CN111321148A - 一种用于子痫前期临床风险评估的标志基因及其应用 - Google Patents
一种用于子痫前期临床风险评估的标志基因及其应用 Download PDFInfo
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Abstract
一种用于子痫前期临床风险评估的标志基因及其应用,属于医学技术领域,以子痫前期患者和正常妊娠孕妇的胎盘组织标本为研究对象,提取胎盘组织滋养细胞中的RNA和蛋白,同时确定子痫前期滋养细胞自噬改变,明确ASPP2在调控滋养细胞自噬中的作用。本发明明确ASPP2作为子痫前期早期预警及诊断的关键靶基因。本发明的蛋白取材来自胎盘,样本获取简便,对患者无任何创伤,普遍容易接受,具有较强的推广性和实施性,在子痫前期的早期诊断与风险评估中具有极大的应用价值。
Description
技术领域
本发明属于医学技术领域,具体地说,涉及一种用于子痫前期临床风险评估的标志基因及其应用。
背景技术
子痫前期(Preeclampsia,PE)是妊娠期最为常见的并发症之一,是孕妇因妊娠后内环境改变导致胎盘功能障碍为主要病理特征的妊娠期特有且常见的胎盘源性疾病。据报道国内PE的发病率为9.4-10.4%,国外为7.5-12.8%,其所造成的孕产妇死亡数约占妊娠相关死亡总人数的10.5-16.8%,是孕产妇死亡的第二大原因。且自从2016年我国二胎政策实施后,全国孕产妇死亡率比去年同期增长30.6%,呈现逐年增多的趋势。除了蛋白尿外,妊娠高血压的孕妇罹患心血管疾病以及死亡的风险很高。相比正常孕妇,这些妊娠高血压母亲的胎儿则具有更高相关病症的发病风险,如胎儿宫内发育迟缓,早产和胎儿宫内死亡等。胎盘作为妊娠期特有的重要临时性器官,承载着母体营养传输和母胎屏障等重要功能,也是妊娠期母胎之间适应性调节的重要纽带,其发育的异常是PE等不良妊娠结局发生的原因。当妊娠终止胎盘娩出后,孕产妇血压及其他指标逐步恢复正常,提示其靶标应该在胎盘。子痫前期目前常用诊断方法有:血液、尿液、眼底检查,但方法传统,主要依靠疾病临床表现诊断,待诊断明确后,孕周较大,如果要消除症状只能通过终止妊娠。在治疗方面,临床上只能通过解痉、降压等对症治疗缓解病情,安全有效的药物极少;在有效治疗方案缺乏的情况下,终止妊娠是彻底治愈的唯一措施,而娩出的未足月儿及低出生体重儿为家庭、社会及国家带来沉重负担。因此,寻找一种子痫前期发生发展的早期分子诊断标志物,进而探讨靶向胎盘的妊娠疾病的防治策略,做到早期发现、早诊断、早治疗,这对全面提高人类生殖健康水平、出生人口素质乃至终生的健康水平都具有深远的意义。
细胞自噬(autophagy)是将细胞内受损、变性和衰老的蛋白质及细胞器运输到溶酶体内进行消化降解的过程,可分为自噬前体形成、自噬泡形成和自噬泡与溶酶体融合完成底物降解等三个阶段,近年来研究观察到滋养细胞自噬水平升高与胎盘功能受损密切相关,且贯穿于子痫前期全过程。关于滋养细胞自噬水平升高引起子痫前期的解释是,滋养细胞自噬功能受损,导致滋养细胞清除受损细胞器及多余蛋白质能力低下,致其取代螺旋动脉内皮细胞、降解血管平滑肌及弹力纤维的能力下降,另一方面,由于滋养细胞的自噬活性过强,使滋养细胞过度死亡,侵入螺旋小动脉能力减弱从而加快子痫前期的进程。同时认为导致胎盘滋养细胞自噬的机制还有氧化应激、母胎免疫反应异常和子宫螺旋小动脉“血管重铸”异常等,造成子宫-胎盘供血不足引发一系列临床症状。由于细胞自噬过程因位置、状态、环境的不同,生物功能也有所不同,并且自噬体膜来源、自噬溶酶体融合机制、自噬相关蛋白功能及自噬对细胞生存与死亡所发挥的双重作用等环节受多种因素调控,其具体情况与疾病的进程有关,且自噬诱导剂替代治疗还处于初期研究阶段,因此如能找到一种调控滋养细胞自噬的关键预警蛋白,可作为子痫前期的防治的有效靶点。
ASPP2(p53凋亡刺激蛋白2)于2001年被发现和鉴定,常通过P53依赖途径促进细胞凋亡而发挥生物学效应,在人类自噬系统的网络组成数据中通过核磁图谱分析推测出ASPP2能与自噬蛋白Beclin-1物理性结合;利用neoR基因(新霉素抗性基因)剔除鼠ASPP2基因的外显子后,发现ASPP2-/-(p53凋亡刺激蛋白2等位基因完全敲除)小鼠因胚胎早期的高死亡率而使存活率降低,ASPP2+/-小鼠胚胎中的成纤维细胞也出现G0/G1(DNA合成前期/DNA合成后期)细胞关卡,病理切片分析显示成纤维细胞自噬明显减少,尽管ASPP2+/-小鼠能够正常存活,但是随着年龄的增长,自发性肿瘤的发生率明显增高,这些研究表明ASPP2可通过调控细胞自噬参与其他疾病的发生发展。我们在子痫前期患者胎盘组织中观察到自噬水平升高,且ASPP2 mRNA(p53凋亡刺激蛋白2的信使单链核糖核酸)表达增加,在滋养细胞中,免疫印迹法检测到ASPP2表达增高,免疫荧光染色和透射电镜分析显示滋养细胞存在细胞自噬水平升高,暗示ASPP2是调控滋养细胞自噬的重要基因,可以作为研究子痫前期的新靶标。且过表达、干扰ASPP2蛋白表达后,滋养细胞自噬显著变化。由此可见,ASPP2是影响胎盘滋养细胞自噬的关键蛋白,可作为早期预警及诊断的靶标,具有重要的潜在价值。
发明内容
本发明的目的在于提供一种用于子痫前期临床风险评估的标志基因及其应用,该蛋白取材来自胎盘,样本获取简便,对患者无任何创伤,普遍容易接受,具有较强的推广性和实施性。
其具体技术方案为:
一种用于子痫前期临床风险评估的标志基因,所述的标志基因为ASPP2,即p53凋亡刺激蛋白2。
该标志基因的培育包括以下步骤:
(1) 查阅患者病历资料,收集正常孕妇及子痫前期孕妇胎盘组织;
(2) 制备胎盘组织电镜标本,观察滋养细胞自噬情况;
(3) 提取胎盘组织蛋白,并检测蛋白浓度,免疫印迹法检测胎盘组织自噬相关基因LC3(微管相关蛋白轻链3)、p62(选择性自噬接头蛋白)及ASPP2表达改变;
(4) 培养人源胎盘滋养细胞,自噬流双标腺病毒转染细胞缺氧处理后,观察自噬情况;
(5) 提取胎盘组织蛋白,并检测蛋白浓度,免疫印迹法检测滋养细胞自噬相关基因LC3、p62及ASPP2表达改变;
(6) 过表达、干扰ASPP2后,免疫印迹法检测ASPP2自身表达改变;
(7) 过表达、干扰ASPP2后,免疫印迹法检测滋养细胞自噬相关基因LC3及p62表达改变;
(8) 过表达、干扰ASPP2后,转染mRFP-GFP-LC3激光共聚焦观察滋养细胞自噬流变化;
进一步,子痫前期患者胎盘中自噬相关蛋白LC3II/I比值增高、p62表达降低,ASPP2表达增高且电镜观察自噬小体显著增加,细胞水平与其保持一致;
进一步,过表达ASPP2后,滋养细胞自噬相关蛋白LC3II/I比值增高、p62表达降低;干扰ASPP2后结果与之相反;
进一步,过表达、干扰ASPP2后,转染mRFP-GFP-LC3(红色荧光蛋白-绿色荧光蛋白-标记的微管相关蛋白轻链3腺病毒)激光共聚焦观察滋养细胞自噬水平增加;
本发明所述特异性子痫前期临床风险评估的标志基因在子痫前期早期预防及诊断过程中的应用。
与现有技术相比,本发明的有益效果:
1、本发明明确ASPP2作为子痫前期早期预警及诊断的关键靶基因。
2、本发明的蛋白取材来自胎盘,样本获取简便,对患者无任何创伤,普遍容易接受,具有较强的推广性和实施性。
附图说明
图1电镜观察子痫前期患者和正常妊娠患者胎盘组织自噬改变示意图。图中,PC为正常妊娠组;PE为子痫前期患者;白色箭头为自噬小体;n为细胞核。
图2为胎盘组织自噬相关基因LC3II/I、p62蛋白表达示意图。图中,PC为正常妊娠组;PE为子痫前期患者;LC3II为自噬体膜型微管相关蛋白轻链3;LC3I为胞浆型微管相关蛋白轻链3;p62为选择性自噬接头蛋白;β-actin为肌动蛋白;LC3II/I为自噬体膜型微管相关蛋白轻链3/胞浆型微管相关蛋白轻链3;Fold Change为变化倍数,与PC组比较,**P<0.01,*P<0.05。
图3为胎盘组织ASPP2蛋白表达示意图。图中,PC为正常妊娠组;PE为子痫前期患者;ASPP2为p53凋亡刺激蛋白2;β-actin为肌动蛋白;mRNA为信使单链核糖核酸;Protein为蛋白;ASPP2 expression levels为p53凋亡刺激蛋白2表达水平;与PC组比较,**P<0.01,*P<0.05。
图4为自噬双标腺病毒转染人源胎盘滋养细胞,电镜观察自噬情况示意图。图中,Normoxia为正常组;Hypoxia为缺氧组;RFP为红色荧光;GFP为绿色荧光;Merge为合并;与PC组比较,**P<0.01,*P<0.05。
图5为滋养细胞自噬相关基因LC3II/I、p62蛋白改变示意图。图中,LC3II/I为自噬体膜型微管相关蛋白轻链3/胞浆型微管相关蛋白轻链3;p62为选择性自噬接头蛋白;Normoxia为正常组;Hypoxia为缺氧组;LC3I为胞浆型微管相关蛋白轻链3;β-actin:肌动蛋白;Fold Change为变化倍数;与PC组(正常妊娠组)比较,**P<0.01,*P<0.05。
图6为过表达、干扰ASPP2后,免疫印迹法检测ASPP2自身表达改变示意图。图中,Control为正常对照组;p-GFP为过表达阴性对照组;p-ASPP2为p53凋亡刺激蛋白2过表达组;Ad-shNC为干扰腺病毒阴性对照组;Ad-shASPP2-1为p53凋亡刺激蛋白2干扰腺病毒1;Ad-shASPP2-2为p53凋亡刺激蛋白2干扰腺病毒2;Ad-shASPP2-3为p53凋亡刺激蛋白2干扰腺病毒3;ASPP2为p53凋亡刺激蛋白2;mRNA为信使单链核糖核酸;Protein为蛋白;RelativeASPP2 levels in cells为细胞中p53凋亡刺激蛋白2的表达;与PC组比较,**P<0.01,*P<0.05。
图7为过表达、干扰ASPP2后,免疫印迹法检测滋养细胞自噬相关基因LC3及p62表达改变示意图。图中,ASPP2为p53凋亡刺激蛋白2; mRFP-GFP-LC3为红色荧光蛋白-绿色荧光蛋白-标记的微管相关蛋白轻链3腺病毒;p-NC为过表达阴性对照组;p-ASPP2为p53凋亡刺激蛋白2过表达组;sh-NC为干扰腺病毒阴性对照组;sh-ASPP2为p53凋亡刺激蛋白2干扰腺病毒;LC3II为自噬体膜型微管相关蛋白轻链3;LC3I为胞浆型微管相关蛋白轻链3;p62为选择性自噬接头蛋白;β-actin为肌动蛋白;LC3II/I为自噬体膜型微管相关蛋白轻链3/胞浆型微管相关蛋白轻链3;LC3B II/I protein expression levels为自噬体膜型微管相关蛋白轻链3/胞浆型微管相关蛋白轻链3 的表达水平;p62 protein expression levels为选择性自噬接头蛋白的表达水平;与PC组比较,**P<0.01,*P<0.05。
图8过表达、干扰ASPP2后,转染mRFP-GFP-LC3激光共聚焦观察滋养细胞自噬流变化示意图。图中,p-NC为过表达阴性对照组;p-ASPP2为p53凋亡刺激蛋白2过表达组;sh-NC为干扰腺病毒阴性对照组;sh-ASPP2为p53凋亡刺激蛋白2干扰腺病毒;RFP为红色荧光;GFP为绿色荧光;Merge为合并;与PC组比较,**P<0.01,*P<0.05。
具体实施方式
下面结合附图和具体实施方案对本发明的技术方案作进一步详细地说明。
1 材料准备
1.1主要仪器与试剂
人源胎盘滋养细胞株(HTR-8/SVneo)购自于上海瑞鹿生物科技有限公司;总RNA提取试剂盒(北京天根生化科技有限公司);全蛋白提取试剂盒、BCA蛋白含量检测试剂盒(南京凯基生物有限公司);ASPP2、LC3、p62抗体(美国Abcam公司);辣根过氧化物酶标记的山羊抗兔IgG(北京中杉金桥公司);超净工作台(中国苏州安泰空气技术有限公司);实时荧光定量PCR仪(上海Funglyn);酶标仪(美国伯腾有限公司);普通PCR仪、凝胶成像系统(美国Bio-Rad有限公司);1640培养基、胎牛血清(美国Gibco有限公司);自噬流双标腺病毒(mRFP-GFP-LC3双标腺病毒)、ASPP2干扰腺病毒(上海汉恒生物科技有限公司);ASPP2过表达质粒(上海吉凯生物科技有限公司)。
2 标志基因的培育方法
2.1临床标本的选择
宁夏国龙妇产医院手术终止妊娠后采集的胎盘组织。所有实验严格按照宁夏医科大学临床研究伦理委员会批准的方案进行,并获得所有患者的知情同意。子痫前期患者(PE,n=45)根据美国妇产科学院临床病理标准进行选择。胎盘组织取自年龄匹配的正常妊娠组(PC, n=40),健康孕妇分娩,无医疗病史或使用药物,并接受产前护理作为对照。排除标准:本研究不包括多胎妊娠、胎儿先天性畸形/染色体异常、近期感染、抗磷脂抗体、孕期创伤、药物或酒精滥用、既往高血压、有血栓性血友病史或接受过抗凝或抗聚集治疗或吸烟的妇女。待患者分娩后,采集胎盘标本,制备电镜标本,提取组织RNA及蛋白,用于后续检测。
2.2人胎盘滋养细胞的培养
用含有10%的胎牛血清及1%的青链霉素双抗的RPMI 1640培养液培养人胎盘滋养细胞(HTR-8/SVneo),置于37℃、含5% CO2的培养箱中。当细胞密度达到70%到80%时,缺氧处理,其中常氧培养的细胞为Normoxia组,缺氧处理的滋养细胞为Hypoxia组,干预细胞48h后收集细胞用于后续实验。
2.3胎盘组织及细胞的电镜标本制备
将胎盘组织切成1×1×1mm左右大小。收集好的HTR-8/SVneo细胞PBS洗涤,胰酶消化,离心收集。然后,用含2%多聚甲醛和2.5%戊二醛的固定缓冲液在0.1M PBS中固定组织和细胞。嵌入后,样品被切成0.12μm部分和沾2%醋酸双氧铀和4%柠檬酸铅。用透射电镜(Zeiss,Oberkochen,德国)观察人胎盘和细胞迷宫,用2%乙酸铀酰和4%柠檬酸铅作对照。
2.3免疫印迹法检测自噬相关蛋白LC3、p62及ASPP2表达改变
取胎盘组织和HTR-8/SVneo细胞,分别在提取缓冲液(NP-40)、100 ng/mlPSMF中裂解,冰裂解10 min。上清液采用BCA蛋白法测定蛋白浓度,冷冻-80℃保存。30µg蛋白质在8%或12%分离聚丙烯酰胺SDS- PAGE凝胶和转移到0.22µmPVDF膜。在添加5%脱脂牛奶的PBST中室温封膜2h, 4℃孵育过夜,在封膜缓冲液中稀释一次抗体。用PBST冲洗膜10 min,共3次,二抗室温孵育2h,并使用增强化学发光法检测蛋白质。采用Image Lab5.0软件进行密度计分析。
2.4 qRT-PCR检测ASPP2mRNA表达
使用RNA分离试剂盒从人胎盘组织和HTR-8/SVneo细胞中分离出总RNA,然后根据制造商的协议,使用逆转录试剂盒进行逆转录。qRT-PCR采用SYBR Green Real-Time PCRMaster Mix试剂盒对ASPP2 mRNA表达量用FTC3000 qRT-PCR仪器进行检测分析。β-actin被用作控制规范化。实验结果的计算按照如下公式:检测目的基因的相对表达量=2-△△Ct,其中,ΔΔCt=[CtGI(检测样品)-CtGAPDH(检测样品)]-[CtGI(校正样本)–CtGAPDH(校正样本)]。GI是指所测目的基因,Ct是指检测到的反应体系中荧光信号强度,校正样本指的是所有被选做能够代表1倍所测目的基因表达量的样本。应用这一方法能直接获得目的基因相对于管家基因的定量结果。
2.5 利用串联MRFP-GFP-LC3评估自噬通量
在感染mRFP-GFP-LC3腺病毒的HTR-8/SVneo细胞中通过LC3聚集监测自噬囊泡的形成。将HTR-8/SVneo细胞接种于共聚焦培养皿上,以60%的合流率感染MRFP-GFP-LC3腺病毒。4h后,移除腺病毒后,缺氧培养细胞。串联MRFP-GFP-LC3蛋白在pH7.0时显示红色(MRFP)和绿色(GFP)荧光。黄色(RFP和GFP)点表示自噬小体的形成,而游离红色点(仅RFP)表示自噬小体与溶酶体融合,其中酸性pH淬灭GFP荧光。以红/黄斑点的相对比值作为自噬通量的指标。
3. 统计学处理
使用GraphPad Prism 6.0进行统计分析。除非另有说明,每个条形图均以均值±标准差(SD)表示。将基因表达归一化为稳定表达的β-肌动蛋白,并计算为相对变化。在选定的实验中,适当地使用单因素方差分析,然后使用Student-Newman-Kaul‘s test(用于多组之间的比较)或unpaired Student’s test(在两组之间)。p值小于0.05被认为是有统计学意义的。
4 结果
4.1子痫前期患者(PE)和正常妊娠患者(PC)胎盘组织微观结构自噬改变
透射电镜观察人体胎盘标本自噬改变,发现在PE妊娠胎盘中的自噬小体结构(双膜外观和液泡内的细胞质内容物)显著增加,而PC妊娠的自噬小体减少(图1)。
4.2胎盘组织自噬相关基因LC3II/I、p62蛋白表达
为了评价子痫前期患者胎盘组织中自噬的发生情况,采用Western blotting检测了LC3B-I转化为LC3B-II(自噬激活标志)和p62表达(胎盘床活检标本中自噬抑制的标志)。与PC相比,PE妊娠胎盘中LC3-II的量增加,而p62的表达降低,差异有显著性(图2)。
4.3胎盘组织ASPP2蛋白表达
考虑到ASPP2(1-123)的N-末端含有与ATG12和LC3结构高度相似的泛素折叠共享基序,我们推测ASPP2可能是PE中自噬的关键调节因子。qRT-PCR和免疫印迹分析均显示缺氧条件下子痫前期胎盘和HTR8/SVneo细胞中ASPP2的表达显著,差异有显著性(图3)。
4.4自噬流观察培养人源胎盘滋养细胞自噬情况
利用pH敏感的串联mRFP-GFP-LC3腺病毒载体感染HTR8/SVneo细胞,以监测自噬诱导的点状形成。与常氧相比,缺氧时HTR8/SVneo细胞中游离红色和黄色斑点均显着增加,表明自噬体和自溶酶体均增加。数据表明,在体外和体内缺氧条件下,滋养细胞的自噬明显被激活(图4)。
4.5滋养细胞自噬相关基因LC3II/I、p62蛋白改变
缺氧可导致HTR8/SVneo细胞中LC3B-II/I水平的升高和p62蛋白表达的显著降低,差异有显著性(图5)。
4.6过表达、干扰ASPP2后,免疫印迹法检测ASPP2自身表达改变
与对照组相比,ASPP2干扰腺病毒质粒3效果最显著,ASPP2蛋白和mRNA水平较对照组显著降低,后续实验都将选用干扰腺病毒3。同时,ASPP2过表达组中ASPP2蛋白和mRNA水平较对照组明显升高,差异有统计学意义(P<0.05)。
4.7过表达、干扰ASPP2后,免疫印迹法检测滋养细胞自噬相关基因LC3及p62表达改变
正如我们所预期的,与阴性对照相比,在ASPP2过表达的HTR8/SVneo细胞中同时观察到LC3BII/I的上调和p62的抑制,当ASPP2敲除时,相较于对照组LC3II/I以及p62蛋白水平变化相反,差异具有统计学意义。
4.8过表达、干扰ASPP2后,转染mRFP-GFP-LC3激光共聚焦观察滋养细胞自噬流变化
此外,用MRFP-GFP-LC3串联标记腺病毒载体感染HTR8/SVneo细胞后观察到斑点形成,以直接监测ASPP2调节的自噬过程。正如预期的那样,ASPP2可以增加自噬-溶酶体融合过程,ASPP2的敲除逆转了这个改变(图8)。
5 结论
本发明的目的在于提供一种用于子痫前期临床风险评估的标志基因及其应用,该蛋白取材来自胎盘,样本获取简便,对患者无任何创伤,普遍容易接受,具有较强的推广性和实施性。
滋养细胞自噬是调控子痫前期发生发展的关键环节,贯穿疾病的始终。由于孕妇的特殊性,无法彻底有效的治疗该疾病,目前,仅在胎儿及其附属物(胎盘)娩出后,临床症状才会缓解消失。而ASPP2作为胎盘中调控滋养细胞凋亡的关键蛋白,能够有效的改善滋养细胞自噬水平,可作为一种用于子痫前期临床风险评估的标志基因及其应用,具有较强的推广性和实施性。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序 列 表
ASPP2引物序列
Forward: 5’- ATTGAATCAAGAGCAGAATGCC-3’
Reverse: 5’- CAGCTCATTAACACGCTTATCC-3’
ASPP2蛋白序列
MMPMFLTVYL SNNEQHFTEV PVTPETICRD VVDLCKEPGE SDCHLAEVWC
GSERPVADNE RMFDVLQRFG SQRNEVRFFL RHERPPGRDI VSGPRSQDPS
LKRNGVKVPG EYRRKENGVN SPRMDLTLAE LQEMASRQQQ QIEAQQQLLA
TKEQRLKFLK QQDQRQQQQV AEQEKLKRLK EIAENQEAKL KKVRALKGHV
EQKRLSNGKL VEEIEQMNNL FQQKQRELVL AVSKVEELTR QLEMLKNGRI
DSHHDNQSAV AELDRLYKEL QLRNKLNQEQ NAKLQQQREC LNKRNSEVAV
MDKRVNELRD RLWKKKAALQ QKENLPVSSD GNLPQQAASA PSRVAAVGPY
IQSSTMPRMP SRPELLVKPA LPDGSLVIQA SEGPMKIQTL PNMRSGAASQ
TKGSKIHPVG PDWSPSNADL FPSQGSASVP QSTGNALDQV DDGEVPLREK
EKKVRPFSMF DAVDQSNAPP SFGTLRKNQS SEDILRDAQV ANKNVAKVPP
PVPTKPKQIN LPYFGQTNQP PSDIKPDGSS QQLSTVVPSM GTKPKPAGQQ
PRVLLSPSIP SVGQDQTLSP GSKQESPPAA AVRPFTPQPS KDTLLPPFRK
PQTVAASSIY SMYTQQQAPG KNFQQAVQSA LTKTHTRGPH FSSVYGKPVI
AAAQNQQQHP ENIYSNSQGK PGSPEPETEP VSSVQENHEN ERIPRPLSPT
KLLPFLSNPY RNQSDADLEA LRKKLSNAPR PLKKRSSITE PEGPNGPNIQ
KLLYQRTTIA AMETISVPSY PSKSASVTAS SESPVEIQNP YLHVEPEKEV
VSLVPESLSP EDVGNASTEN SDMPAPSPGL DYEPEGVPDN SPNLQNNPEE
PNPEAPHVLD VYLEEYPPYP PPPYPSGEPE GPGEDSVSMR PPEITGQVSL
PPGKRTNLRK TGSERIAHGM RVKFNPLALL LDSSLEGEFD LVQRIIYEVD
DPSLPNDEGI TALHNAVCAG HTEIVKFLVQ FGVNVNAADS DGWTPLHCAA
SCNNVQVCKF LVESGAAVFA MTYSDMQTAA DKCEEMEEGY TQCSQFLYGV
QEKMGIMNKG VIYALWDYEP QNDDELPMKE GDCMTIIHRE DEDEIEWWWA
RLNDKEGYVP RNLLGLYPRI KPRQRSLA
序列表
<110> 宁夏医科大学总医院
<120> 一种用于子痫前期临床风险评估的标志基因及其应用
<140> 2020100231514
<141> 2020-01-09
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3432
<212> DNA
<213> 凋亡刺激蛋白2(human)
<400> 1
ggggccgcag tctggccacc cgcttccatg cggttcgggt ccaagatgat gccgatgttt 60
cttaccgtgt atctcagtaa caatgagcag cacttcacag aagttccagt tactccagaa 120
acaatatgca gagacgtggt ggatctgtgc aaagaacccg gcgagagtga ttgccatttg 180
gctgaagtgt ggtgtggctc tgaacgtcca gttgcggata atgagcgaat gtttgatgtt 240
cttcaacgat ttggaagtca gaggaacgaa gttcgcttct tccttcgtca tgaacgcccc 300
cctggcaggg acattgtgag tggaccaaga tctcaggatc caagtttaaa aagaaatggt 360
gtaaaagttc ctggtgaata tcgaagaaag gagaacggtg ttaatagtcc taggatggat 420
ctgactcttg ctgaacttca ggaaatggca tctcgccagc agcaacagat tgaagcccag 480
caacaattgc tggcaactaa ggaacagcgc ttaaagtttt tgaaacaaca agatcagcga 540
caacagcaac aagttgctga gcaggagaaa cttaaaaggc taaaagaaat agctgagaat 600
caggaagcta agctaaaaaa agtgagagca cttaaaggcc acgtggaaca gaagagacta 660
agcaatggga aacttgtgga ggaaattgaa cagatgaata atttgttcca gcaaaaacag 720
agggagctcg tcctggctgt gtcaaaagta gaagaactga ccaggcagct agagatgctc 780
aagaacggca ggatcgacag ccaccatgac aatcagtctg cagtggctga gcttgatcgc 840
ctctataagg agctgcagct aagaaacaaa ttgaatcaag agcagaatgc caagctacaa 900
caacagaggg agtgtttgaa taagcgtaat tcagaagtgg cagtcatgga taagcgtgtt 960
aatgagctga gggaccggct gtggaagaag aaggcagctc tacagcaaaa agaaaatcta 1020
ccagtttcat ctgatggaaa tcttccccag caagccgcgt cagccccaag ccgtgtggct 1080
gcagtaggtc cctatatcca gtcgtctact atgcctcgga tgccctcaag gcctgaattg 1140
ctggtgaagc cagccctgcc ggatggttcc ttggtcattc aggcttcaga ggggccgatg 1200
aaaatacaga cactgcccaa catgagatct ggggctgctt cacaaactaa aggctctaaa 1260
atccatccag ttggccctga ttggagtcct tcaaatgcag atcttttccc aagccaaggc 1320
tctgcttctg tacctcaaag cactgggaat gctctggatc aagttgatga tggagaggtt 1380
ccgctgaggg agaaagagaa gaaagtgcgt ccgttctcaa tgtttgatgc agtagaccag 1440
tccaatgccc caccttcctt tggtactctg aggaagaacc agagcagtga agatatcttg 1500
cgggatgctc aggttgcaaa taaaaatgtg gctaaagtac cacctcctgt tcctacaaaa 1560
ccaaaacaga ttaatttgcc ttattttgga caaactaatc agccaccttc agacattaag 1620
ccagacggaa gttctcagca gttgtcaaca gttgttccgt ccatgggaac taaaccaaaa 1680
ccagcagggc agcagccgag agtgctgcta tctcccagca taccttcggt tggccaagac 1740
cagacccttt ctccaggttc taagcaagaa agtccacctg ctgctgccgt ccggcccttt 1800
actccccagc cttccaaaga caccttactt ccacccttca gaaaacccca gaccgtggca 1860
gcaagttcaa tatattccat gtatacgcaa cagcaggcgc caggaaaaaa cttccagcag 1920
gctgtgcaga gcgcgttgac caagactcat accagagggc cacacttttc aagtgtatat 1980
ggtaagcctg taattgctgc tgcccagaat caacagcagc acccagagaa catttattcc 2040
aatagccagg gcaagcctgg cagtccagaa cctgaaacag agcctgtttc ttcagttcag 2100
gagaaccatg aaaacgaaag aattcctcgg ccactcagcc caactaaatt actgcctttc 2160
ttatctaatc cttaccgaaa ccagagtgat gctgacctag aagccttacg aaagaaactg 2220
tctaacgcac caaggcctct aaagaaacgt agttctatta cagagccaga gggtcctaat 2280
gggccaaata ttcagaagct tttatatcag aggaccacca tagcggccat ggagaccatc 2340
tctgtcccat catacccatc caagtcagct tctgtgactg ccagctcaga aagcccagta 2400
gaaatccaga atccatattt acatgtggag cccgaaaagg aggtggtctc tctggttcct 2460
gaatcattgt ccccagagga tgtggggaat gccagtacag agaacagtga catgccagct 2520
ccttctccag gccttgatta tgagcctgag ggagtcccag acaacagccc aaatctccag 2580
aataacccag aagaaccaaa tccagaggct ccacatgtgc ttgatgtgta cctggaggag 2640
taccctccat acccaccccc accataccca tctggggagc ctgaagggcc cggagaagac 2700
tcggtgagca tgcgcccgcc tgaaatcacc gggcaggtct ctctgcctcc tggtaaaagg 2760
acaaacttgc gtaaaactgg ctcagagcgt atcgctcatg gaatgagggt gaaattcaac 2820
ccccttgctt tactgctaga ttcgtctttg gagggagaat ttgaccttgt acagagaatt 2880
atttatgagg ttgatgaccc aagcctcccc aatgatgaag gcatcacggc tcttcacaat 2940
gctgtgtgtg caggccacac agaaatcgtt aagttcctgg tacagtttgg tgtaaatgta 3000
aatgctgctg atagtgatgg atggactcca ttacattgtg ctgcctcatg taacaacgtc 3060
caagtgtgta agtttttggt ggagtcagga gccgctgtgt ttgccatgac ctacagtgac 3120
atgcagactg ctgcagataa gtgcgaggaa atggaggaag gctacactca gtgctcccaa 3180
tttctttatg gagttcagga gaagatgggc ataatgaata aaggagtcat ttatgcgctt 3240
tgggattatg aacctcagaa tgatgatgag ctgcccatga aagaaggaga ctgcatgaca 3300
atcatccaca gggaagacga agatgaaatc gaatggtggt gggcgcgcct taatgataag 3360
gagggatatg ttccacgtaa cttgctggga ctgtacccaa gaattaaacc aagacaaagg 3420
agcttggcct ga 3432
<210> 2
<211> 22
<212> DNA
<213> 上游引物(human)
<400> 2
agcttgatcg cctctataag ga 22
<210> 3
<211> 26
<212> DNA
<213> 下游引物(human)
<400> 3
ccctcagctc agctcattaa cacgct 26
Claims (4)
1.一种用于子痫前期临床风险评估的标志基因,所述的标志基因为ASPP2,即p53凋亡刺激蛋白2。
2.一种用于子痫前期临床风险评估的标志基因,其特征在于,该标志基因培育包括以下步骤:
(1) 收集人体胎盘标本,明确胎盘组织自噬改变;
(2) 复制细胞模型,缺氧模拟人体病理环境;
(3) 提取组织及细胞的RNA及蛋白,检测ASPP2的表达改变;
(4) 转染、干扰ASPP2过表达质粒及干扰腺病毒,检测自噬相关蛋白的表达,并检测自噬流的变化情况,明确ASPP2对滋养细胞自噬的调控作用。
3.根据权利要求1所述的一种用于子痫前期临床风险评估的标志基因,其特征在于,子痫前期患者胎盘组织ASPP2蛋白显著增高,自噬相关蛋白LC3BII/I增加、p62降低。
4.根据权利要求1所述一种用于子痫前期临床风险评估的标志基因的应用,标志基因ASPP2应用在子痫前期临床风险评估中。
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