摘  要

随着我国社会经济发展，人口老龄化加快，心血管疾病（Cardiovascular disease, CVD）患病率正逐年递增，由心血管疾病导致的死亡人数在2020年已达到458万人，在居民死亡构成比中占据首位。随之而来，用于心血管疾病诊疗的医疗产品的需求也在增高。人工血管、人工心脏、血管补片等组织工程修复材料是治疗心血管疾病的有效手段。目前临床上已有牛心包、猪主动脉瓣来源的生物瓣膜解决瓣膜疾病的问题，但生物瓣的耐久性和钙化问题一直是心血管医用材料研究领域需攻克的难点。动物源材料因其天然ECM成分、良好的生物相容性等优势，一直以来在原材料的开发上有着明显的优势。鱼鳔作为鱼体内的弹性器官，具有良好的机械性能，且相比于牛心包材料，钙化水平更低，在心脏瓣膜和人工血管的应用研究中展现了巨大潜力。为进一步实现鱼鳔源生物瓣膜的转化，保证其作为与血液接触的三类医疗器械使用的安全性，其免疫原性与血液相容性验证不可或缺。基于以上背景，验证鱼鳔源材料的免疫原性与血液相容性，为其在瓣膜病的治疗中提供应用依据。研究内容主要包括以下三部分：

鱼鳔源材料的免疫原性研究：根据 ISO 10993-20指导原则，利用体外与体内实验通过将鱼鳔与牛心包、假手术组动物对比，以探究其对机体的的免疫反应。首先，利用淋巴细胞增殖来评价材料对淋巴细胞的功能影响。然后利用小鼠皮下材料埋植，通过血常规、免疫器官质量系数、淋巴细胞增殖、淋巴细胞亚型分析、组织病理学等方法共同评价材料在体内的免疫原性。结果表明，体内实验中，培养3天后，与假手术组相比，鱼鳔组的淋巴细胞增殖、小鼠免疫器官质量系数无明显差异；使用鱼鳔材料浸提液的淋巴细胞增殖率较低，且各淋巴细胞亚型比例正常，与假手术组无明显差异，说明鱼鳔材料不会引起小鼠机体的细胞免疫反应；血常规显示，白细胞、中性粒细胞、淋巴细胞等无异常；埋植部位无明 显炎症反应，除此以外，HE染色显示，各个脏器无肉芽组织、纤维斑块、坏死物等异常反应。

鱼鳔源材料的血液相容性研究：根据GB/T 16886.4-2022《医疗器械生物学评价——第4部分：与血液相互作用的试验选择》，利用体外与体内实验共同验证鱼鳔源材料的血液相容性。体外实验中，通过溶血实验、血浆复钙实验、APTT以及PTT实验验证两种材料对血液的影响。体内实验中，将两种材料制备成的补片，于新西兰白兔颈静脉实施修补术，术后给予低分子肝素，并持续超声监测血管是否有堵塞狭窄的状况。术后1个月，3个月取材，观察组织病理情况。结果显示，体外实验表明，鱼鳔组溶血率均小于5%，且二者间无显著差异；通过凝血时间、血浆复钙实验等将材料对内源性凝血和外源性凝血的作用阐释，鱼鳔和ePTFE均可以激活内源性凝血途径，鱼鳔材料激活更明显。体内实验结果显示，两个时间点下，两组材料修补的血管均通畅无堵塞无狭窄，这为今后的应用研究增强了信心。

鱼鳔源材料的成分研究：使用液相质谱的方法（LC-MS/MS）来了解鲢鱼、草鱼、牛心包、猪二尖瓣、猪主动脉瓣的蛋白质组成成分。结果表明鲢鱼、草鱼、牛心包、猪二尖瓣以及猪主动脉瓣中，胶原蛋白、糖蛋白和多糖是占比最丰富的三大类。其中胶原蛋白占比最高，弹性蛋白占比最少。另外，鱼鳔胶原主要分型为Ⅰ型胶原，牛心包和猪二尖瓣、猪主动脉瓣的Ⅵ型胶原比鱼鳔表达更高一些。与此同时，我们发现，鱼鳔、猪瓣膜以及牛心包的ELN基因表达较低，POSTN和EMILIN1表达较高，且猪二尖瓣膜、猪主动脉瓣和牛心包的表达高于鱼鳔，推测出，鱼鳔瓣膜相较于牛心包瓣膜抗钙化性能优异可能与该基因的表达低有关。

综上，鱼鳔材料含有丰富的胶原蛋白，其中Ⅰ型胶原蛋白含量占比最高，与猪主动脉瓣、二尖瓣及牛心包组分相似。通过以上各项免疫原性检测表明，鱼鳔材料均不会引起机体明显免疫排斥反应，且不会引起体内各脏器的损害。通过以上各项血液相容性结果显示，鱼鳔源材料与市售的ePTFE材料的血液相容性相当，在静脉系统中表现出良好的血液相容性，为鱼鳔源材料作为血液接触材料的临床应用打下坚实基础。

Abstract

The incidence of cardiovascular disease (CVD) is increasing in recent years due to population aging and the changes in dietary habit. In China, the number of deaths caused by CVD has reached 4.58 million in 2020, occupying the highest proportion of residents' deaths. Consequently, the demand for medical products for the diagnosis and treatment of cardiovascular diseases is also increasing. Medical devices such as artificial blood vessels, artificial hearts and vascular patches are effective treatments for cardiovascular diseases. At present, the sources of clinical valve prosthesis commonly derived from bovine pericardium or porcine aortic valve, however, in the development of new medical devices, the durability and anti-calcification property of bioprosthesis are still too difficult to overcome. Medical devices utilizing animal tissues and their derivatives have obvious advantages in the exploitation of raw materials, due to their natural ECM composition, good biocompatibility and other advantages. Fish swim bladder, as an elastic organ, has good mechanical properties and lower calcification compared with bovine pericardium; it shows great potential in the application of heart valves and artificial blood vessels. In order to further realize the transformation of swim bladder-derived biological valve, and ensure its safety as a third-class medical device in contact with blood, it is indispensable to verify its immunogenicity and blood compatibility.Based on above background, the immunogenicity and blood compatibility of swim bladder-derived materials were verified to provide a basis for their application in the treatment of valvular diseases. The research mainly includes the following three parts:

Immunogenicity assessment of swim bladder-derived biomaterials. According to ISO 10993-20 "Principles and methods for immunotoxicology testing of medical devices", we explored the immune response of the swim bladder to the organism by comparing it with bovine pericardium and the sham group in vivo and vitro. First, lymphocyte proliferation was used to evaluate the functional effect of materials on lymphocytes. Then the materials were implanted subcutaneously in mice, and the immunogenicity of the materials in vivo was evaluated by routine blood test, immune organ coefficient, lymphocyte proliferation, lymphocyte subtype analysis, and histopathology. The results showed that, there was no significant difference in lymphocyte proliferation and immune organ coefficient between the bladder and the sham at 3 days in vivo. The lymphocyte proliferation rate of the bladder group was low, and the proportion of the lymphocyte subtype was normal, which was not significantly different from the sham group, indicating that the swim bladder material did not cause specific immunity in mice. Blood routine examination showed that white blood cells, neutrophils, and lymphocytes were in normal state. There was no obvious inflammatory reaction in the implantation site. In addition, HE staining showed that there was no granulation tissue, plaque, necrosis and other abnormal reactions in various organs.

Blood compatibility assessment of swim bladder-derived biomaterials. According to GB/T 16886.4-2022 "Biological Evaluation of Medical devices -- Part 4: Selection of Tests for Interaction with Blood", the blood compatibility of fish swim bladder-derived materials was verified by in vitro and in vivo experiments. The effects of swim bladder on blood were verified by hemolysis test, plasma recalcification test, APTT and PTT experiments. In the in vivo study, the jugular vein of New Zealand white rabbits was surgically treated with a patch made of swim bladder, followed by administration of low molecular weight heparin (LMWH) and continuous ultrasound monitoring for blockage of stenosis. And then take out of the specimens at 1 month and 3 months post-operation to observe the histopathological changes. The results showed that the hemolysis rate of the bladder group was less than 5%, and there was no significant difference compared with ePTFE group. The effects of the materials on endogenous coagulation and exogenous coagulation were explained by clotting time and plasma recalcification experiments. Both the swim bladder and the ePTFE can activate the endogenous coagulation pathway, and the activation of the bladder material was more obvious. The results showed that the blood vessels repaired by swim bladder were unobstructed and free of stenosis in in vivo experiments, which strengthened the confidence for future application research.

Composition of swim bladder-drived materials. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to investigate the protein composition of silver carp, grass carp, bovine pericardium, porcine mitral valve and porcine aortic valve. The results showed that collagen, glycoprotein and polysaccharide were the mutual most abundant proteins in all above materials. Among them, collagen accounted for the highest proportion, and elastin accounted for the least. In addition, the main type collagen in swim bladder was type Ⅰ collagen, and the expression of type Ⅵ collagen in bovine pericardium, porcine mitral valve and porcine aortic valve was higher than that in swim bladder. Besides, we found that the expression of ELN gene was low in swim bladder, porcine mitral valve, porcine aortic valve and bovine pericardium. But the expression of POSTN and EMILIN1 in the porcine mitral valve, porcine aortic valve and bovine pericardium was higher than the swim bladder. Therefore, we speculate that the superior anti-calcification performance of the swim bladder valve may be related to the low expression of POSTN gene compared with the bovine pericardial valve.

In conclusion, the swim bladder contains abundant collagen, among which the content of type Ⅰ collagen is the highest, which is similar to that of porcine aortic valve, porcine mitral valve and bovine pericardium. Above immunogenicity tests showed that the swim bladder material did not cause obvious immune rejection, and did not cause damage to the organs in the body. Above results of blood compatibility showed that the blood compatibility of swim bladder material was similar to that of the commercial ePTFE material, and it showed good blood compatibility in the venous system, which layed a solid foundation for the clinical application of the swim bladder material as a blood contact material.