摘要
人生长激素是(hGH)促进人体身体生长以及蛋白同化作用的重要激素,近年来重组人生长激素(rhGH)被滥用在竞技体育中以提高运动员竞技能力,但是其检测一直是反兴奋剂研究领域的重大难题。目前,关于生长激素的检测方法主要包括直接检测法和间接检测法。直接检测法以生长激素体内的不同亚型为基础；间接检测法是针对与生长激素相关的其他生物学指标开展的检测工作。直接检测法是目前被WADA指定的检测方法。但两种方法各有优缺点,其基础方法、检测技术以及应用前景均需更深一步的研究与探讨。
本论文首先针对中国男性健康人群开展了rhGH受试研究,25名年轻男性志愿者被分为3组进行随机、双盲、对照受试。皮下注射rhGH连续14天,剂量为0.1IU/kg/d。共采血20次,分别为第一针注射前1小时,注射后2、4、8、12、23、34小时,以及第4、6、8、10、12、14、15、16、17、18、21、28、35天。
直接检测法结果显示,单针注射后,RecGH/PitGH在2-4小时达到顶峰,12小时检出率为63.2%,23小时己无法检出；累积注射时,注射后10小时检出率77.2%,21小时检出率仅5.3%,其检测窗口仅10-16小时。此外,检测结果显不PitGH检测结果个体差异性较大,推测是由PitGH中亚型种类多,其免疫反应结合不稳定导致的。根据我实验室rhGH的检测数据以及WADA的技术文件,建议降低判定限并改进试剂盒的检测抗体。
关于间接检测法,IGF-Ⅰ受rhGH的影响显著,受试第2天开始上升,第10天达到峰值,21天降至基础值。IGFBP3在注射rhGH后其变化趋势与IGF-Ⅰ相近,但变化幅度小且有明显的昼夜节律性,不宜作为检测指标。P-Ⅲ—P受rhGH的影响滞后,第6天浓度开始上升,第16天达到峰值,第35天血样浓度降至基础值。本研究纳入IGF-Ⅰ和P-Ⅲ-P两个检测指标,构建了判定公式,根据该判定公式检测窗口可达72小时。但是,各研究项目得到的判定公式无法互代。
综合分析直接检测法和间接检测法的特点,建议在今后研究中应改进目前直接法检测试剂盒的抗体及判定限,并针对不同人群及实验室建立间接检测法判定公式或判定限,同时建议联合应用直接和间接检测法的开展rhGH检测工作。
Human Growth hormone (hGH) is one of the most important hormones, which promotes the human body growth and protein assimilation. Recent years, recombinant human growth hormone (rhGH) is abused in sports to improve athletic ability, meanwhile, the detection of recombinant human growth hormone abuse is a huge challenge in antidoping research. There are two main detection methods:the direct method and indirect method. Direct method is the isoform approach, at the basis of differentiation of GH isoforms in human body. While indirect method is a bio-markers approach, which monitors certain blood parameters effected by the administration of hGH. Isoform approach is the only method authorized by World Anti-Doping Agency (WADA). Both of the two methods have advantages and disadvantages, based on method of detection technology and potential applications are required to further research and discussion.
For healthy male Chinese population,25healthy male volunteers were randomly divided into3groups. Treatment was administered by subcutaneous injection for14days,0.1IU/kg body weight/day. Blood samples were taken fasting at baseline (1hour before the first injection) and at2,4,8,12,23,34hours after injection, as well as4,6,8,10,12,14,15,16,1718,21,28,35days.
Direct method results showed that after a single injection, RecGH/PitGH peaked at2-4hours, the adverse percent at hour12was63.2%, no positive sample at hour23. For continuous injection, the adverse percent at hour10was77.2%, at hour21was5.3%. The detection window was only10-16hours. In addition, the result indicated that the high individual viaration in PitGH is due to unstable antigen-antibody reaction of different isofoms of PitGH. According to the analytical data and the WADA technical document, we suggest that the decision limit of kit1for male should be decreased, as well as perfect the antibody of the detetion assay.
On the indirect method, IGF-1viarated significantly after the rejection of rhGH, it increased at day2, reached the peak at day10, retuned to the baseline at day21.The viaration of IGFBP3is unsignificantly cross correlated to the administration, and also the daily viaration is very high, so it is not appropriate to be used as a marker for detection of doping with rhGH. P-Ⅲ-P increased from day6, reached the peak at dayl6, retuned to the baseline at day35. The discriminant formula was constructed wih IGF-Ⅰ and P-Ⅲ-P. According to the formula, the detection window can delay to72hours. However the formulas from different research cannot be iter-used.
Combined the direct and indirect method, we suggest that the antibody of the direct detection assay and decision limit should be improved; the discriminant formula or decision limit should be adjusted in different ethnicity or laboratories. At last, according the different characteristic of two detection method, we suggest that using two methods for different purposes, the direct methond for taching the cheat, while the indirect method more for targeting the athlete and deterent.
引文
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