京尼平对去细胞化猪肝脏组织材料免疫原性影响的研究

发布时间：2018-05-10 20:32

本文选题：去细胞化猪肝 + 交联　；参考：《四川医科大学》2015年硕士论文

【摘要】：目的:制备具有低免疫原性的去细胞化猪肝脏组织材料,探讨经交联剂京尼平修饰的去细胞化猪肝脏组织材料的免疫原性,为进一步利用去细胞化肝脏生物支架材料重建肝脏器官并实现临床异体移植奠定实验基础。方法:①去细胞化猪肝脏组织材料的制备及其修饰:1).随机选取体重为12-15kg雄性巴马小型猪4只,将其深度麻醉,取其完整肝脏,并随机选取其中3个肝脏,经门静脉插管后,以1%SDS+1%Triton X-100方案灌注对完整猪肝脏进行去细胞化处理,制成去细胞化的猪肝脏生物组织材料;另一只未经去细胞化的猪肝脏作为对照组。对去细胞组及未去细胞组材料进行病理切片并用苏木精和伊红染色(HE),Masson’s trichrome染色,免疫组织化学染色,DNA含量分析及PCR检测抗原基因表达以评价去细胞化效果。2).将去细胞化的猪肝脏组织材料切成直径约为1.5cm的方形块,并将其随机分为三组(每组约30块),其中两组分别给予浓度为0.625%的京尼平(GP)和浓度为的0.625%戊二醛(GA)进行交联,另一只未去细胞肝脏亦切成相同大小方形块,作为对照组。然后对各组材料(GP修饰去细胞组,GA修饰去细胞组,未修饰去细胞组及未去细胞组)进行形态学观察,病理切片HE染色检测其显微结构及细胞成分清除情况,并且通过电子显微镜观察各组细胞外基质三维立体结构。②去细胞化及去细胞化后修饰的肝脏组织材料体外诱导单核细胞迁移及异体植入大鼠皮下局部反应实验:1).以u-937人单核细胞系进行体外迁移实验,运用碾磨器碾磨材料并高速离心方法分别提取gp修饰去细胞组,ga修饰去细胞组,未修饰去细胞组及未去细胞组的猪肝脏组织材料中的蛋白,利用带有pet膜的六孔板行体外迁移试验检测人单核细胞分别对各组蛋白提取物的迁移反应。2).将各组已经切好的等体积小块组织材料分别植入sd大鼠皮下,并分别于植入后第3,7,14及28天取出植入的材料及其周围包裹组织,进行病理切片及he染色,并在高倍视野下对材料周围浸润的粒细胞/淋巴细胞及单核巨噬细胞分别进行记数,检测材料组织在动物体内的免疫排斥反应。结果:①去细胞化及交联修饰后的检测:1).he及masson’strichrome染色显示去细胞化的肝脏组织材料中细胞成分被清除干净,并保留了原组织的细胞外基质三维立体结构,该三维立体结构成分包括胶原蛋白、弹性蛋白和硫酸粘多糖(gags)等成分;琼脂糖凝胶电泳显示去细胞化后的肝脏组织材料中未观察到dna残留片段,pcr表明组织中的α-1,3半乳糖-β-1,4半乳糖-n-乙酰氨基葡萄糖抗原、猪白细胞抗原(sla)、猪内源性逆转录病毒(perv)等与免疫反应有关的成分均被清除;免疫组化提示细胞外基质特定位置的gal抗原表位明显减少。2).经京尼平和戊二醛交联后的去细胞化肝脏组织材料外观分别表现为深蓝色和褐色,体积较前有所变小,质地变硬。经修饰后材料的he染色显示去细胞化肝脏组织材料仍未见细胞成分残留,并且未改变其细胞外基质的三维立体支架结构;扫描电镜(sem)观察到去细胞化的肝脏组织材料表现出较未去细胞化的肝脏组织材料更丰富的相互交联的多孔结构,经京尼平及戊二醛交联并不会影响其多孔性。②免疫原性检测:1).体外人单核细胞迁移实现表明GP修饰去细胞组,GA修饰去细胞组,未修饰去细胞组及未去细胞组的猪肝脏组织材料提取蛋白分别作为迁移实验底物时,分别有(34.67±4.33)×103,(31.67±2.906)×103,(64.33±6.936)×103,(108.8±15.33)×103个U-937细胞自由迁移到下腔室,对比未去细胞的肝脏组织提取蛋白,去细胞后的肝脏组织材料提取物能明显的降低人单核细胞迁移率(P0.05),且经交联剂修饰后能进一步降低迁移率(P0.05)。2).将材料植入大鼠皮下,并定时将材料及其周围包裹组织取出行病理切片及HE染色提示,植入后第3天,未去细胞组及未修饰去细胞组肝脏组织材料即出现明显的急性炎症排斥反应,并于植后第14天材料明显变薄,第28天均被降解吸收;经GA交联修饰后的肝脏组织材料于植入后第28天可见炎性细胞的浸润包裹,出现了免疫反应,无明显降解;然而,在整个观察期间,经京尼平修饰后的材料组只见少许炎性细胞浸润包裹。结论:①.1%SDS+1%Triton X-100灌注方案能有效地清除掉完整猪肝脏组织器官中的细胞及抗原成分。?.交联剂GP及GA均不会改变去细胞化猪肝脏组织材料的多孔性及三维立体结构。?.交联剂京尼平能有效的降低去细胞化猪肝脏组织材料的免疫原性。
[Abstract]:Objective: to prepare a porcine liver tissue material with low immunogenicity, and to explore the immunogenicity of the porcine liver tissue modified by genipin, a cross-linking agent, and to lay an experimental basis for further using the scaffold material to reconstruct the liver organs and realize the clinical allograft. The preparation and modification of liver tissue materials: 1) 1 male Bama miniature pigs were randomly selected and 4 of them were randomly selected to take the complete liver, and 3 of them were selected randomly. After the portal vein was intubated by the portal vein, the whole pig liver was treated with 1%SDS+1%Triton X-100 scheme, and the porcine liver was prepared. Biological tissue materials; another undecellularized pig liver as a control group. Pathological sections of the cell and uncell group materials were histopathologically sectioned and treated with hematoxylin and eosin staining (HE), Masson 's trichrome staining, immunohistochemical staining, DNA content analysis and PCR detection of antigen gene expression to evaluate the effect of de cytochemical effect.2). The cytochemical porcine liver tissue materials were cut into square blocks with a diameter of about 1.5cm, and they were randomly divided into three groups (each group of about 30 pieces), of which two groups were given crosslinking with a concentration of 0.625% and a concentration of 0.625% glutaraldehyde (GA), and the other was cut into the same size block as a control group. The morphological observation was carried out on all kinds of materials (GP modified cell group, GA modified cell group, unmodified cell group and non cell group). The microscopic structure and cell composition were detected by HE staining in pathological sections, and the three-dimensional structure of extracellular matrix was observed by electron microscope. The liver tissue materials used to induce monocyte migration and allogenic subcutaneous local reaction in vitro: 1). In vitro migration experiments were carried out in U-937 human mononuclear cell lines. GP modified cell group, GA modified cell group, unmodified cell group and uncell group were extracted with mill grinding material and high speed centrifugation. The protein in the pig liver tissue material was used to test the migration response of human mononuclear cells with the six pore plate with PET membrane in vitro. The migratory response of human mononuclear cells to each group of protein extracts was.2 respectively. The subcutaneously inserted small tissue materials were implanted into the subcutaneous tissue of SD rats respectively, and the implanted materials were removed for 3,7,14 and 28 days after implantation, respectively. The surrounding tissue, pathological section and he staining, and the number of granulocytes / lymphocytes and mononuclear macrophages infiltrated around the material under high magnification, the immune rejection of material tissue in animals was detected. Results: (1) the detection of cell and cross-linking repair: 1).He and Masson 'strichrome staining The cellular components of the liver tissue were cleaned, and the three-dimensional structure of the extracellular matrix of the original tissue was retained, including collagen, elastin and mucopolysaccharide (GAGs), and the agarose gel electrophoresis showed no DNA in the liver tissue materials after the cells were decellularized. Residual fragments, PCR showed that alpha -1,3 galactose - beta -1,4 semi lactose -n- acetylglucosamine antigen, porcine leukocyte antigen (SLA), porcine endogenous retrovirus (PERV), and other components related to the immune response were removed; immunohistochemistry suggested that the gal antigen epitopes of the specific location of the extracellular matrix decreased.2). Genipin and amyl two The appearance of the acellular liver tissue materials after the crosslinking of aldehydes was dark blue and brown respectively, and the volume was smaller than before, and the texture became hard. The HE staining of the modified material showed that there was no residual cell composition in the cellular liver tissue material, and the three-dimensional scaffold structure of the extracellular matrix was not changed, and the scanning electron microscope (SEM) view was not changed. The cellular structure of liver tissue showed more abundant cross-linked porous structure than uncellular liver tissue. Cross-linked by genipin and glutaraldehyde did not affect its porosity. (2) immunogenicity detection: 1) human mononuclear cell migration in vitro showed that GP modified cell group, GA modified cell group, unrepaired. When the porcine liver tissue materials extracted from the cell group and the non cell group were used as the substrate for the transfer of the experiment, there were (34.67 + 4.33) x 103, (31.67 + 2.906) x 103, (64.33 + 6.936) x 103, and (108.8 + 15.33) * * 103 U-937 cells migrated freely to the lower chamber, compared with the non cell liver tissue to extract protein and the liver group after cell removal. The fabric extract could significantly reduce the mobility of human monocyte (P0.05), and further reduce the mobility (P0.05).2 after the crosslinking agent. The material was implanted subcutaneously into the rat, and the pathological sections of the material and its surrounding tissue were selected for pathological section and HE staining. Third days after implantation, the cell group and the unmodified cell group liver were not removed. The material of the dirty tissue appeared obvious acute inflammatory rejection, and the material became thinner at fourteenth days after implantation, and was degraded and absorbed on twenty-eighth days. The liver tissue material after GA crosslinking was covered with infiltration of inflammatory cells on the twenty-eighth day after implantation, and the immune response was not degraded. However, during the whole observation period, genipin was observed. Conclusion: (1) the.1%SDS+1%Triton X-100 perfusion scheme can effectively remove the cell and antigen components in the whole pig liver tissue. The cross-linking agent GP and GA do not change the porous and three-dimensional structure of the porcine liver tissue materials. It can effectively reduce the immunogenicity of acellular pig liver tissue materials.

【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3

【参考文献】