电镜技术分析毛细血管的新生方式，并通过半定量计分法计算机体生长过程及多孔丝素材料修复肌肉组织各阶段中缺氧诱导因子(HIF-1α)和内皮细胞生长因子(VEGF)的表达程度。研究结果显示，在大鼠肌肉生长过程中的初期既有出芽新生也有套叠新生，但出芽的现象较少；随着大鼠年龄的增长，中后期肌肉组织中的毛细血管主要以套叠新生为主。同时发现在生长过程的初期，HIF-1α及VEGF表达均比较强烈，而中后期HIF-1α及VEGF表达明显减弱。通过丝素材料植入创伤肌肉组织中的实验发现，在植入材料的早期即13天以前材料周边主要以出芽方式进行血管新生来满足材料中组织细胞的供氧需求；而后期即13天以后则主要以套叠式血管新生来重塑与调整已有血管网。并且在植入股肌肉的早期，多孔丝素材料内HIF-1α及VEGF强烈表达，随着时间的推移，中后期时HIF-1α及VEGF表达渐渐变弱。由此推测HIF-1α及VEGF的表达与血管新生方式之间有密切的联系，当HIF-1α及VEGF在血管新生过程中的表达程度由强变弱时，标志着血管新生从初期的出芽为主逐渐转变为以套叠为主。
此外，结合文献报道及实验结果，从血管新生过程中的缺氧程度差异、生长因子的表达以及内皮细胞对血管内皮生长因子的响应以及新生血管的构建等几个环节出发，根据组织中新生血管生长前期与后期的差异以及出芽新生与套叠新生的差异，对大鼠肌肉组织生长过程及多孔丝素材料中血管新生模式的发生机制作出了分析及探索，并对材料植入大鼠腿部肌肉后的血管新生过程以及新生血管网络构建模式进行了思考与讨论。
通过对多孔丝素材料中血管新生模式及其机制的研究，为阐明材料中血管化的进程及其生物学机制提供基础的工作，同时也为适应性更宽泛的医用生物材料的开发提供理论依据及实验基础。
Because of excellent biological properties, silk materials receive much attention fromresearchers in the research area of biomedical materials in recent years. During the processof biomaterials repairing tissues, in order to make biomaterials scaffold form acircumstance that is suitable for tissue regeneration and cell survival, it requires rapid andeffective neovascularization of biomaterials after implantation, thus neovascularization ofbiomaterials is also the key subject of study on the biomaterials repairing tissues. However,in the field of biomedical materials, the problem about neovascularization of biomaterialsin vitro or in vivo is not fully settled, which is still a bottleneck problem of tissueengineering and biomaterial application. As an important step of realizing theneovascularization process, angiogenesis includes various modes that are predominantlysprouting angiogenesis and intussusceptive angiogenesis, and so far these kinds of modeshave been found under different physiological and pathological conditions, but problemson the mechanism of angiogenesis mode have not been completely clarified. The researchon the capillary angiogenesis mode is the front topic of angiogenesis, and related researchwork has received much concern from researchers in the last twenty years. With regard tothe angiogenesis mode in biomaterials, the past research reports are few. Based on thepresent situation, this paper aims at exploring the mechanism of angiogenesis modes inporous silk fibroin materials, and the angiogenesis courses under these two conditions ofbody growth process and porous silk fibroin materials repairing muscle were respectivelyinvestigated and studied.
In this paper, the angiogenesis processes under these two conditions of both bodygrowth process and porous silk fibroin materials repairing muscle were observed andanalyzed by methods of HE staining and immunohistochemical assay. In addition, thequantity of the newly formed capillaries in the material was analyzed. Meanwhile, theangiogenesis mode was analyzed by TEM technology. Moreover, the expression degrees ofhypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) inthe each stage of body growth process and porous silk fibroin materials repairing musclewere calculated by semi-quantitive scoring method. The results showed that both sprouting and intussusception occurred at the early stage of tissue growth, but less sproutingphenomena were found; and with the increase of the age of the SD rat, intussusception isthe main angiogenesis mode at the middle and late stage of tissue growth. Correspondingly,there were strong expressions of both HIF-1α and VEGF at the early stage of growthprocess, while the expressions of both HIF-1α and VEGF weakened markedly at themiddle and late stage. On the other hand, according to the experiment that silk fibroinmaterials were implanted into the wound muscle of SD rats, it was found that capillarieswere mainly newly formed by sprouting before day13to supply oxygen for the cells,while by intussusception after day13to remodel and adjust the existing vascular network.Accordingly, at the early stage of implantation, there were strong expressions of bothHIF-1α and VEGF in the materials, while at the middle or latter stage of implantation, theexpressions of HIF-1α and VEGF gradually weakened. Therefore, it was speculated thatthere are close relations between the expressions of HIF-1α, VEGF and angiogenesismodes in materials. That is, when the expression levels of HIF-1α and VEGF during theangiogenesis process change from strong to weak, the main mode of capillariesangiogenesis also turn from sprouting to intussusception.
In additon, by combining the literature reports and experimental results, this paperstretched from several aspects such as differences of hypoxia degree, growth factorexpression and endothelial cells in response to VEGF, and in accordance with thedifferences between the former stage and the late stage of the growth of newly formedcapillaries as well as the differences between sprouting angiogenesis and intussusceptiveangiogenesis, the mechanism of the angiogenesis modes was analyzed and explored underthe conditions of body growth process and porous silk fibroin materials repairing tissue,moreover, the angiogenesis process and capillary network construction mode in thematerials were thought and discussed after the implantation.
This paper provided the basis for clarifying the neovascularization process andbiological mechanism of the materials as well as the theoretical basis and experimentalfoundation for the exploitation of biomedical materials with wider adaptability throughstudying on the angiogenesis mode and its mechanism in the porous silk fibroin materials.
引文
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