血清非甲基化胰岛素DNA检测方法的建立

Development of a method to detect serum unmethylated insulin DNA

目的建立血清非甲基化胰岛素DNA的检测方法，并初步评价其临床应用价值。方法对文献报道的Akirav法和Husseiny法反应体系进行验证和改良。同时选取2014年2月至2015年2月中南大学湘雅二医院门诊或病房诊断为1型糖尿病（T1DM）且病程1年以内的患者6例，以及年龄相匹配的健康对照8例，采用改良Akirav法检测其血清中非甲基化胰岛素DNA水平，初步评价临床应用价值。本研究方案经中南大学湘雅二医院伦理委员会批准[（2012）伦理审第（S12）号]。结果Akirav法的甲基化非依赖性PCR（MIP）反应特异性较差，无法获得目标产物；Husseiny法的甲基化特异性定量PCR（qMSP）引物扩增效率为（76．98±2．88）％，低于推荐的80％，结果可信度差。利用Akirav法的主要思路，结合Husseiny法中提供的引物，建立的改良Akirav法灵敏度较高，检测T1DM患者血清中的非甲基化胰岛素DNA水平高于正常对照[非甲基化指数（DI）分别为0．039（0．028—0．083）和0．016（0．003—0．029），P=0．005]。结论改良Akirav法能有效检测血清非甲基化胰岛素DNA水平，T1DM患者血清中非甲基化胰岛素DNA水平高于健康人。

Objective To develop a specific and sensitive method to detect serum unmethylated insulin （INS） DNA for monitoring pancreatic beta cell death and evaluate its clinical practice value. Methods This study validated and modified the Akirav method and the Husseiny method. Six type 1 diabetes mellitus （T1DM） patients, who were diagnosed with T1DM in the Second Xiangya Hospital, Central South University between February 2014 and February 2015, and another 8 sex- and age-matched healthy controls, were enrolled in the study. Patients＇ serum samples were examined by the modified Akirav method. Results Methylation-independent PCR of the Akirav method cannot amplify the specific product. The efficiency of primers for quantitative methylation-speeific PCR used in Husseiny method was （76. 98 ＋ 2. 88）%, which was lower than 80% （recommend efficiency）, resulting in poor reliability. This study applied primers for the Husseiny method into the procedures of the Akirav method. The results showed that the level of unmethylated INS DNA in T1DM group is significantly higher than that in the healthy controls [ demethylation index： 0. 039（0. 028 -0. 083 ） vs 0. 016（0. 003 -0. 029）, P =0. 005 ]. Conclusions The modified Akirav method can effectively detect the level of unmethylated INS DNA. The level of unmethylated INS DNA in T1DM patients is higher than that in the healthy controls.