造血干细胞移植中HLA-Ⅱ类基因分型影响因素的探讨  

Study of the influencial factors of HLA class Ⅱ genotyping methods in hematopoietic stem cell transplantation

作　　者：屠敏[1] 孔繁华[1] 奚永志[1] 孙玉英[1] 郑黎燕[1] 金荔[1] 刘楠[1] 陈兴国[1] 郭斯启[1] 张惠丽[1] 

机构地区：[1]军事医学科学院附属医院免疫学研究室暨国家生物医学分析中心免疫分析实验室,北京100039

出　　处：《中国免疫学杂志》2000年第7期369-370,共2页Chinese Journal of Immunology

基　　金：国家自然科学基金!3990187

摘　　要：目的：为了准确地进行造血干细胞移植供－受体的ＨＬＡ－Ⅱ类配型，对ＨＬＡ－Ⅱ类基因分型实验的影响因素进行了探讨。方法：采用美国莱姆达公司提供的微量ＳＳＰ＾ＴＭＨＬＡ－Ⅱ类ＰＣＲ－ＳＳＰ分型试剂盒对４００例造血干细胞移植供－受体进行基因分型。结果：采用０．５％ＥＤＴＡ抗凝的血标本与肝素抗凝的血标本相比，前者的护增效果好。ＤＮＡ终浓度为２５～２００ｎｇ／ｕｌ，最佳浓度为１００ｎｇ／ｕｌ。Objective: In order to type HLA class Ⅱ of hematopoietic stem cell transplantation donor-recipient pairs accurately, the authors studied the factors impacting on HLA class Ⅱ genotyping methods. Methods: Genotyping methods of HLA-Ⅱ PCR-SSP (Lambda, Inc)were employed to genotype 400 cases of related hematopoietic stem cell transplant donors and recipients. Results: The results indicated that amplified results were much better in 0. 5% EDTA anticoagulated peripheral bled compared with heparin samples. Final DNA concentration should be 25 ~ 200 ng/μl(100ng/μl is optimal) with A260/A280 ratio between 1 .65 ~ 1.80. PCR reaction parameters is also very important in HLA class Ⅱ genotyping. The usage and the activity of Taq DNA polymerase directly affect typing results. DMIX tube should be stored at -65℃, and avoid frequently freezing and melting, otherwise, it might decrease the output of amplification. Conclusion: The proper processing of specimen and optimization d reaction conditions for HLA class Ⅱ genotyping are the key factors for good results.

关 键 词：HLA-Ⅱ类 基因分型 造血干细胞移植 

分 类 号：R457[医药卫生—治疗学] R392.4[医药卫生—临床医学]