线粒体DNA拷贝数检测方法的建立及在肾脏疾病诊断中的应用  

The real-time PCR assay for mitochondrial DNA copy number and its applications in kidney disease diagnosis

作　　者：何静[1] 夏虹[2] 张昌明[2] 刘志红[2] 施少林[2] 

机构地区：[1]南京大学医学院临床学院 [2]南京军区南京总医院国家肾脏疾病临床医学研究中心全军肾脏病研究所,南京210016

出　　处：《肾脏病与透析肾移植杂志》2014年第4期342-348,共7页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：国家自然科学基金(81370827);国家重点基础研究发展计划(973计划)(2012CB517606);江苏省自然科学基金(BK20131324)

摘　　要：目的:建立基于实时定量PCR的线粒体DNA(mt DNA)拷贝数的检测方法,并利用所建立的方法初步检测和探讨mt DNA在肾脏疾病诊断中的应用。方法:设计mt DNA PCR扩增引物,PCR扩增获得mt DNA片段,与T载体质粒连接,构建mt DNA定量标准品。采用SYBR Green-Ⅰ染料法实时定量PCR方法建立mt DNA定量的标准曲线,检验定量方法的重复性与特异性。利用所建立的方法检测局灶节段性肾小球硬化(FSGS)患者血清mt DNA和尿液微囊泡(MV)mt DNA的变化;此外,还对体外嘌呤霉素氨基核苷(PAN)刺激的足细胞所分泌的MV中mt DNA进行了检测。结果:(1)选取mt DNA特异性引物,通过SYBRGreen-Ⅰ实时定量PCR建立mt DNA拷贝数绝对定量的检测方法具有良好的特异度、灵敏度和重复性;(2)检测10例FSGS患者和6例正常对照血清mt DNA水平发现,FSGS患者血清mt DNA水平明显低于正常对照(P<0.05);(3)检测5例FSGS患者与5例健康对照一定体积尿液中MV mt DNA含量发现,FSGS患者高于健康对照,但FSGS患者尿液MV的含量也显著高于健康对照;(4)受PAN刺激的足细胞,其分泌的MV中mt DNA含量下降,与足细胞内mt DNA含量变化相一致。结论 :本方法为探索mt DNA作为肾脏疾病诊断的生物标志物的可能性提供了有效的实验手段。Objective： To establish a method to quantitate mitochondrial DNA （mtDNA） and to explore its potential as biomarker of kidney disease. Methodology ： A specific mtDNA fragment was amplified by PCR and cloned to a T vector plasmid. This construct was used for generating a standard curve that relates copy nubmers with Ct values. The producibility and sensitivity were optimized using the mtDNA construct. The preliminary study to examine mtDNA content in the serum samples or urine microvesicles from the patients with focal segmental glomerulosclerosis （FSGS） was performed. The mtDNA content in the microvesicles from human immortalized podocytes treated with or without puromycin aminonucleosides （PAN） was also detected. Results： （ 1 ） The mtDNA copy nubmer assay by SYBR Green ｜ -based real- time PCR was successfully set up with excellent reproducibility and sensitivity. （2） With this method, the serum levels of 10 FSGS patients and 6 healthy controls were examined, and there were significant lower serum mtDNA levels in FSGS patients compared with that in controls （ P 〈 0.05 ）. （ 3 ） The copy numbers of MV mtDNA in a given volume of urine samples from 5 FSGS patients and 5 healthy controls were also measured, respectively, and there was higher in patients with FSGS than that in controls ; meanwhile the MV content in the urine samples of the FSGS patients was also higher than that of controls. （4） The mtDNA content in the microvesicles from human immortalized podocytes treated with or withoutPAN was compared, and the mtDNA content was lower in the MV of PAN-treated podocytes than that of untreated cells ; in addition, the intracellular mtDNA content in PAN-treated podocytes was also lower. Conclusion：This method should be able to find its use in exploring the potential of mtDNA as a biomarker for kidney diseases.

关 键 词：线粒体DNA 实时定量PCR 肾脏疾病 生物标志物 微囊泡 

分 类 号：R692[医药卫生—泌尿科学]