CN116574792A - Anril在制备慢性肾脏病相关心血管病变诊断试剂盒中的应用 - Google Patents
Anril在制备慢性肾脏病相关心血管病变诊断试剂盒中的应用 Download PDFInfo
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Abstract
本发明公开了一种ANRIL在制备慢性肾脏病相关心血管病诊断试剂盒中的应用,属于慢性肾脏病诊断技术领域,本发明发现ANRIL在慢性肾脏病患者血浆中表达增高,其表达水平与肾功能负相关,与血管内皮功能状态负相关,在一定程度上可以反映CKD患者心血管病变的严重程度。同时,进一步探讨并验证其参与介导内皮细胞功能障碍的作用及机制。该检查方法简单易行,对受试者创伤性小,受试者顺从性更佳,同时为靶向性药物的研发奠定了理论基础。
Description
技术领域
本发明涉及慢性肾脏病诊断技术领域,更具体地说是涉及ANRIL在制备慢性肾脏病相关心血管病诊断试剂盒中的应用。
背景技术
慢性肾脏病(chronic kidney disease,CKD)是影响全球人类健康的重要疾病,具有高发病率、高致死率、低知晓率的流行病学特点。我国成人慢性肾脏病的患病率高达10.8%,意味着患病人数达1.195亿之众,但其知晓率却仅有12.5%。糖尿病、高血压、肥胖和老龄化的增加,极大的推动CKD患病率的增加。同时,2020年WHO数据显示CKD在全球死因排行中已上升至第11位。心血管疾病是CKD患者的主要死因,所致死亡率约占慢性肾脏病患者总死亡率的50%。CKD是心血管疾病的独立危险因素,研究证实,肾小球滤过率(estimated glomerular filtration rate,eGFR)与心血管事件发生率和死亡率呈负相关;且随着肾功能下降,心血管疾病如动脉粥样硬化等发病率明显上升。心血管事件预测及早期诊断是降低CKD患者不良预后需解决的关键问题。
长链非编码RNA(long non-coding RNAs,lncRNAs)是一类核苷酸数量大于200,且不具备蛋白质编码功能的RNA。可通过组蛋白修饰、染色质重塑、DNA甲基化和去甲基化、RNA干扰等多种机制在表观遗传学水平、转录水平、转录后水平对细胞的增殖、代谢、迁移和侵袭等产生重要的调控作用。血清血浆中的LncRNAs性质稳定、含量丰富、易于定量检测,且存在显著的疾病特异性,可以作为疾病的生物标志物。然而LncRNAs在CKD心血管事件中的作用仍未知,临床上尚缺乏用于CKD心血管事件预测的较为稳定的生物标志物的报道。
因此,如何提供一种可以作为CKD心血管事件发病诊断的标志物并对其进行应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明将ANRIL作为诊断慢性肾脏病的标志物,ANRIL在慢性肾脏病患者血浆中表达增高,其表达水平与肾功能负相关,与血管内皮功能状态负相关,在一定程度上可以反映CKD患者心血管病变的严重程度。
为了实现上述目的,本发明采用如下技术方案:
ANRIL(INK4基因座的反义非编码RNA)在制备慢性肾脏病相关心血管病诊断试剂盒中的应用。优选地,ANRIL作为一种慢性肾脏病的血清标志物,与慢性肾脏病血管内皮功能障碍、线粒体裂变异常相关,患者血浆中ANRIL水平升高。
优选地,以血清中ANRIL为检测指标,检测程序为:
作为与上述技术方案相同的发明构思,本发明还请求保护一种诊断试剂盒,包括:检测引物和检测试剂;其中,所述引物序列如SEQ ID NO.1~SEQ ID NO.4所示;
ANRIL-F:5’-TACATCCGTCACCTGACACG-3’,如SEQ ID NO.1所示;
ANRIL-R:5’-ACGAGGGGAGCCAGGAATAA-3’,如SEQ ID NO.2所示;
β-actin-F:5’-GAAGAGCTACGAGCTGCCTGA-3’,如SEQ ID NO.3所示;
β-actin-R:5’-CAGACAGCACTGTGTTGGCG-3’,如SEQ ID NO.4所示。
优选地,检测试剂包括:
作为与上述技术方案相同的发明构思,本发明还请求保护一种引物,如SEQ IDNO.1~SEQ ID NO.4所示。
作为与上述技术方案相同的发明构思,本发明还请求保护以ANRIL为作用靶点在制备治疗慢性肾脏病药物中的应用。
优选地,以ANRIL为作用靶点具体为:抑制ANRIL表达可改善尿毒症毒素IS所致的内皮细胞损伤、改善尿毒症毒素IS所致的内皮细胞线粒体裂变异常、逆转分裂关键蛋白DRP-1和融合相关蛋白MFN2表达的异常。
经由上述的技术方案可知,与现有技术相比,本发明提供lncRNAANRIL在慢性肾脏病心血管事件预测中的作用。发现ANRIL与肾功能负相关,与血管内皮功能状态负相关;进一步研究证明,ANRIL可以介导血管内皮细胞功能相关蛋白表达异常,线粒体裂变异常,从而引起其功能障碍。因此,基于ANRIL为标志物,作为诊断慢性肾脏病心血管并发症的诊断标准之一,为临床上对慢性肾脏病心血管并发症的检测提供了简便快捷的方法,也为慢性肾脏病心血管并发症的靶向性药物提供了理论基础。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1为ANRIL与慢性肾脏病血管内皮功能障碍相关统计图;A为real-timePCR检测显示,与健康人群对比,CKD患者血浆ANRIL水平明显增高,B为Spearman相关分析显示,ANRIL表达与肾小球滤过率(eGFR)呈负相关,C为ANRIL表达与血管内皮功能状态评价指标FMD水平负性相关,D为ELISA检测显示与健康人群对比,CKD患者血浆BDNF浓度明显降低,E为Spearman相关分析显示,CKD患者血浆BDNF浓度与肾小球滤过率(eGFR)呈负相关,F为ANRIL表达与BDNF水平负性相关,*表示与健康对照组相比P<0.05;
图2为ANRIL表达水平示意图;A为real-timePCR检测在尿毒症患者血清刺激的细胞模型中ANRIL表达水平,B为FISH检测在尿毒症患者血清刺激的细胞模型中ANRIL表达及分布,C为real-timePCR检测采用不同浓度尿毒症毒素硫酸吲哚酚(IS)、马尿酸(HA)、吲哚-3-乙酸(IAA)、同型半胱氨酸(Hcy)刺激下内皮细胞ANRIL表达水平变化,*表示与对照组相比P<0.05;
图3为基因调控效率验证图;A.real-timePCR检测ANRIL的敲低效率,B.real-timePCR检ANRIL的过表达效率,C.westernblot检测EZH2siRNA调控效率,D.westernblot检测BDNF过表达质粒调控效率,*P<0.05vsControl;
图4为ANRIL介导内皮细胞功能障碍及线粒体裂变异常示意图;A为抑制ANRIL表达可改善尿毒症毒素IS所致的内皮细胞损伤,具体为IS诱导内皮细胞功能相关蛋白eNOS表达降低,VCAM-1、vWF表达增加;Sh-ANRIL抑制内皮细胞中ANRIL的表达,可逆转上述蛋白表达异常,B为抑制ANRIL表达可改善尿毒症毒素IS所致的内皮细胞线粒体裂变异常,具体为线粒体分裂关键蛋白DRP-1表达增加,融合相关蛋白MFN2表达减少;抑制ANRIL表达可逆转上述蛋白表达异常,*表示与对照组相比P<0.05,#表示与IS刺激组相比P<0.05;
图5为ANRIL通过下调BDNF调控内皮细胞功能障碍示意图;A为抑制ANRIL表达可改善尿毒症毒素IS所致的BDNF表达下调,B为抑制ANRIL表达可逆转ANRIL过表达所致的BDNF表达下调,C为上调BDNF可改善ANRIL过表达引起的内皮细胞功能障碍:具体为ANRIL过表达可诱导内皮细胞功能相关蛋白eNOS表达降低,VCAM-1、vWF表达增加;转染BDNF过表达质粒,可逆转上述蛋白表达异常,D为westernblot检测显示上调BDNF可改善ANRIL过表达引起的线粒体裂变异常,E为上调BDNF可改善ANRIL过表达引起的线粒体ROS积聚,*表示与对照组相比P<0.05,#表示与IS刺激组或ANRIL过表达组相比P<0.05;
图6为ANRIL募集转录因子EZH2,调控BDNF表达示意图;A.为RNApull-down结合WesternBlot检测显示ANRIL结合EZH2,B.RIP检测ANRIL与EZH2结合,结果显示ANRIL过表达可使其与EZH2结合增加,C.WesternBlot检测调控ANRIL及EZH2表达后内皮细胞H3K27me3水平,D.为WesternBlot检测调控ANRIL及EZH2表达后内皮细胞BDNF表达水平,E.为CHIP检测BDNF启动子区H3K27me3水平,F为CHIP检测BDNF启动子区EZH2结合水平,*表示与null组相比P<0.05,#表示与ANRIL过表达组相比P<0.05;
图7为验证ANRIL通过EZH2/BDNF调控内皮细胞功能障碍示意图;A.WesternBlot检测内皮细胞相关蛋白及线粒体相关蛋白表达变化,B.免疫荧光检测VCAM-1表达水平,C.免疫荧光检测MFN2表达水平,D.MitoSOXRed染色显示内皮细胞线粒体ROS水平,Scalebars=50μm,*表示与对照组相比P<0.05,#表示与ANRIL过表达组相比P<0.05。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1临床研究
本发明实施例纳入于山东第一医科大学附属省立医院就诊的成年CKD患者共60例及性别、年龄匹配的健康成年人45例,收集性别、年龄、血压等基本资料及血脂、肾功等生化指标,并用无创超声测定右肱动脉血流介导的血管舒张功能(Flow-mediateddilatation,FMD)评估入组者的血管内皮功能;同时,研究表明脑源性神经营养因子(BDNF)可能是心血管疾病的新型重要预测因子,ELISA方法检测其血浆水平。具体样本信息如表1所示。采集空腹血标本检测血浆中ANRIL表达水平,并分析ANRIL表达水平与肾功能、血管内皮功能之间的相关性;深入探讨ANRIL在CKD内皮功能损伤中的作用。具体过程及结果如下述实施例。
其中,CKD患者纳入标准:
(1)出现肾损伤标志大于三个月,如:蛋白尿、肾脏病理异常、影像学检查肾结构异常;
(2)或肾小球滤过率下降:eGFR<60ml/min/1.73m2大于3个月。
排除标准:
(1)无法提供知情同意;
(2)正在参与干预性临床试验;
(3)妊娠期或哺乳期妇女;
(4)排除急性肾损伤患者,包括:48小时内血肌酐升高≥26.5μmol/l,确认或推测7天内血肌酐较基础值升高≥50%;
(5)仅单纯性血尿:即尿沉渣镜检:红细胞≥3个/高倍镜视野,且为肾小球源性血尿,并不伴高血压、蛋白尿(包括微量白蛋白尿)且eGFR≥60mL/min/1.73m2;
(6)NYHA心力衰竭分级为III级或IV级,即有基础心脏疾病,体力活动明显受限,小于一般体力活动甚至休息状态下即出现疲乏、心悸、气喘或心绞痛症状;
(7)肝硬化、HIV感染或艾滋病;
(8)既往接受器官或骨髓移植;
(9)既往2年接受肿瘤化疗或烷化剂治疗;既往6个月内接受免疫抑制剂治疗;
(10)既往接受多于一月的透析治疗;
(11)排除系统性自身免疫性疾病所致肾炎;排除多囊肾等遗传性肾脏疾病;
(12)排除既往有糖尿病、高脂血症病史。
健康对照纳入人群纳入标准
既往无糖尿病、冠心病、高血压等慢性疾病病史,肾功、血脂、血糖、肝功等化验指标均在正常范围内。年龄、性别匹配。
该研究经山东第一医科大学附属山东省立医院伦理委员会批准,所有入组对象均签署知情同意书。
血管内皮功能检测
采用血流介导的内皮依赖性舒张功能(Flow-mediateddilatation,FMD)检测。步骤如下:采用彩色多普勒超声诊断仪测量肱动脉充血反应前后内径,探头频率14MH。CKD组和对照组人员仰卧位,右上肢裸露并肌肉放松、掌心向上外展15°;将高频超声探头置于上臂肘上3至7cm处,取肱动脉纵轴切面,当血管前后壁内膜显示最清楚时,测量肱动脉前后壁内膜之间的距离,测量5次,取平均值,得出静息状态下肱动脉舒张末内径基础值D0。做好标记,以备稍后充血反应后检测。于研究对象肘关节下2-3cm处缚一血压计,将血压计袖带充气加压至受试者的收缩压以上50mmHg,完全阻断血流5分种后迅速松开袖带,于30-180s内连续测量肱动脉反应性充血后内径值,取3个最大测量值,求平均值为D1。计算血流介导的肱动脉内径扩张率FMD%=(D1-D0)/D0×100%。
血浆lncRNA ANRIL检测
血浆RNA提取
应用游离RNA提取试剂盒(BIOG cfRNA Easy Kit,百代生物)提取血浆中RNA。步骤如下:
1)洗涤液准备:洗涤液A:21ml加入无水乙醇9ml,混匀;洗涤液B:9ml加入无水乙醇21ml,混匀。
2)取无RNA酶的1.5ml离心管,标记,加入200μl血浆样本,4μl RNA carrier混匀,继续加入300μl裂解液及20μl消化液,振荡混匀,56℃水浴10min;
3)加入1ml无水乙醇,颠倒混匀;
4)将吸附柱放于收集管内,标记,转移3)中溶液760μl至吸附柱内,静置2min,12000rpm4℃离心1min,弃掉收集管内废液;
5)转移3)中剩余溶液至收集管,重复上述步骤;
6)将吸附柱放回收集管内,加500μl洗涤液A至吸附柱内,12000rpm4℃离心1min,弃废液;
7)将吸附柱放回收集管内,加500μl洗涤液B至吸附柱内,静置2min,12000rpm4℃离心1min,弃废液;
8)将吸附柱放回收集管内,12000rpm4℃离心2min,弃废液;
9)另取1.5ml无RNA酶离心管,标记,将上述吸附柱防御其中,加入30μl洗脱液,静置3min,12000rpm4℃离心2min,收集RNA溶液;
10)超微量分光光度计测量RNA浓度。
逆转录
逆转录试剂盒:Evo M-MLV反转录试剂盒(AG,China),步骤如下:
1)去除基因组DNA,体系如下:
5x gDNA Clean Buffer | 2.0μl |
gDNA Clean Reagent | 1.0μl |
RNA and RNase Free dH2O | 7.0μl |
混匀,上机,42℃2min。
2)逆转录反应,体系如下:
逆转录程序:
所得cDNA于-20℃冰箱保存用于后续real-timePCR检测。
上述RTPrimerMix为OligoDt(18T)Primer与Random6mersPrimer混合物。
.real-timePCR
real-timePCR试剂盒:SYBRGreenProTaqHS预混型Qpcr试剂盒目的基因与内参扩增引物
real-timePCR反应体系
反应体系:
Real-timePCR反应程序如下:
lncRNA表达水平计算:对表达量RV=lg2(-ΔCt),ΔCt=Ct(lncRNA)-Ct(β-actin)
血浆BDNF检测
BDNF血浆浓度检测参照BDNFEnzyme-linkedimmunosorbentassay(ELISA)试剂盒(Elabscience,China)说明书进行。
操作步骤:
1)分别设定标准孔、空白孔和样本孔。标准孔加入100μL倍比稀释的标准品,空白孔加入100μL标准品&样本稀释液,其余孔加入100μL待测样本。给酶标板覆膜,37℃孵育90分钟。
2)甩尽孔内液体,每个孔中加入生物素化抗体工作液100μL,酶标板加上覆膜,37℃温育1小时。
3)甩尽孔内液体,在洁净的吸水纸上拍干。每孔加洗涤液350μL,浸泡1分钟,吸去或甩掉酶标板内的液体,拍干。重复此洗板步骤3次。
4)每孔加酶结合物工作液100μL,酶标板加上覆膜,37℃温育30分钟。
5)甩尽孔内液体,洗板5次。
6)每孔加底物溶液(TMB)90μL,酶标板加上覆膜,37℃避光孵育15分钟。
7)每孔加终止液50μL,终止反应。立即用酶标仪在450nm波长测量各孔的光密度(OD值)。根据标准曲线计算BDNF的浓度。BDNF检测灵敏度为18.75pg/ml。
统计分析
应用SPSS17.0进行统计学分析,符合正态分布的计量资料用均值±标准差(x±s)表示,t检验分析两组间统计学差异;不符合正态分布的计量资料采用四分位数间距(Q1-Q3)表示,非参数检验分析两组间统计学差异。计数资料用百分率(%)表示,卡方检验分析统计学差异。两变量之间相关性用Spearman相关分析。p<0.05时认为差异有统计学意义。
研究结果
本研究共纳入成年CKD患者60例及性别、年龄匹配的健康成年人45例。纳入人群临床资料详见表1。不同组别中血浆lncRNAANRIL水平、BDNF浓度、ANRIL表达与eGFR等结果如图1所示,结果显示,与对照组人群对比,CKD患者血浆中ANRIL水平明显增高(图1A),p=0.02,差异有统计学意义。Spearman相关分析检测CKD人群血浆ANRIL水平与eGFR关系,结果显示ANRIL表达与eGFR负相关(图1B)(r=-0.284,p=0.032);通过FMD检测无创评估血管内皮功能,CKD患者FMD检测结果较健康对照组低(5.52±3.79vs7.79±4.68)。进一步Spearman相关分析检测CKD患者血浆ANRIL水平与FMD相关性,结果显示ANRIL水平与FMD负性相关(图1C)(r=-0.464,p=0.002),提示ANRIL与CKD患者内皮功能障碍相关。
通过ELIS检测血浆BDNF水平,CKD患者血浆BDNF浓度也较低(813.94pg/mLvs2179.06pg/mL)(图1D),并与eGFR呈正相关(r=0.465,p<0.001)(图1E)。此外,CKD患者BDNF浓度与ANRIL水平呈负相关(r=-0.385,p=0.003)(图1F)。提示ANRIL可能与CKD患者的内皮功能障碍有关,BDNF可能在这一过程中发挥重要作用。
表1.纳入人群临床资料
注:SBP:systolicpressure,收缩压;DBP:diastolicpressure,舒张压;eGFR:estimatedglomerularfiltrationrate,估测肾小球滤过率;HDL-c:highdensitylipoproteincholesterol,高密度脂蛋白胆固醇;LDL-c:lowdensitylipoproteincholesterol,低密度脂蛋白胆固醇;FMD:Flow-mediateddilatation,血流介导的内皮依赖性舒张功能。BDNF:brain-derivedneurotrophicfactor,脑源性神经营养因子。
实施例2CKD患者血清或尿毒症毒素诱导ANRIL高表达
细胞培养:所用细胞为脐静脉内皮细胞。血清刺激:收集入组CKD患者血清及健康对照组人群血清,分别构建血清池,以基础培养基稀释至20%,用以作用于内皮细胞,刺激48小时后realtimePCR检测细胞中ANRIL表达水平(图2A),荧光原位杂交(FISH)检测ANRIL在细胞中定位(图2B)。
尿毒症毒素刺激:吲哚-3-乙酸(IAA)、高同型半胱氨酸(Hcy)、硫酸吲哚酚(IS):分析天平称取适量试剂,以双蒸水配制,0.22μm滤器过滤除菌;马尿酸(HA)以DMSO配制。以不同浓度毒素刺激内皮细胞,realtimePCR检测细胞中ANRIL表达水平
具体研究方法
real-timePCR
①细胞RNA提取
(1)取处理结束细胞,PBS冲洗2次,吸尽PBS,加入1mlTrzol,冰上静置7min;
(2)裂解结束转入已标记好的无RNA酶EP管中;
(3)加入200μl氯仿,上下颠倒混匀,冰上静置5min,待其分层;
(4)4℃离心12000rpm,15min,转移上层水相至新管;
(5)加入300μl异丙醇,涡旋,冰上静置5min;
(6)4℃离心12000rpm,10min;
(7)弃上清,加入1ml75%乙醇,涡旋震荡,4℃离心,7500rpm,5min;
(8)弃上清,加入1ml无水乙醇,涡旋震荡,4℃离心,7500rpm,5min;
(9)弃上清,室温晾干,根据沉淀大小,加适量体积DEPC水溶解沉淀;
(10)超微量分光光度计检测RNA浓度。
②反转录:同前。
③real-timePCR:同前。
real-timePCR相关引物
荧光原位杂交(FISH)
(1)六孔板预接种细胞爬片并完成刺激因素处理;
(2)固定:弃掉六孔板中培养基,PBS洗1遍,4%多聚甲醛固定15min,PBS洗三次,每次3min;
(3)通透:0.5%tritonX-100室温通透5min,PBS洗三次,每次3min。
(4)预杂交液37℃孵育30min。
(5)探针工作液孵育:避光条件下,取2μl20mMlncRNA探针加至100μl杂交液中。避光,弃去预杂交液,加探针工作液37℃过夜孵育。
(6)洗爬片,42℃预热杂交洗液;
避光,42℃杂交洗液Ⅰ(4xSSC),洗三次,每次5min;
避光,42℃杂交洗液Ⅱ(2xSSC),洗一次;
避光,42℃杂交洗液Ⅲ(1xSSC),洗一次;
避光,PBS洗细胞,常温,5min;
(7)DAPI染色,避光DAPI染核5min,PBS洗三次,每次5min;
(8)封片、观察:抗荧光淬灭封片剂封片,荧光显微镜观察。
基因调控效率验证
为研究ANRIL在内皮细胞损伤中的作用,构建特异性ANRILshRNA(Sh-ANRIL)慢病毒载体及ANRIL过表达载体(ANRIL),转染内皮细胞,进一步提取RNA,real-timePCR检测显示获得较好的敲低及过表达效率(图3A,B)。
为研究其作用机制,构建EZH2siRNA(si-EZH2),转染内皮细胞,westernblot检测其调控效率(图3C)。构建BDNF过表达质粒,转染内皮细胞,westernblot检测其调控效率(图3D)。
具体研究方法细胞转染:
慢病毒转染:
ANRIL过表达与敲低慢病毒载体由赛业生物科技公司(广州,中国)构建。
ANRIL敲低载体及其干扰载体序列如下:
ANRIL敲低载体(Sh-ANRIL):5’-CTCCGCTCCTCTTCTAGATTT-3’,如SEQ ID NO.5所示;
干扰载体(Scramble):5’-CCTAAGGTTAAGTCGCCCTCG-3’,,如SEQ ID NO.6所示;
细胞慢病毒感染:参照病毒说明书,选取荧光率80%以上且细胞形态及增殖状况正常所对应的MOI值为50。正式转染:
(1)细胞预先接种至六孔板内,转染当天细胞密度达到40%;
(2)胰蛋白酶消化其中一孔细胞,细胞计数;
(3)计算所需病毒体积:根据预实验所得最适MOI值及所计数细胞数目计算每孔所需病毒体积;
(4)六孔板换液,换为1ml新鲜培养基;
(5)对照病毒及目的基因病毒冰上融解,吹打混匀,按计算所需体积加至细胞中;
(6)混匀后,每孔分别加入polybrene,使其终浓度为5μg/ml,混匀,转入培养箱培养;
(7)6小时后观察细胞形态,可换液;若细胞状态尚可,可次日换液;
(8)48小时后高内涵成像仪观察细胞内荧光表达情况,并加嘌呤霉素进行药物筛选,获得稳转细胞株。
小干扰转染:
EZH2小干扰载体由吉满生物科技(上海,中国)有限公司构建合成,序列如下:
EZH2小干扰载体序列:Si-EZH2:5’-GAGGGAAAGUGUAUGAUAA-3’如SEQ ID NO.7所示;5’-UUAUCAUACACUUUCCCUC-3’如SEQ ID NO.8所示,
EZH2阴性对照载体序列:si-NC:5’-UUCUCCGAACGUGUCACGU-3’,如SEQ ID NO.9所示;5’-ACGUGACACGUUCGGAGAA-3’,如SEQ ID NO.10所示。
转染步骤
(1)si-RNA溶解:si-RNA为冻干粉状态,根据说明书加入DEPC水溶解,分装保存。
(2)细胞预先接种至六孔板内,转染当天细胞密度达到40%;
(3)稀释转染试剂:取高压灭菌1.5ml EP管标记后,加入125μl opti-MEM减血清培养基,继而加入3.75μl Lipofectamine 3000Transfection Reagent,混匀;
(4)稀释si-RNA:取高压灭菌1.5ml EP管标记后,分别加入125μl opti-MEM,继而分别加入5μl siRNA,轻柔混匀;
(5)将4)中悬液与3)混匀,室温孵育10-15min;
(6)将5)中悬液加至细胞中,混匀;转入培养箱中培养48-72小时后检验转染效率。
过表达质粒载体转染
BDNF过表达载体由吉玛生物公司构建并合成。
转染步骤
1)细胞预先接种至六孔板内,转染当天细胞密度达到40%;
2)稀释转染试剂:取高压灭菌1.5ml EP管标记后,加入125μl opti-MEM,继而加入3.75μl Lipo fectamine 3000Transfection Reagent,混匀;
3)稀释质粒:取高压灭菌1.5ml EP管标记后,分别加入125μl opti-MEM,继而分别加入2500ng质粒和5μL P3000试剂,混匀;
4)将2)中悬液与3)混匀,室温孵育10-15min;
5)将5)中悬液加至细胞中,混匀;转入培养箱中培养48-72小时后检验转染效率。
real-time PCR:步骤同前
western blot
(1)细胞总蛋白提取
将离心机提前预冷至4℃。弃掉6孔板里的培养基,预冷的PBS缓冲液润洗细胞2次,弃掉PBS,加入按照蛋白裂解液(RIPA裂解液:蛋白酶抑制剂:磷酸酶抑制剂为100:1:1),冰上裂解30min。用细胞刮取细胞后转移至1.5mL EP管中,4℃12000rpm离心20-30min。取离心后的上清至新1.5mL EP管中。
(2)蛋白浓度测定:BCA蛋白浓度测定试剂盒(碧云天,中国)
(3)蛋白变性
加入1/3体积4x蛋白上样缓冲液,混匀,95℃孵育5min变性,-20℃保存备用。
(4)根据目的蛋白分子量大小制胶。
(5)加样、电泳:80V恒压电泳30min后改为110V电泳
(6)转膜(湿转)
1)PVDF膜经甲醇浸泡激活,将黑白转膜架置于转膜液中,黑面为下,自下而上依次放好海绵、滤纸、凝胶、PVDF膜、滤纸、海绵,关闭转膜夹。将转膜夹放入转印框中,220mA恒流转膜适宜时间。
(7)封闭:转膜结束后,取出PVDF膜,置于5%脱脂牛奶中,室温摇床封闭1小时。
(8)封闭结束,TBST洗膜,5min/次,共三次,将条带转至已配好的抗体中,4℃摇床过夜。所用一抗:Anti-BDNF antibody(Abcam,ab108319,1:2000),Anti-KMT6/EZH2antibody(Abcam,ab228697,1:1000),β-actin Monoclonal Antibody(Proteintech,66009-1-Ig,1:5000)。
(9)二抗室温摇床孵育1小时,TBST洗膜,每次8min,共三次。
HRP-conjugated Affinipure Goat Anti-Rabbit IgG(SA00001-2,Proteintech),HRP-conjugated Affinipure Goat Anti-Mouse IgG(SA00001-1,Proteintech).
(10)显影:按1:1比例配制ECL发光液(Millipore),并由Amersham Imager 680(GE,Boston,MA,USA)成像
(11)条带分析:用Image J分析条带灰度值,对比目的蛋白条带与内参条带灰度值,分析不同处理组目的分子表达情况。
实施例3ANRIL介导内皮细胞功能障碍及线粒体裂变异常。
IS是诱导CKD内皮功能障碍的重要毒素,且其致ANRIL增加效果较为明显,故而选用IS进行后续机制探讨。为明确ANRIL在IS致内皮功能障碍中的作用,用Sh-ANRIL敲低内皮细胞中ANRIL的表达,继而用IS刺激内皮细胞,realtime PCR与western blot检测内皮细胞功能相关蛋白eNOS、VCAM-1、vWF表达水平;western blot检测内皮细胞线粒体分裂融合相关蛋白Drp1、Mfn2表达水平。结果显示,与对照组相比,IS刺激后内皮细胞功能相关蛋白eNOS表达降低,VCAM-1、vWF表达增加,线粒体分裂融合相关蛋白Drp-1表达增加,Mfn2表达减少;而Sh-ANRIL抑制内皮细胞中ANRIL的表达,可逆转上述蛋白表达异常(图4)。
具体步骤:
细胞转染:同前
realtime PCR:步骤同前。
real-time PCR相关引物
western blot:步骤同前。
所用一抗:eNOS(D9A5L)Rabbit mAb(Cell Signaling,32027,1:1000),Anti-VonWillebrand Factor antibody(Abcam,ab174290,1:2000),Anti-VCAM1antibody(Abcam,ab134047,1:1000),Anti-Drp1 antibody(Abcam,ab184247,1:1000),Anti-Mitofusin2antibody(Abcam,ab124773,1:1000),Anti-BDNF antibody(Abcam,ab108319,1:2000),β-actin Monoclonal Antibody(Proteintech,66009-1-Ig,1:5000)。
所用二抗:HRP-conjugated Affinipure Goat Anti-Rabbit IgG(Proteintech,SA00001-2,1:5000),HRP-conjugated Affinipure Goat Anti-Mouse IgG(Proteintech,SA00001-1,1:5000)。
实施例4ANRIL通过下调BDNF调控内皮细胞功能障碍。
Western blot检测IS刺激或ANRIL过表达后BDNF表达水平,结果显示IS刺激或ANRIL过表达均可使BDNF表达下调,用Sh-ANRIL敲低内皮细胞中ANRIL的表达可逆转其表达异常(图5A,B)。进一步检测BDNF作用,内皮细胞单独过表达ANRIL或者同时共表达ANRIL及BDNF,继而Westernblot检测调控表达后细胞中蛋白表达水平,结果显示,BDNF过表达可显著逆转内皮细胞以及线粒体关键蛋白的异常表达(图5C,D)。线粒体ROS荧光染色结果显示,BDNF质粒共转染细胞上调BDNF表达后,线粒体ROS的产生也减少(图5E)。提示ANRIL通过下调BDNF调控内皮细胞功能障碍。
具体步骤:
细胞转染:同前
realtimePCR:步骤同前。
westernblot:步骤同前。
线粒体ROS染色
1)试剂配制:线粒体超氧化物红色荧光探针Mitosox为粉末状固体,50μg每管;储存液配制:加入13μlDMSO溶解试剂;
2)六孔板预接种细胞爬片并完成刺激因素处理;
3)HBSS恢复至室温,润洗爬片1次;
4)固定:4%多聚甲醛固定15min,HBSS润洗爬片3次,每次4min;
5)封闭:封闭液(1%BSA并含0.05%Tween-20)室温封闭1小时;
6)封闭过程中配制工作液:储存液以HBSS按1:1000稀释;
7)HBSS润洗爬片2次,每次4min,加工作液,37℃避光孵育20min;
8)HBSS润洗爬片3次,每次4min,DAPI染核5min;
9)HBSS润洗爬片5次,每次4min;
10)抗荧光淬灭封片剂封片,荧光显微镜观察。
实施例5ANRIL通过募集EZH2调控BDNF表达。
通过RNApull-down结合WesternBlot检测ANRIL结合蛋白,结果显示,生物素标记的ANRIL序列可以下拉出EZH2,而ANRIL反义序列则无此效应,提示ANRIL可以与EZH2直接结合(图6.A)。RNA免疫沉淀(RIP)实验进一步验证其相互作用,结果显示EZH2抗体可以沉淀分离出ANRIL,且与空载组相比,过表达组ANRIL与EZH2结合明显增加(图6.B)。
EZH2是多梳蛋白PRC2复合物的重要组分,通过介导靶基因启动子区域组蛋白H3第27位赖氨酸的三甲基化(H3K27me3)修饰增加而诱导基因转录抑制,从而下调靶基因表达。本研究通过westernblot检测内皮细胞H3K27me3水平,结果显示ANRIL过表达可引起H3K27me3水平增加,而转染si-EZH2后,可逆转这一改变(图6C)。同时westernblot检测显示,ANRIL过表达可引起BDNF表达下调,干扰EZH2表达可使BDNF表达恢复(图6.D)。进一步通过染色质免疫共沉淀(ChIP)检测BDNF启动子区EZH2结合水平及组蛋白甲基化水平,结果显示上调ANRIL表达后,BDNF启动子区H3K27me3水平明显增加,且该启动子区EZH2结合明显增加(图6.E,F)。提示ANRIL通过募集EZH2调控BDNF抑制。
具体步骤:
RNA Pulldown
体外转录所用全长ANRIL表达载体由GenePharma Technology构建。使用MAXIscriptTMSP6/T7转录试剂盒(Invitrogen,AM1320)体外转录lncRNA。使用PierceTMRNA3’End Desthiobiotinylation Kit(Thermo Fisher Scientific,20163)进行RNA探针标记。使用IP裂解缓冲液(Thermo Fisher Scientific)提取蛋白。继而使用PierceMagnetic RNA-Protein Pull-Down Kit(Thermo Fisher Scientific,20164)进行RNApull down,RNA结合蛋白洗脱后变性并经Western blot检测目的蛋白富集情况。
Western blot所用一抗:Anti-KMT6/EZH2antibody(Abcam,ab228697,1:1000),GAPDH Monocl onal antibody(Proteintech,60004-1-Ig,1:5000)
所用二抗:HRP-conjugated Affinipure Goat Anti-Rabbit IgG(Proteintech,SA00001-2,1:5000),HRP-conjugated Affinipure Goat Anti-Mouse IgG(Proteintech,SA00001-1,1:5000)。
RIP
参照Magna RIP RNA Binding Protein Immunoprecipitation Kit(Millipore,17-701)说明手册进行RIP实验。
步骤:PBS润洗、刮取细胞,1500rpm4℃离心5min,收集沉淀,加入裂解液重悬(裂解液为RIP lysis buffer 100μl+proteinase inhibitor cocktail 0.5μl+RNaseinhibitor0.25μl,现用现配)。冰上孵育5min,-80℃冰箱过夜。制备磁珠(相应添加抗体EZH2、IgG均为5μg/每组沉淀样本)。取冻融裂解细胞,快速解冻,14000rpm4℃离心10min;取上清,加入所制备抗体/磁珠悬液,4℃摇动过夜孵育;另取10μl上清,标记“input”,-80℃保存。次日,蛋白酶K缓冲液消化蛋白,并进一步纯化RNA。Real time PCR检测RNA富集情况,RNA富集情况以“%input”表示。ANRIL引物同前。
细胞转染:同前
western blot:步骤同前。
所用一抗:Anti-Histone H3antibody(Abcam,ab1791,1:1000),Anti-Histone H3(tri methyl K27)antibody(Abcam,ab192985,1:1000),Anti-BDNF antibody(Abcam,ab108319,1:2000),β-actin Mon oclonal Antibody(Proteintech,66009-1-Ig,1:5000)。
所用二抗:HRP-conjugated Affinipure Goat Anti-Rabbit IgG(Proteintech,SA00001-2,1:5000),HRP-conjugated Affinipure Goat Anti-Mouse IgG(Proteintech,SA00001-1,1:5000)。
染色质免疫共沉淀(ChIP)
参照EZ-MagnaCHIPTMA/G试剂盒(Millipore,17-10086)说明手册进行ChIP测定。
步骤:将细胞于1%甲醛中固定10分钟以使蛋白质与DNA交联。用含有1X ProteaseInhibitor Cocktail II的细胞裂解缓冲液裂解细胞,4℃,800g离心5分钟,收集细胞沉淀。加入Nuclear Lysis Buffer重悬,超声处理以获取合适大小的染色体片段。4℃、12000g离心10min,取上清,分装并标记IP管,加入Dilution buffer,取其中1%作为“input”于4℃暂存,分别加免疫沉淀抗体(IgG、EZH2或H3K27me3)和20μL完全重悬的蛋白A/G磁珠,4℃摇动孵育过夜。次日,将洗脱缓冲液加至IP管和输入管中,在62℃孵育2小时继而95℃孵育10分钟来逆转蛋白质-DNA交联。通过Spin Columns纯化DNA。Real time PCR结合BDNF启动子区特异引物检测所沉淀DNA富集情况,DNA富集情况以“%input”表示。引物序列如下:
BDNF promoter(-753to-480):Forward-CACAGGGAGATGCAAGTTGA,如SEQ ID NO.19所示;
reverse-GAAAGGCACTCCCATTTCAG,如SEQ ID NO.20所示;
实施例6验证ANRIL通过EZH2/BDNF调控内皮细胞功能障碍。
通过细胞转染调控内皮细胞ANRIL、EZH2及BDNF表达,结果显示,干扰EZH2表达或上调BDNF表达,可逆转ANRIL高表达引起的eNOS及Mfn2低表达,且降低VCAM-1、vWF与Drp-1表达水平(图7A-C)。且与ANRIL过表达组相比,干扰EZH2表达或上调BDNF表达后,线粒体ROS水平降低(图7D)。
具体步骤
细胞转染:同前
western blot:步骤同前。
免疫荧光
1)种六孔板爬片,细胞刺激结束后,行细胞免疫荧光染色;
2)固定:弃掉六孔板中培养基,PBS洗一遍,加4%多聚甲醛固定15min;
3)弃掉多聚甲醛,PBS洗细胞,每次3min,共三次;
4)破膜:0.3%tritonX-100室温通透5min,PBS洗细胞,每次3min,共三次;
5)封闭:弃掉PBS,加5%BSA室温封闭30min;
6)一抗过夜:用PBS或5%BSA配制一抗,一抗4℃孵育过夜;
7)自4℃取出于室温静置半小时复温,PBS洗爬片三次;
8)二抗孵育:根据一抗选择适宜二抗,避光配制,37℃孵育避光90min,PBS润洗三次;
9)DAPI染核5min,PBS洗爬片五次,每次5min;
10)封片,抗荧光淬灭封片剂封片,荧光显微镜观察。
所用一抗:Anti-VCAM1antibody(Abcam,ab134047,1:1000),Anti-Mitofusin2antibody(Abcam,ab124773,1:1000)
所用二抗:Alexa594-conjugatedGoatAnti-RabbitIgG(Abcam,ab150080,1:200)
线粒体ROS染色:同前。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
1.ANRIL在制备慢性肾脏病相关心血管病变诊断试剂盒中的应用。
2.根据权利要求1所述的应用,其特征在于,ANRIL作为一种慢性肾脏病的血清标志物,与慢性肾脏病血管内皮功能障碍、线粒体裂变异常相关,患者血浆中ANRIL水平升高。
3.根据权利要求1所述的应用,其特征在于,以血清中ANRIL为检测指标,检测程序为:
预变性95℃5min
变性95℃10s
退火58℃10s
45个循环,扩增
延伸72℃10s
溶解曲线65-95℃5min。
4.一种慢性肾脏病诊断试剂盒,其特征在于,包括:检测引物和检测试剂;其中,所述引物序列如SEQ ID NO.1~SEQ ID NO.4所示;
ANRIL-F:5’-TACATCCGTCACCTGACACG-3’,如SEQ ID NO.1所示;
ANRIL-R:5’-ACGAGGGGAGCCAGGAATAA-3’,如SEQ ID NO.2所示;
β-actin-F:5’-GAAGAGCTACGAGCTGCCTGA-3’,如SEQ ID NO.3所示;
β-actin-R:5’-CAGACAGCACTGTGTTGGCG-3’,如SEQ ID NO.4所示。
5.根据权利要求4所述的一种慢性肾脏病诊断试剂盒,其特征在于,检测试剂包括:
6.一种引物,其特征在于,如SEQ ID NO.1~SEQ ID NO.4所示。
7.以ANRIL为作用靶点在制备治疗慢性肾脏病相关心血管并发症药物中的应用。
8.根据权利要求7所述的应用,其特征在于,以ANRIL为作用靶点具体为:抑制ANRIL表达可改善尿毒症毒素IS所致的内皮细胞损伤、改善尿毒症毒素IS所致的内皮细胞线粒体裂变异常、逆转分裂关键蛋白DRP-1和融合相关蛋白MFN2表达的异常。
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