CAJ | 학술논문

目的采用2-D/质谱电泳方法,比较孕17d正常组胎鼠和全反式维甲酸诱导的显性脊柱裂胎鼠羊水蛋白表达谱的变化,筛查先天性显性脊柱裂相关羊水标记物.方法孕10d大鼠,采用全反式维甲酸胃管灌饲的方法建立先天显性脊柱裂动物模型24只(脊柱裂组),正常对照组9只(正常组).孕17d晨,麻醉孕鼠,显微镜下获取每个胎囊的羊水及胎鼠,并检查胎鼠畸形情况.提取羊水蛋白质,merck试剂盒去除高丰度白蛋白和IgG,行2-D、胶体染色、图像扫描分析及质谱检测,Western-blot进一步验证2-D结果.结果鉴定出6种蛋白质(其中1种蛋白质可能对应多个蛋白点):甲胎蛋白(AFP)、转铁蛋白(transferrin,TF)、信号识别颗粒受体B亚基(srprb)55kDa、srprb 77kD、α-1抗蛋白酶及载脂蛋白A4(Apo A4).其中,Apo A4和Srprb 77 kDa在脊柱裂组表达下调,而TF、Srprb 55 kDa和a-1抗蛋白酶在脊柱裂组表达上调.46个蛋白质点被鉴定为AFP及其片段,11个AFP蛋白点在脊柱裂组表达降低,35个蛋白点在脊柱裂组表达升高.通过Western-blot共发现69 kDa、52 kDa、36 kDa、24 kDa和14 kDa 5种分子量的AFP片段,其中69 kDa的AFP在脊柱裂组和正常组含量均为最高.脊柱裂组与正常组比较,AFP上调42％,而Apo A4下调44％,组间差异有统计学意义(P＜0.05).结论本研究发现羊水中6种蛋白质与显性脊柱裂畸形发生有关,进一步研究这些差异表达蛋白,可以为了解脊柱裂的发病机制和产前诊断提供实验依据.
목적 채용2-D/질보전영방법,비교잉17d정상조태서화전반식유갑산유도적현성척주렬태서양수단백표체보적변화,사사선천성현성척주렬상관양수표기물.방법 잉10d대서,채용전반식유갑산위관관사적방법건립선천현성척주렬동물모형24지(척주렬조),정상대조조9지(정상조).잉17d신,마취잉서,현미경하획취매개태낭적양수급태서,병검사태서기형정황.제취양수단백질,merck시제합거제고봉도백단백화IgG,행2-D、효체염색、도상소묘분석급질보검측,Western-blot진일보험증2-D결과.결과 감정출6충단백질(기중1충단백질가능대응다개단백점):갑태단백(AFP)、전철단백(transferrin,TF)、신호식별과립수체B아기(srprb)55kDa、srprb 77kD、α-1항단백매급재지단백A4(Apo A4).기중,Apo A4화Srprb 77 kDa재척주렬조표체하조,이TF、Srprb 55 kDa화a-1항단백매재척주렬조표체상조.46개단백질점피감정위AFP급기편단,11개AFP단백점재척주렬조표체강저,35개단백점재척주렬조표체승고.통과Western-blot공발현69 kDa、52 kDa、36 kDa、24 kDa화14 kDa 5충분자량적AFP편단,기중69 kDa적AFP재척주렬조화정상조함량균위최고.척주렬조여정상조비교,AFP상조42％,이Apo A4하조44％,조간차이유통계학의의(P＜0.05).결론 본연구발현양수중6충단백질여현성척주렬기형발생유관,진일보연구저사차이표체단백,가이위료해척주렬적발병궤제화산전진단제공실험의거.
Objective To explore the potential biomarker for early diagnosis of spina bifida aperta by analyzing of proteomic profile of amniotic fluid in rats with spina bifida aperta (SBA) induced by all-trans retinoic acid (ATRA).Methods The pregnant rats were randomly divided into 2 groups:SBA group (24 rats) and normal group (9 rats).SBA was induced by a single intragastric administration of ATRA to the pregnant rats on gestational day 10 (E10).All pregnant rats were sacrificed by an overdose chloral hydrate on E17 day,and fetuses and amniotic fluid were collected.Albumin and IgG were removed using Protein A/G kit to increase the concentration of target proteins.The proteomic profiles of amniotic fluid were analyzed by two-dimensional gel electrophoresis (2-DE)/mass spectrometry (MS).Results Six proteins were found to be differentially expressed in the amnioticfluid of SBA rats.Of the 6 proteins,transferrin,alpha-1 antiproteinase and signal recognition particle receptor were unregulated,and apolipoprotein A Ⅳ and Srprb 77 kDa were down-regulated.Additionally,46 protein fragments in SBA amniotic fluid were found,including 11 were upregulated and 35 were downregulated.A Western blot analysis confirmed the 2-DE/MS data,indicating the 6 isoforms of AFP (69-,52-,36-,24-and 14-kDa) were associated with SBA.Moreover,the 69-kDa AFP was the most abundant protein in the amniotic fluid of SBA,which was increased by 1.42-fold (P＜0.05).Conclusions Six proteins in amniotic fluid are correlated with spinal bifida aperta,and may be used as biomarkers for early diagnosis of spina bifida aperta.