CN110229877A - 一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用 - Google Patents
一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用 Download PDFInfo
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Abstract
本发明涉及一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用,具体涉及一种为通过检测血清中miRNA的表达水平来诊断血脂异常肝郁证的试剂的应用,所述试剂基于定量PCR方法检测血脂异常肝郁证样本中miRNA的靶基因的表达情况,所述miRNA的靶基因的表达情况为15个上调miRNA及9个下调miRNA。本发明所述试剂可应用于预防或诊断由肝郁脾虚证引发的血脂异常。
Description
技术领域
本发明涉及分子生物学领域,具体涉及一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用。
背景技术
血脂是血浆中所有脂质的总称,高脂血症(hyperlipidemia)是由于脂肪代谢或运转异常导致血浆中血脂水平过高,可表现为高胆固醇血症(hypercholesterolemia)、高甘油三酯血症(hypertriglyceridemia)和两者皆有(混合性高脂血症)。另外,高密度脂蛋白降低也是一种病理状态。与上述血脂代谢紊乱统称为血脂异常(dyslipidemia)。如果血脂过多,容易造成“血稠”,在血管壁上沉积,逐渐形成小斑块,这些“斑块”增多、增大,逐渐堵塞血管,使血流变慢,严重时血流被中断。这种情况如果发生在心脏,就引起冠心病;发生在脑,就会出现脑中风;如果堵塞眼底血管,将导致视力下降、失明;如果发生在肾脏,就会引起肾动脉硬化,肾功能衰竭;发生在下肢,会出现肢体坏死、溃烂等。此外,高血脂9可引发高血压、诱发胆结石、胰腺炎,加重肝炎、导致男性性功能障碍、老年痴呆等疾病。最新研究提示高血脂可能与癌症的发病有关。血脂异常是现代社会中常见疾病,大量文献研究发现,高脂血症证素分布与“肝”密切相关,本课题组前期通过对广东地区高脂血症中医证候分布规律进行分析,发现广东地区血脂异常人群中“肝郁脾虚证”最为常见,占36.1%[1]。
miRNA是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子,它们在动植物中参与转录后基因表达调控。近年来研究发现,多种miRNA参与调节脂质合成、分解及转运等过程,并在血脂异常患者中表达量发生变化,在脂质代谢相关疾病研究中发挥重要作用。从分子水平揭示肝郁脾虚证的发病机制,可为及早发现因“肝郁脾虚证”引发的血脂异常的危险人群、预防血脂异常的发生提供帮助。
发明内容
本发明的目的在于提供miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用。
本发明公开了一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用,进一步地,所述试剂通过检测血清中miRNA的表达水平来诊断血脂异常肝郁证的产品。
进一步地,所述试剂基于定量PCR方法检测血脂异常肝郁证样本中miRNA的靶基因的表达情况。
更进一步的,所述血脂异常肝郁证样本中miRNA的靶基因的表达情况为15个上调miRNA miR-382-5p、miR-432-5p、let-7f-5p、miR-454-3p、miR-374a-5p、miR-126-5p、let-7a-5p、miR-142-3p、miR-26a-5p、miR-27b-3p、miR-27a-3p、let-7d-5p、miR-652-3p、miR-148a-3p、miR-30e-5p,有9个下调miRNA miR-34c-5p、miR-181c-5p、miR-193b-3p、miR-129-2-3p、miR-149-5p、miR-542-5p、miR-133a、miR-133b、miR-124-3p。
进一步的,所述定量PCR方法检测miRNA表达情况包括如下步骤:
步骤1,利用miRNA逆转录试剂盒(Exiqon,Danmark)将20-25ng的总RNA反转录成cDNA;
步骤2,在定量PCR仪上对cDNA进行实时定量PCR扩增,Real-time PCR循环条件:
PCR反应条件为:95℃预变性10min,然后95℃10s,60℃60s,进行40个循环;最后,在PCR结束后进行融解曲线分析。通过研究发现,95℃至60℃降温过程应设置为1.6℃每秒。
步骤3,数据分析:
a.计算每个处理组中的每个通路相关基因的ΔCt。ΔCt(group 1)=average Ct–average of HK genes’Ct for group 1array;ΔCt(group 2)=average Ct–average ofHK genes’Ct for group 2array;
b.计算2个PCR Array(或两组)中每个基因的ΔΔCt。ΔΔCt=ΔCt(组2)-ΔCt(组1);
c.通过2-ΔΔCt计算组2与组1对应基因的表达差异。
更进一步的,所述步骤2之前还包括如下步骤:
S1、准备Real-time PCR试剂
将制备的cDNA模板,nuclease free water和SYBRTMGreen master mix置于冰上溶解15-20分钟,SYBRTMGreen master mix应避光溶解,使用前应倒置混匀,其他试剂震荡混匀。所有试剂应离心后使用。所有试剂和反应液应始终与冰上操作。
S2、稀释cDNA模板
将RT反应获得的cDNA模板用nuclease free water稀释110倍。
S3、混合所有反应试剂,操作如下:
A.将PCR板简单离心后,移去封膜;
B.将110倍稀释的cDNA模板与2×SYBR Green master mix按照1:1混合(例如2200ul 2×master mix和2200ul稀释的cDNA模板混合);
C.倒置混匀反应液并离心;
D.将混合反应液根据下表的建议加入板中的每个孔;
E.重新封好PCR板:
有益效果在于:
本发明首次利用定量PCR芯片研究了血脂异常症肝郁证患者和健康志愿者血清miRNA表达谱,发现差异表达的24个血清miRNA。进一步功能注释显示,两组差异表达的miRNA的靶基因显著富集到包括神经元和神经组织的发育、存活和凋亡、蛋白和糖代谢、信号转导通路、病原体入侵、脂肪酸代谢等在内的多个KEGG通路。基于此,本发明首次发现了miRNA基因表达与血脂异常症肝郁证相关,通过检测受试者中miRNA基因表达,可以判断受试者是否患有血脂异常症肝郁证的风险,相比传统的检测手段,基因诊断更及时和灵敏。
附图说明
图1血脂异常肝郁证患者与健康志愿者之间的差异表达miRNA;
图2差异表达miRNA和样本聚类分析,其中4个差异miRNA的表达水平能够将血脂异常肝郁证患者和健康志愿者分成两类,而且上调和下调miRNA也被分为两类;
图3差异表达miRNA靶基因预测和功能注释,其中A为上调miRNA,B为下调miRNA。
具体实施方式
下面结合附图和实施例对本发明作进一步详细的说明,以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
实施例1
1、血脂异常症临床诊断标准
参考中华心血管病杂志编委会血脂异常防治对策专题组“血脂异常防治建议”,确定血脂异常症临床诊断标准,同时也将血清HDL-C降低作为血脂异常症诊断标准之一。诊断标准具体如下:正常饮食情况下,检测隔夜禁食12-14h后的血脂水平,不同日期血脂符合下列条件一项或多项3次以上即可诊断为血脂异常:(1)TC≥5.72mmol/L(220mg/dL);(2)TG≥1.7mmol/L(150mg/gL);(3)LDL-C≥3.64mmol/L(140mg/gL);(4)HDL-C≤0.91mmol/L(35mg/dL)。
2、中医辨证分型标准
参照《中药新药临床研究指导原则》制定肝郁证(本文特指肝郁气结证)的诊断标准。中医辨证分型由2位中医师单独进行,对中医证型有分歧,则与第3位人员讨论后确定。如有兼夹证候,脾虚证必须占全部证候的2/3以上。
3、血脂异常症肝郁证患者的纳入及排除标准
纳入标准:年龄42-58岁,同时符合血脂异常症诊断标准和肝郁证的辨证分型标准,并签署知情同意书。排除标准:合并有除血脂异常症外的其他内分泌或心血管疾病者;合并有感染和炎性疾病者;合并有精神病者;合并有心、脑、肝、肾和造血系统等严重器质性病变者;妊娠或哺乳期女性。
4、健康志愿者的纳入标准
健康志愿者由主任医师(洪敏)通过健康体检和会诊确定。纳入标准:年龄42-58岁,无明显西医疾病,无超重和家族糖尿病,无典型中医证候,并签署知情同意书。
5、10例受试者均来自2015年4月-2016年4月广东药科大学附属第一医院中医科的就诊患者或本院体检中心的健康检查者。3例肝郁证患者,其中2例女性和1例男性,年龄43-58岁,平均(48±6.1)岁;病程3-8年,平均(4.5±5.6)年,全部为高胆固醇血症;3名健康志愿者,其中2名女性,1名男性,年龄42-58岁,平均(50±7.4)岁。两组年龄、比较,差异无统计学意义(P>0.05)。本研究在获得广东药科大学附属第一医院伦理委员会的授权下进行,伦理审批号为[2013]临审(15)号。
6、血清样品的采集
所有受试者在早晨7:00-9:00的空腹状态下,用无抗凝剂的采集管,抽取3mL静脉血,4℃凝血,1000r/min离心10分钟,收集血清。血清标本被分装在无RNA酶的冻存管中,-80℃保存备用。整个过程在采血后2小时内完成。
7、血清miRNA定量PCR芯片实验
取1mL血清加入适量的Trizol(Invitrogen life technologies,USA)试剂,再通过酚氯仿的方法提取总RNA。总RNA经质控后,被用于miRNA定量PCR芯片试验。本研究选择Exiqon公司(Danmark)的血清/血浆miRNA定量PCR芯片,该芯片包含人类372个成熟miRNA,用于检测血脂异常症肝郁证患者的血清miRNA表达谱。
(1)准备Real-time PCR试剂
将制备的cDNA模板,nuclease free water和SYBRTMGreen master mix置于冰上溶解15-20分钟。SYBRTMGreen master mix应避光溶解,使用前应倒置混匀,其他试剂震荡混匀。所有试剂应离心后使用。所有试剂和反应液应始终与冰上操作。
(2)稀释cDNA模板
将RT反应获得的cDNA模板用nuclease free water稀释110倍(例如,向20μl反应液中加入2180ul nuclease free water)。这是根据每个RT反应使用22ng总RNA的量计算得到并考虑了10%加样损失。
(3)混合所有反应试剂
建议步骤:
A.将PCR板简单离心后,移去封膜。
B.将110倍稀释的cDNA模板与2×SYBR Green master mix按照1:1混合(例如2200ul 2×master mix和2200ul稀释的cDNA模板混合)。
C.倒置混匀反应液并离心
D.将混合反应液根据下表的建议加入板中的每个孔
E.重新封好PCR板:
(4)将PCR板简单低温离心
(5)Real-time PCR扩增
根据下表中的反应条件进行Real-time PCR扩增和溶解曲线分析。
Real-time PCR循环条件:
(6)数据分析:采用ΔΔCt方法
使用GenEx qPCR分析软件(www.exiqon.com/mirna-pcr-analysis)对数据进行标准和深入的数据分析。
a.计算每个处理组中的每个通路相关基因的ΔCt。
ΔCt(group 1)=average Ct–average of HK genes’Ct for group 1array
ΔCt(group 2)=average Ct–average of HK genes’Ct for group 2array
b.计算2个PCR Array(或两组)中每个基因的ΔΔCt。
ΔΔCt=ΔCt(组2)-ΔCt(组1)
备注:通常组1是对照,组2是实验组。
c.通过2-ΔΔCt计算组2与组1对应基因的表达差异。
数据分析具体的包括如下步骤:
①差异表达miRNA的筛选本研究选择各样品中表达稳定的看家基因SNORD38B和SNORD49A作为内参,对于≥38个Ct值的数据在进行数据处理时默认为38个Ct值。最后利用2-ΔΔCt的方法,计算血脂异常症肝郁证患者与与健康志愿者之间每个miRNA的相对表达值,即表达倍数。对于表达倍数<1.00的数据进行负倒数处理。两组之间miRNA表达量的差异采用t检验。本研究差异表达miRNA的筛选标准为≥2.0,且P<0.05。表达倍数为负值表示该miRNA在肝郁证患者血清中表达下调,正值则表达上调。数据分析利用GenEx qPCR(Exiqon,Danmark)和SPSS18.0(IBM,USA)软件进行。
②差异miRNA生物信息学分析
2.1、层次聚类分析将10个临床样本的差异表达miRNA和表达倍数导入MeV4.9(TM4,USA)软件,并利用中位数中心法校正miRNA/行,然后选择欧式距离测度和全连接聚类的连接方式,构建样本和miRNA的层次聚类图。
2.2、miRNA靶基因预测和功能注释利用综合数据库miRSystem(version20150312)对miRNA进行靶基因预测和功能注释。该数据库整合有TarBase和miRecord2个实验验证数据库,和Diana-microT、miRanda、miRBridge、PicTar、PITA、RNA22、Targetscan等7个预测数据库,用于靶基因的预测。此外,该数据库整合有KEGG(Kyoto Encycolpedia of Genes andGenomes)、GO(Gene Ontology)分子功能、Biocarta等5个数据库,用于靶基因的功能注释。而且该数据库还提供了O/E比值、超几何和经验P值等统计学方法,用于鉴定富集通路和生物学功能的显著性。本研究将miRNA及其表达倍数上传到数据库中,对被实验验证或被3个以上预测数据库鉴定的靶基因,进行KEGG通路和GO分子功能注释。而且功能注释中的总基因数设定为25-500个基因,O/E比值设定为≥2.0,P<0.05。
结果:
1、如图1所示,血脂异常肝郁证患者和健康志愿者差异表达血清的miRNA;
本研究发现24个差异表达的miRNA,其中15个miRNA在肝郁证患者的血清中表达上调,9个miRNA表达下调。
2、如图2所示,差异表达miRNA和样本聚类分析;
24个差异miRNA的表达水平能够将血脂异常肝郁证患者和健康志愿者分成两类,而且上调和下调miRNA也被分为两类。注:聚类分析图示两组之间miRNA表达量的差异倍数≥2.0,且P<0.05。颜色深的表示上调miRNA,颜色浅的表示下调miRNA;HLM表示血脂异常肝郁证患者,CON表示健康志愿者。
3、如图3所示,差异表达miRNA靶基因预测和功能注释,其中A为上调miRNA;B为下调miRNA。
miRSystem数据库对miRNA靶基因预测结果显示,血脂异常肝郁证患者血清中表达上调的15个miRNA调控159个靶基因,下调的9个miRNA调控108个靶基因。功能注释结果,发现多个富集的KEGG通路,其中上调miRNA的靶基因富集10个KEGG通路,下调miRNA的靶基因富集10个KEGG通路。上调miRNA富集的KEGG通路主要包括细胞信号转导、神经元和神经组织的发育、存活和凋亡、多能性干细胞、蛋白和糖代谢、基底细胞癌5个方面。下调miRNA富集的KEGG通路主要包括细菌和杆菌等病原体入侵、脂肪酸代谢、增殖和凋亡相关的信号通路、长时间压抑4方面。
Claims (6)
1.一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用。
2.如权利要求1所述的一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用,其特征在于,所述试剂为通过检测血清中miRNA的表达水平来诊断血脂异常肝郁证的产品。
3.如权利要求2所述的一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用,其特征在于,所述试剂可基于定量PCR方法检测血脂异常肝郁证样本中miRNA的靶基因的表达情况。
4.如权利要求3所述的一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用,其特征在于,所述miRNA的靶基因的表达情况为15个上调miRNA miR-382-5p、miR-432-5p、let-7f-5p、miR-454-3p、miR-374a-5p、miR-126-5p、let-7a-5p、miR-142-3p、miR-26a-5p、miR-27b-3p、miR-27a-3p、let-7d-5p、miR-652-3p、miR-148a-3p、miR-30e-5p,有9个下调miRNA miR-34c-5p、miR-181c-5p、miR-193b-3p、miR-129-2-3p、miR-149-5p、miR-542-5p、miR-133a、miR-133b、miR-124-3p。
5.如权利要求4所述的一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用,其特征在于,所述定量PCR方法检测miRNA表达情况包括如下步骤:
步骤1,利用miRNA逆转录试剂盒(Exiqon,Danmark)将20-25ng的总RNA反转录成cDNA;
步骤2,在定量PCR仪上对cDNA进行实时定量PCR扩增,Real-time PCR循环条件:
PCR反应条件为:95℃预变性10min,然后95℃10s,60℃60s,进行40个循环;
步骤3,数据分析:
a.计算每个处理组中的每个通路相关基因的ΔCt:ΔCt(group 1)=average Ct–average of HK genes’Ct for group 1 array;ΔCt(group 2)=average Ct–average ofHK genes’Ct for group 2 array;
b.计算2个PCR Array(或两组)中每个基因的ΔΔCt:ΔΔCt=ΔCt(组2)-ΔCt(组1);
c.通过2-ΔΔCt计算组2与组1对应基因的表达差异。
6.如权利要求5所述的一种miRNA在制备预防或诊断血脂异常肝郁证试剂中的应用,其特征在于,所述定量PCR方法检测miRNA表达谱包括如下步骤:
S1、准备Real-time PCR试剂
将制备的cDNA模板,nuclease free water和SYBRTMGreen master mix置于冰上溶解15-20分钟,SYBRTMGreen master mix应避光溶解,使用前应倒置混匀,其他试剂震荡混匀,所有试剂应离心后使用,所有试剂和反应液应始终与冰上操作;
S2、稀释cDNA模板
将RT反应获得的cDNA模板用nuclease free water稀释110倍;
S3、混合所有反应试剂,操作如下:
A.将PCR板简单离心后,移去封膜;
B.将110倍稀释的cDNA模板与2×SYBR Green master mix按照1:1混合(例如2200ul 2×master mix和2200ul稀释的cDNA模板混合);
C.倒置混匀反应液并离心;
D.将混合反应液根据下表的建议加入板中的每个孔;
E.重新封好PCR板:
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