Fah－/－Rag2－/－IL2Rg－/－小鼠肝脏原位异种重建模型的建立及其评价

doi:10.13481/j.1671-587X.20230533

摘要/Abstract

摘要：

目的构建猪-小鼠肝细胞嵌合模型，探讨猪肝细胞在无T淋巴细胞、B淋巴细胞和自然杀伤细胞（NK细胞）介导排斥条件下的移植及再生的相关条件，评估猪肝细胞在异种受体中的定居、增殖和功能因子分泌情况，为提高猪源化小鼠模型中猪肝细胞的重建效率提供依据。方法采用脾脏内注射法将猪肝细胞移植至Fah^－/－Rag2^－/－IL2Rg^－/－（FRG）小鼠体内，构建移植模型。将16只FRG小鼠随机分为对照组、空白对照组、实验组和绿色荧光蛋白（GFP）基因实验组，每组4只。空白对照组小鼠停止给予2-（2-硝基-4-三氟甲基苯甲酰基）-1，4-环己二酮（NTBC），监测体质量；对照组4只小鼠注射猪肝细胞，移植前1周停止给予NTBC，移植后监测体质量，当体质量下降20%时，给予NTBC 3 d；实验组小鼠注射猪肝细胞，移植前1周停止给予NTBC，移植后监测体质量，当体质量下降20%时，给予NTBC 3 d；GFP基因实验组小鼠注射携带GFP基因猪肝细胞，移植前1周停止给予NTBC，移植后监测体质量，当体质量下降20%时，给予NTBC 3 d。观察各组小鼠体质量、生存情况。检测各组小鼠血清中丙氨酸氨基转移酶（ALT）水平，流式细胞术检测各组小鼠外周血中CD19+、CD3+和NK1.1+细胞表达情况，ELISA法检测各组小鼠血清中猪白蛋白表达水平，免疫组织化学法检测各组小鼠猪肝细胞再生效率。结果与剪碎后再以胶原酶消化的方式比较，两步灌注法消化过程更温和、对细胞损伤更小，细胞活性可以达到92%。停止给予NTBC后，对照组小鼠体质量进行性降低，生命活力逐渐降低。连续停药第28天小鼠开始出现死亡现象。小鼠肝组织中出现肝细胞肿大、细胞浆疏松透亮和细胞核溶解等不可逆细胞损伤。空白对照组小鼠停药后血清中ALT水平逐渐升高。对照组停药小鼠重新给予NTBC后，体质量逐渐恢复至正常水平。流式细胞术，停止给予NTBC后，小鼠外周血中CD19+、CD3+和NK1.1+细胞完全缺失。免疫组织化学染色，实验组小鼠肝组织中未见深色Fah+阳性细胞，移植小鼠肝组织中可见深色Fah+阳性细胞，猪肝细胞再生效率为（11±4）%。GFP基因实验组小鼠可见同一视野经三色激光分别激发，猪肝细胞再生效率为（10±2）%。结论成功构建了猪源化FRG小鼠肝脏嵌合模型，建立了长效稳定扩增猪肝细胞的小鼠模型。

关键词: 肝细胞异种移植, Fah, 肝细胞, 猪-小鼠肝细胞嵌合模型, 免疫缺陷小鼠

Abstract:

Objective To construct a pig-mouse liver cell chimeric model，and to discuss the relevant conditions for the transplantation and regeneration of the porcine hepatocytes without T lymphocyte， B lymphocyte， and NK lymphocyte mediated rejection， and to evaluate the settlement， proliferation， and secretion of functional factors of the porcine hepatocytes in xenograft receptors， and to provide the basis for improving the reconstruction efficiency of the porcine hepatocytes in pig derived mouse models. Methods The porcine hepatocytes were transplanted into the Fah^－/－Rag2^－/－ IL2Rg ^－/－（FRG） mice by using intrasplenic injection method to construct the transplantation model. Sixteen FRG mice were randomly divided into control group， blank control group， experimental group， and green fluorescence protein（GFP） gene experimental group， and there were four mice in each group.The administration of nitisinone 2-2-nitro-4-（trifluoromethyl）cyclohexane-1，3-dione（NTBC） of the mice in blank control group was stopped，and the body weight was monitored； the mice in control group were injected with the porcine hepatocytes， the administration of NTBC was stopped one week before transplantation， and the body weight was monitored after transplantation. When the body weight was decreased by 20%， the NTBC was administered for 3 d. The mice in experimental group were injected with the porcine hepatocytes， and the administration of NTBC was stopped one week before transplantation； the body weight was monitored after transplantation， and when the body weight was decreased by 20%， the NTBC was administered for 3 d. The mice in GFP gene experimental group were injected with the porcine hepatocytes carrying the GFP gene， the administration of NTBC was stopped one week before transplantation， and the body weight was monitored after transplantation； when the body weight was decreased by 20%， the NTBC was administered for 3 d. The body weights and survival status of the mice in various groups were observed. The levels of alanine aminotransferase （ALT） in serum of the mice in various groups were detected；the expressions of CD19+， CD3+， and NK1.1+cells in peripheral blood of the mice in various groups were detected by flow cytometry；the expression levels of porcine albumin in serum of the mice in various groups were detected by ELISA assay； the regeneration efficiencies of the porcine hepatocytes of the mice in various groups were detected by immunohistochemistry. Results Compared with method of collagenase digestion after shearing， the digestion process of the two-step perfusion method was milder and had less damage to cells， and the cell activity achieved 92%. After stopping the administration of NTBC， the body weight of the mice in control group was gradually decreased and the vitality was gradually decreased. Death began to occur in the mice on the 28th day after continuous stopping the administration.The irreversible cellular damage in liver tissue of the mice could be seen such as liver cell enlargement， cytoplasmic looseness and transparency， and nuclear lysis. The ALT levels in serum of the mice in blank control group was gradually increased after stopping the administration. After re-administration of NTBC， the body weight of the mice in control group was gradually returned to the normal levels. The flow cytometry results showed that the CD19+， CD3+， and NK1.1+cells in peripheral blood of the mice could not be seen after stopping the administration of NTBC. The immunohistochemistry staining results showed that no dark Fah+positive cells were found in liver tissue of the mice in experimental group， while the dark Fah+positive cells were observed in liver tissue of the transplanted mice； the regeneration efficiency of porcine hepatocytes was （11 ± 4）%. The regeneration efficiency of porcine hepatocytes of the mice in GFP gene experimental group was （10 ± 2）% when stimulated by three color lasers in the same field of view. Conclusion The pig derived FRG mouse liver chimeric models are successfully constructed， and the mouse models for long-term stable expansion of the porcine hepatocytes are established.

Key words: Hepatocyte xenotransplantation, Fah, Hepatocyte, Pig-mouse liver cell chimeric model, Immunodeficient mice

中图分类号:

R575

张蒙,周琪,胡正. Fah^－/－Rag2^－/－IL2Rg^－/－小鼠肝脏原位异种重建模型的建立及其评价[J]. 吉林大学学报(医学版), 2023, 49(5): 1358-1365.

Meng ZHANG,Qi ZHOU,Zheng HU. Establishment of liver in situ xenograft reconstruction model of Fah^－/－Rag2^－/－IL2Rg^－/－ mice and its evaluation[J]. Journal of Jilin University(Medicine Edition), 2023, 49(5): 1358-1365.

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