去细胞人羊膜基质的制备及免疫原性

doi:10.16098/j.issn.0529-1356.2016.04.021

解剖学报 ›› 2016, Vol. 47 ›› Issue (4): 557-562.doi: 10.16098/j.issn.0529-1356.2016.04.021

去细胞人羊膜基质的制备及免疫原性

路欣¹袁洁² 郭昕悦²李卫红¹ 尚宏伟¹张立新¹ 黄晓武³ 张海燕^2*

1. 首都医科大学基础医学院基础医学实验教学中心，北京 100069; 2. 首都医科大学基础医学院细胞生物学系，北京 100069; 3. 首都医科大学附属复兴医院宫腔镜诊治中心，北京 100038

收稿日期:2015-12-15 修回日期:2016-01-29 出版日期:2016-08-06 发布日期:2016-08-06
通讯作者: 张海燕 E-mail:culture@ccmu.edu.cn
基金资助:
人脂肪间充质干细胞定向肝实质细胞分化的研究

Preparation for acellular human amniotic membrane matrix and assessment the immunogenic property

LU Xin¹YUAN Jie²GUO Xin-yue²LI Wei-hong¹SHANG Hong-wei¹ ZHANG Li-xin¹ HUANG Xiao-wu³ ZHANG Hai-yan ^2*

1. Experimental Center for Basic Medical Teaching, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China; 2. Department of Cell Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China; 3. Hysteroscopic Diagnosis and Treatment Center, Fuxing Hospital, Capital Medical University, Beijing 100038, China

Received:2015-12-15 Revised:2016-01-29 Online:2016-08-06 Published:2016-08-06
Contact: ZHANG Hai-yan E-mail:culture@ccmu.edu.cn

摘要/Abstract

摘要：

目的制备去细胞人羊膜基质（AHAM）并分析其移植至小鼠体内后的免疫反应，评价去细胞人羊膜基质作为细胞移植支架的免疫原性。方法利用胰蛋白酶-EDTA消化人羊膜（HAM)，去除人羊膜上皮细胞，制备新鲜去细胞人羊膜基质；新鲜去细胞人羊膜基质置于含硫酸软骨素及甘油的冷冻保护液中，-80℃保存；使用前经PBS复水处理后即冷冻去细胞人羊膜基质；免疫细胞化学分析去细胞人羊膜基质中人类白细胞抗原的表达情况；将冷冻的去细胞人羊膜基质移植至BALB /c小鼠肝脏内4周后，分析脾脏组织中CD4⁺ 、CD8⁺T淋巴细胞比例的变化。结果新鲜及冷冻保存后的去细胞人羊膜基质均不表达人类白细胞抗原；冷冻的去细胞人羊膜基质移植至小鼠体内后，脾脏组织中CD4⁺ 、CD8⁺T淋巴细胞无明显变化。结论去细胞人羊膜基质的免疫原性较低，移植后不会引起由T细胞介导的特异性排斥反应，可以作为免疫安全性的生物支架用于疾病的细胞移植治疗。

关键词: 去细胞人羊膜基质, 免疫原性, 移植, T淋巴细胞, 小鼠

Abstract:

Objective To prepare of the acellular human amniotic membrane matrix (AHAM) and to assess the immunogenic property of AHAM as a scaffold for cell transplantation. Methods To prepare the AHAM, the HAM was peeled from the placenta, cut into pieces and incubated in trypsin with EDTA for decellularization. For generating cryopreserved AHAM, the fresh AHAM pieces were placed in dishes with a 1∶1 mixture of glycerol and chondroitin sulfate in MEM-NEAA and stored at -80℃. Before using, the cryopreserved AHAMs were rehydrated with sterile PBS. The expression of human leucocyte antigen in HAM and AHAM was determined by immunofluorescence. The ratio of CD4⁺and CD8⁺ T cells in mouse spleen after AHAM transplantation for four weeks was assessed by immunofluorescence and flow cytometry. Results Immunofluorescence analysis confirmed that the fresh and cryopreserved AHAM were negative for human leucocyte antigen antibody. Immunofluorescence and flow cytometry analysis confirmed that the ratio of CD4⁺and CD8⁺ T cells in mouse spleen after AHAM transplantation was not changed. Conclusion The immunogenic property of AHAM is relatively low and does not cause the T cells mediated immunological rejection after transplantation in the mouse model. These results support the potential of AHAM as a cell delivery platform to treat and manage disease.

Key words: Acellular human amniotic membrane matrix, Immunogenicity, Transplantation, T lymphocyte, Mouse

路欣袁洁郭昕悦李卫红尚宏伟张立新黄晓武张海燕. 去细胞人羊膜基质的制备及免疫原性[J]. 解剖学报, 2016, 47(4): 557-562.

LU Xin YUAN Jie GUO Xin-yue LI Wei-hong SHANG Hong-wei ZHANG Li-xin HUANG Xiao-wu ZHANG Hai-yan. Preparation for acellular human amniotic membrane matrix and assessment the immunogenic property[J]. Acta Anatomica Sinica, 2016, 47(4): 557-562.

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去细胞人羊膜基质的制备及免疫原性

Preparation for acellular human amniotic membrane matrix and assessment the immunogenic property

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