检测大鼠血浆游离DNA的定量PCR方法的建立及意义

Establishment of real time PCR for detecting plasma cell free DNA of rats and its significance

目的:过度训练是体育界非常重要的问题,目前用多个指标的综合评定来判断是否有过度训练,尚无灵敏、特异的监控指标和简便的评价方法。本文建立检测大鼠血浆游离DNA(cell free DNA,cf DNA)的实时荧光定量PCR方法,并探讨血浆cf DNA水平能否成为反映大鼠过度训练的新监控指标。方法:12只雄性SD大鼠随机分为2组(n=6):安静组和过度训练组。过度训练组进行5周递增负荷的过度训练,每周运动6 d,运动的最后一天运动至力竭。力竭运动后36 h采集静脉血,检测大鼠血浆cf DNA、睾酮(T)和皮质酮(Cort)、总抗氧化能力(T-AOC)、丙二醛(MDA)和肌酸激酶(CK)的水平以及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性。结果:1所建立的方法能特异扩增大鼠血浆cf DNA;2与对照组相比,过度训练大鼠的血浆cf DNA水平显著增加(约为安静组的5.43倍);3血浆cf DNA与血浆T、Cort、T/C比值和MDA显著相关(相关系数分别为-0.729,0.854,-0.655和0.720);与T-AOC、GSH-Px、SOD和CK无相关性。结论:1建立检测大鼠血浆cf DNA的定量PCR方法;2过度训练大鼠的血浆cf DNA显著增加,且cf DNA与血浆T、Cort和T/C比值高度相关,提示血浆cf DNA是过度训练的新监控指标;3过度训练大鼠血浆cf DNA的增加可能与过度训练所致的氧化应激增强有关,可能与骨骼肌损伤关系不大。

Objective： Overtraining is a serious problem in sports, assessed by comprehensive multi-index evaluation,but so far there is still no sensitive, specific monitoring indicator or simple evaluation method to evaluate it. This research established a method for detecting plas- ma cell free DNA （cfDNA） of rats by real time PCR and discuss edits significance： a new molecular marker of overtraining？ Methods： Twelve male SD rats were randomly divided into control group and overtraining group. The overtraining group rats were undertaken overtraining on a motor-driven treadmill for 5 weeks, while the control group rats kept quiescent.All the rats were drawn blood at pre-and after-5 weeks to detect plasma levels of cfDNA, testosterone （T） and corticosterone （Cort） as well as peroxidation/antioxidation parameters （T-AOC, MDA, SOD, GSH-Px） and creatin kinase （CK）. Results：①Plasma cfDNA of rat was detected specifically by our real time PCR. ②Compared with control group rats, the plasnm cfDNA of overtraining rats increased obviously （about 5.43 fold）. QPlasma cfDNA was related to plasma T, Cort, T/ C ratio and MDA （ corfelation eoefficent were -0.729, 0. 854, -0.655 and 0. 720, respectively） rather than plasma T- AOC, GSH-Px, SOD and CK. Conclusion： ①A real time PCR method was established successfully to determine plasma cfDNA of rat. ②A remarkable raises of plasma levels of efDNA were found in ovemaining rats which were associated with T, Curt and T/C, suggested that plasma cfDNA might be a new molecular marker of ovemaining. ③The increase of plasma efDNA of overtraining rat might correlate with enhanced oxidative stress in- duced by overtraining instead of muscle damage.