中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (31): 5782-5785.doi: 10.3969/j.issn.1673-8225.2010.31.020

• 移植与免疫 transplantation and Immunology • 上一篇    下一篇

供体特异性体内免疫状态的定量检测

蒋泽生，高  毅   

  1. 南方医科大学附属珠江医院肝胆二科，南方医科大学再生医学研究所，广东省广州市    515282
  • 出版日期:2010-07-30 发布日期:2010-07-30
  • 通讯作者: 高毅，博士，教授，南方医科大学附属珠江医院肝胆二科，广东省广州市 515282 gaoyi6146@163.com
  • 基金资助:

    国家自然科学基金资助项目(30972825)，课题名称：供体抗原特异性体内免疫状态体内定量检测的基础研究。

Specific quantitative immunological assay to monitor the immune status in vivo

Jiang Ze-sheng, Gao Yi   

  1. Second Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou  510282, Guangdong Province, China
  • Online:2010-07-30 Published:2010-07-30
  • Contact: Gao Yi, Doctor, Professor, Second Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China gaoyi6146@163.com
  • Supported by:

     the National Natural Science Foundation of China, No. 30972825*

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425

被引次数

5

摘要：

背景：由于免疫反应的复杂性，目前的免疫学检测方法均有其局限性，虽然各种检测技术精确程度不断提高，但是单一指标不足以判断机体复杂的免疫状态。建立一种在多指标基础上的联合评估模式来监测移植后受体免疫状态是当前需要解决的难题。
目的：建立具有供体抗原特异性的体内免疫状态定量检测方法，探索该方法的流式检测技术参数、荧光染色条件、输注细胞数及确定最佳测试时间点。
方法：取C57及BALB/c小鼠脾脏制备脾细胞单细胞悬液，分别用不同浓度活细胞染料羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)染色后制备1∶1混合细胞悬液，按混合细胞输注C57→BALB/c小鼠皮肤移植排斥模型，分别于输注后1，2，4，10 h，1，2，3，6 d应用流式细胞仪检测外周血两种荧光细胞的比例变化，同时设未移植BALB/c小鼠为对照，探索CFSE荧光染色条件、输注细胞数及确定最佳测试时间点，按公式计算供体特异性细胞杀伤率。
结果与结论：供体特异性体内免疫状态定量检测方法流式检测设定在淋巴细胞门，两种细胞CFSE的染色浓度差控制在20倍左右，输注细胞总数在一定范围内对结果没有影响，输注细胞后2~4 h为最佳检测时间点。提示体内细胞毒性试验是一种简单、准确、稳定的免疫状态体内定量监测方法，可以全面反映小鼠皮肤移植后受体的免疫状态。

关键词: 皮肤移植, 小鼠, 免疫状态, 定量免疫检测, 荧光流式技术

Abstract:

BACKGROUND: Currently, the immunological detection method has limitation due to complexity of immunoreactions. It is difficult to evaluate body immune status by a single index. Thus, it is urgent to establish a joint evaluation pattern to monitor the immune status of recipients following transplantation.   
OBJECTIVE: To establish a method of donor specific cytotoxicity assay in vivo for quantitatively detecting the immune status of the recipients, and to explore technical parameter of flow cytometry, conditions for fluorescence staining, number of transfused cells, and to determine the optimal testing times.  
METHODS: C57 and Balb/c splenocytes were prepared for cell suspension, labeled by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) with different final concentrations. A skin transplantation model was established with the recipients BALB/c mice and the donors C57BL/6 mice. Balb/c mice without skin transplantation served as controls. After intravenous transfusion of the CFSE-labeled donor/recipient splenocytes mixture, the following examinations were carried out at 1, 2, 4, 10 hours and 1, 2, 3, 6 days. A quantitative estimate of the ratio of donor/recipient splenocytes labeled with CFSE in host peripheral blood. The percent specific lysis was calculated using the equation.
RESULTS AND CONCLUSION: The flow cytometry was setting lymphocyte gate, and the CRSE concentration was controlled within 20-fold. The number of infused cells had no effects on results in certain range. It is optimal to detect at 2-4 hours after cell transfusion. The results demonstrate that in vivo cell toxicity test is simple, precise, reliable and well suited for quantitatively detecting the immune status of skin transplant recipients in vivo.

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