本文選題：去細胞化豬肝 + 交聯(lián)��； 參考：《四川醫(yī)科大學》2015年碩士論文

【摘要】：目的:制備具有低免疫原性的去細胞化豬肝臟組織材料,探討經(jīng)交聯(lián)劑京尼平修飾的去細胞化豬肝臟組織材料的免疫原性,為進一步利用去細胞化肝臟生物支架材料重建肝臟器官并實現(xiàn)臨床異體移植奠定實驗基礎。方法:①去細胞化豬肝臟組織材料的制備及其修飾:1).隨機選取體重為12-15kg雄性巴馬小型豬4只,將其深度麻醉,取其完整肝臟,并隨機選取其中3個肝臟,經(jīng)門靜脈插管后,以1%SDS+1%Triton X-100方案灌注對完整豬肝臟進行去細胞化處理,制成去細胞化的豬肝臟生物組織材料;另一只未經(jīng)去細胞化的豬肝臟作為對照組。對去細胞組及未去細胞組材料進行病理切片并用蘇木精和伊紅染色(HE),Masson’s trichrome染色,免疫組織化學染色,DNA含量分析及PCR檢測抗原基因表達以評價去細胞化效果。2).將去細胞化的豬肝臟組織材料切成直徑約為1.5cm的方形塊,并將其隨機分為三組(每組約30塊),其中兩組分別給予濃度為0.625%的京尼平(GP)和濃度為的0.625%戊二醛(GA)進行交聯(lián),另一只未去細胞肝臟亦切成相同大小方形塊,作為對照組。然后對各組材料(GP修飾去細胞組,GA修飾去細胞組,未修飾去細胞組及未去細胞組)進行形態(tài)學觀察,病理切片HE染色檢測其顯微結構及細胞成分清除情況,并且通過電子顯微鏡觀察各組細胞外基質三維立體結構。②去細胞化及去細胞化后修飾的肝臟組織材料體外誘導單核細胞遷移及異體植入大鼠皮下局部反應實驗:1).以u-937人單核細胞系進行體外遷移實驗,運用碾磨器碾磨材料并高速離心方法分別提取gp修飾去細胞組,ga修飾去細胞組,未修飾去細胞組及未去細胞組的豬肝臟組織材料中的蛋白,利用帶有pet膜的六孔板行體外遷移試驗檢測人單核細胞分別對各組蛋白提取物的遷移反應。2).將各組已經(jīng)切好的等體積小塊組織材料分別植入sd大鼠皮下,并分別于植入后第3,7,14及28天取出植入的材料及其周圍包裹組織,進行病理切片及he染色,并在高倍視野下對材料周圍浸潤的粒細胞/淋巴細胞及單核巨噬細胞分別進行記數(shù),檢測材料組織在動物體內的免疫排斥反應。結果:①去細胞化及交聯(lián)修飾后的檢測:1).he及masson’strichrome染色顯示去細胞化的肝臟組織材料中細胞成分被清除干凈,并保留了原組織的細胞外基質三維立體結構,該三維立體結構成分包括膠原蛋白、彈性蛋白和硫酸粘多糖(gags)等成分;瓊脂糖凝膠電泳顯示去細胞化后的肝臟組織材料中未觀察到dna殘留片段,pcr表明組織中的α-1,3半乳糖-β-1,4半乳糖-n-乙酰氨基葡萄糖抗原、豬白細胞抗原(sla)、豬內源性逆轉錄病毒(perv)等與免疫反應有關的成分均被清除;免疫組化提示細胞外基質特定位置的gal抗原表位明顯減少。2).經(jīng)京尼平和戊二醛交聯(lián)后的去細胞化肝臟組織材料外觀分別表現(xiàn)為深藍色和褐色,體積較前有所變小,質地變硬。經(jīng)修飾后材料的he染色顯示去細胞化肝臟組織材料仍未見細胞成分殘留,并且未改變其細胞外基質的三維立體支架結構;掃描電鏡(sem)觀察到去細胞化的肝臟組織材料表現(xiàn)出較未去細胞化的肝臟組織材料更豐富的相互交聯(lián)的多孔結構,經(jīng)京尼平及戊二醛交聯(lián)并不會影響其多孔性。②免疫原性檢測:1).體外人單核細胞遷移實現(xiàn)表明GP修飾去細胞組,GA修飾去細胞組,未修飾去細胞組及未去細胞組的豬肝臟組織材料提取蛋白分別作為遷移實驗底物時,分別有(34.67±4.33)×103,(31.67±2.906)×103,(64.33±6.936)×103,(108.8±15.33)×103個U-937細胞自由遷移到下腔室,對比未去細胞的肝臟組織提取蛋白,去細胞后的肝臟組織材料提取物能明顯的降低人單核細胞遷移率(P0.05),且經(jīng)交聯(lián)劑修飾后能進一步降低遷移率(P0.05)。2).將材料植入大鼠皮下,并定時將材料及其周圍包裹組織取出行病理切片及HE染色提示,植入后第3天,未去細胞組及未修飾去細胞組肝臟組織材料即出現(xiàn)明顯的急性炎癥排斥反應,并于植后第14天材料明顯變薄,第28天均被降解吸收;經(jīng)GA交聯(lián)修飾后的肝臟組織材料于植入后第28天可見炎性細胞的浸潤包裹,出現(xiàn)了免疫反應,無明顯降解;然而,在整個觀察期間,經(jīng)京尼平修飾后的材料組只見少許炎性細胞浸潤包裹。結論:①.1%SDS+1%Triton X-100灌注方案能有效地清除掉完整豬肝臟組織器官中的細胞及抗原成分。?.交聯(lián)劑GP及GA均不會改變去細胞化豬肝臟組織材料的多孔性及三維立體結構。?.交聯(lián)劑京尼平能有效的降低去細胞化豬肝臟組織材料的免疫原性。
[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.

【學位授予單位】：四川醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R657.3

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本文編號：1870760