CN111172161B - 一种长非编码rna及其在诊断/治疗子痫前期中的应用 - Google Patents
一种长非编码rna及其在诊断/治疗子痫前期中的应用 Download PDFInfo
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Abstract
本发明属于基因工程领域，特别涉及PDIA3P1在制备诊断子痫前期及靶点药物治疗中的应用；在子痫前期孕妇胎盘组织中PDIA3P1的下调与子痫前期的发生发展有关，低表达水平的PDIA3P1与子痫前期发病机制有着密切的关系，通过改变PDIA3P1的表达对子痫前期孕妇滋养细胞的增殖、凋亡、侵袭、迁移等产生影响，增强PDIA3P1表达能够促进滋养细胞增殖、侵袭和迁移。
Description
技术领域
本发明属于基因工程领域，特别涉及长非编码RNA-PDIA3P1在诊断及制备治疗子痫前期药物的应用。
背景技术
子痫前期是全球最常见妊娠期合并症之一，是妊娠期并发症相关死亡的主要原因，世界卫生组织统计报道表明，子痫前期或者子痫直接导致全球每年约14%(约8500)的孕产妇死亡，其是胎儿早产和胎儿生长受限以及孕产妇死亡最常见的原因之一。尽管目前内科治疗得到了长足的发展，但病人总体发生率依然较高。随着测序技术和分子生物学的快速发展，基因诊断和分子靶向治疗成为子痫前期治疗中一个热点问题。因此，研究参与子痫前期的发生发展和转移的的分子机制，对制定特定的诊断方法和个性化的治疗策略至关重要。
过去的十年里，基于快速出现的高通量测序的基因表达分析技术和生物信息学推动了大规模的人类基因组学的研究，进而发现了非编码RNAs。人类基因组中只有2%的编码转录成蛋白，而绝大多数转录成非编码RNAs，包括小分子核糖核酸、长非编码RNA(lncRNAs)和假基因。最近，miRNA在细胞进程的各个方面的作用已经得到证实，然而，lncRNAs的功能研究不是很透彻。ENCODE计划中GENECODE研究组新数据显示有成千上万的lncRNAs，但只有其中的一些有生物学功能。有趣的是，这些lncRNAs通过染色质重塑参与调控多种细胞进程，包括重组干细胞多能性，父母的印记和肿瘤细胞的扩散和转移以及表观遗传修饰和吸附miRNAs。
最近，大量的研究显示，异常lncRNAs表达发挥不同的生物学机制参与多种人类疾病发生。例如，lncRNA ROR可通过抑制甲基转移酶G9A，促进TESC的启动子区H3K9去甲基化参与肿瘤发生。同时，AOC4P通过与波形蛋白绑定和促进其降解，抑制上皮间质转化（EMT）从而抑制肝细胞癌转移。此外，上调的SPRY4-IT1通过与HUR绑定，抑制子痫前期滋养细胞增殖，迁移和血管形成能力。这些发现表明lncRNAs在人类疾病尤其是子痫前期的发生发展过程中扮演至关重要的角色。因此，发现更多的子痫前期相关的lncRNAs并研究他们的生物功能和机制，对更好地理解子痫前期发生发展的的分子生物学有重要的意义。
PDIA3P1是一条长度为2099nt的lncRNA，其序列如序列1所示，其位于人类染色体1q21.1。我们发现PDIA3P1在子痫前期孕妇胎盘组织中较正常孕妇胎盘中表达量下调。在过表达或敲除PDIA3P1后，研究了PDIA3P1在子痫前期发生和发展的作用并研究了PDIA3P1在子痫前期孕妇滋养细胞中的相关靶基因的功能。
发明内容
本发明的目的是提供PDIA3P1在诊断子痫前期及在制备治疗子痫前期药物中的应用。
本发明涉及一种长链非编码RNA，其核苷酸序列为SEQ ID NO: 1所示。
一种长链非编码RNA在制备治疗子痫前期药物中的应用；
一种长非编码RNA在制备诊断子痫前期试剂中的应用；
一种检测RP11-7K24.3的引物，即：SEQ ID NO: 2 R PDIA3P1 FATGGGCCTGTGAAGGTAGTG，SEQ ID NO: 3：PDIA3P1 R GTGGCATCCATCTTGGCTAT；
一种干扰RP11-7K24.3的siRNA，即：SEQ ID NO: 4 si- PDIA3P1 1#CATTAGTGATAAAGATGCCTCTATA，SEQ ID NO: 5 si- PDIA3P1 2#GATAACGGAGATGGTATCATCTTAT；
一种试剂盒，包括所述引物；
一种包括所述长链非编码RNA的药物组合物；
所述引物在制备诊断子痫前期试剂中的应用；
所述药物组合物在制备治疗子痫前期药物中的应用。
所述药物组合物，其中还包括辅料。辅料包括：（lip2000, Opti-mem培养液, PBS磷酸缓冲盐溶液）。
所述试剂盒，所述引物中的浓度分别为10mol/L。
技术方案
通过qPCR筛查临床组织中PDIA3P1的差异表达，发现在子痫前期孕妇胎盘组织中PDIA3P1的表达量较正常孕妇胎盘中表达量低。猜想：PDIA3P1是否参与了子痫前期疾病的发病过程。
随后选用国际认可的正常滋养细胞作为实验研究对象，设计PDIA3P1的干扰序列，以lip2000为转染载体将干扰序列转入细胞后模拟子痫前期疾病疾病的发病过程。通过检测在干扰序列转入细胞后细胞的功能如增殖，凋亡，血管形成能力等。从而证明在正常的滋养细胞HTR-8/SVneo中敲低PDIA3P1的表达，影响了细胞的功能，诱导或加速子痫前期疾病的发病进程。相反，构建PDIA3P1质粒，正反验证PDIA3P1在滋养细胞HTR-8/SVneo中的功能。
通过转录组测序，检测PDIA3P1的可能参与细胞功能（如增殖，凋亡或血管形成）的相关下游靶基因，随后对PDIA3P1调控机制的初步探讨，通过FISH等手段发现PDIA3P1更多的存在于滋养细胞的细胞核中，考虑PDIA3P1可能在转录后水平调控相应的靶基因，通过RNA免疫共沉淀和DNA免疫共沉淀实验检测并验证PDIA3P1通过招募JMJD2A促进下游靶基因DCN的表达。
转染过程所需的各种试剂，
（1）lip2000，一种多用途的脂质体转染试剂，适用于DNA、RNA和寡核苷酸的转染，对大多数真核细胞具有很高的转染效率。其独特的配方使其可直接加入培养基中，血清的存在不会影响转染效率，进而将PDIA3P1干扰序列转进到细胞内。
（2）Opti-mem培养液，含HEPES，2400mg/l碳酸氢钠，次黄嘌呤，胸腺嘧啶，丙酮酸钠，L-谷氨酰胺，微量元素，生长因子，以及减量至1.1mg/l的酚红，作为转染试剂的辅料，其本身对细胞无任何害处，而更好更有效的转进细胞中，以获得预期目的。
（3）PBS 磷酸缓冲盐溶液（phosphate buffer saline）一般作为溶剂，起溶解保护试剂的作用。它是生物化学研究中使用最为广泛的一种缓冲液，主要成分为Na2HPO4、KH2PO4、NaCl和KCl，由于Na2HPO4和KH2PO4它们有二级解离，缓冲的pH值范围很广；而NaCl和KCl主要作用为增加盐离子浓度。排除其本身对实验对象的影响。
组织收集
我们收集了30对2017年至2018年在江苏省人民医院，江苏省妇幼保健院接受剖宫产手术，诊断患有子痫前期的孕妇胎盘组织和不含有任何基础疾病的正常孕妇胎盘组织。并记录临床的特点：包括孕妇年龄，有无吸烟史，孕周数，收缩压，舒张压以，蛋白尿以及胎儿体重。组织样本收集的均为第一时间液氮或储存在-80°C,直至RNA提取。该研究经过南京医科大学伦理委员会批准。获得所有病人的书面知情同意。
细胞系
选取滋养细胞(HTR-8/SVneo) 来自加拿大女皇大学提供。HTR-8/SVneo细胞用RPMI 1640培养基培养;培养基中均含有5%的胎牛血清、100U/ml的青霉素和100mg/ml的链霉素。5% CO2的37℃恒温培养箱中常规培养。每2-3天更换新鲜培养基，当细胞融合度达到80%-90%时传代。所有细胞系被短串联重复序列的DNA分析验证。
RNA提取和定量PCR分析
根据试剂的使用说明，用Trizol试剂分离总RNA。逆转录反应应用TaKaRa PrimeScript试剂盒（TaKaRa, 大连,中国）。逆转录试剂盒对0.8μg总RNA进行逆转录，最终体积为20μl。结果分析：分析引物的特异性及扩增效率，根据溶解曲线判断引物的反应特异性。根据扩增曲线得到Ct值，采用相对量法与内参GAPDH进行目的基因相对表达量的分析。计算公式为：2^(-△Ct)，△Ct=Ct gene-Ct control。
质粒构建
合成人全长PDIA3P1 cDNA，并将其***真核表达载体pCDNA 3.1中，构建PDIA3P1过表达载体质粒，随后将上述质粒转化大肠杆菌，摇菌、培养、挑选单克隆菌培养扩增。
细胞转染
用于转染的质粒载体（pCDNA3.1- PDIA3P1、si-PDIA3P1和空载体质粒），均用去除内毒素的质粒提取试剂盒（DNA Midiprep试剂盒，Qiagen）提取。PDIA3P1的干扰序列及乱序对照（si-NC）均购自Invitrogen公司（Invitrogen公司，CA，USA）。将细胞HTR-8/SVneo按每孔2×105个细胞种于6孔培养板，待细胞贴壁后，于转染前6 h吸弃原有培养基，换成无双抗培养基；取10 μL 脂质体稀释于240 μL的OPTI-MEM中，温和吹打混匀室温下孵育5 min；取100pmol siRNA, si-NC或3ug质粒载体分别稀释于250μL OPTI-MEM中，吹打混匀室温下孵育5min；将孵育好的脂质体与siRNA或质粒稀释液混合，温和吹打混匀。于室温下继续孵育20min；将上述混合物均匀滴入事先加好1.5 mL OPTI-MEM的6孔培养板中，轻轻混匀。37℃,5% CO2培养箱中继续培养6 h后，换完全培养基。转染后36 h，收集细胞提取RNA或蛋白进行实时定量RT-PCR或免疫印迹分析。
细胞增殖活性检测
MTT实验，将处理后的细胞按每孔2000-3000个细胞接种于96孔培养板。待细胞80%贴壁后，细胞同步化12 h，弃去原有培养基。每个样本设置6个复孔，每孔总反应体积为200μl。每孔加入20μl的MTT反应液(5 mg/ml，溶于PBS)，37 ℃避光孵育4 h。弃去上清液，每孔加入150μl二甲亚砜(DMSO)，震荡10 min，酶标仪测定490 nm波长处的吸光度。
克隆形成实验，将处理后的细胞按每孔600，800，1000个细胞接种于6孔培养板。每孔加入2ml的完全培养基，培养10-14天后处理，弃掉完全培养基后加入4%多聚甲醛固定30分钟，弃掉多聚甲醛后加入结晶紫染色2h。最后用流动的PBS冲洗。晾干，拍照。
流式细胞术
凋亡检测，用胰酶消化收集转染48小时后的HTR-8/SVneo细胞，随后根据FITCAnnexin V凋亡检测试剂盒（BD）及其使用说明予以Annexin V-FITC荧光探针和碘化丙锭(PI)染色。流式细胞仪检测和分析。
细胞周期检测，根据说明书使用CycleTESTTM PLUS DNA 试剂盒（BD）予以PI染色，随后用FACScan分析。
亚细胞结构定位（核质分离）
根据使用说明书使用PARIS 试剂盒（Life Technologies，USA）分离HTR-8/SVneo细胞的细胞核和细胞质。使用qPCR方法检测PD1A3P1、GAPDH和U1在细胞质和细胞核中的分布。GAPDH为细胞质参照，U1为细胞核参照。以总RNA百分比呈现PDIA3P1、GAPDH和U1在细胞质和细胞核中的表达情况。
原位杂交技术（FISH）
根据PDIA3P1基因转录本的特点，设计相对应的探针（由上海博谷生物科技公司合成），将HTR-8／SVneo细胞种植于含有15mm爬片6孔板里，待细胞至80%左右时，弃培养基用PBS清洗两遍后，加2ml的甲醇固定30min后送样，由上海博谷生物科技公司进行接下来的处理，选用倒置银光显微镜拍照，定性检测PDIA3P1在细胞中的亚定位，进一步验证核质分离实验结果。
RNA-seq（转录组测序）
将细胞种植于六孔板中，待细胞长至80%左右后给于10ul 的lip2000 si-PDIA3P1和si-NC处理，48h后用Trizol 处理收集细胞，送样，由北京基因检测机构实施，选用Illumina进行随后的实验，得到并处理相应的数据。
RIP-qPCR 实验
将超大皿中滋养细胞刮下，RIP 裂解缓冲液裂解；在4℃下，全细胞提取物与JMJD2A抗体的磁珠温育8小时，其中选用mIgG 作为对照；在各种洗涤缓冲液洗涤磁珠后，将复合物与0.1％SDS/0.5mg/ml蛋白酶K（在55℃下30 分钟）温育以除去蛋白质；纯化的RNA进行qRT-PCR 分析以证明PDIA3P1可与JMJD2A抗体结合。
ChIP-qPCR
分析检测JMJD2A 绑定于DCN 基因的启动子区，表观调控DCN的表达。简而言之，加550μl 的37%多聚甲醛到含20mL 培养基的大皿中；摇床上室温孵育；加入10X 甘氨酸到培养皿中；摇床上摇晃均匀室温孵育；培养皿放冰上待用；吸取培养基；加入20ml 的PBS 清洗细胞；离心；移去上清。冰上孵育15 分钟，每5 分钟轻轻的涡旋细胞裂解液；离心；0.5ml 的细胞核裂解液重悬细胞；冰上超声，离心；将上清每50μl 分装到EP 管中；准备IP 反应，每个反应管中加入450μl 的Dilution buffer；将抗体和20μl 混匀的磁珠加入反应液中，4℃孵育到过夜；用buffer 涡旋清洗磁珠-抗体，结合抗体-靶蛋白-DNA 复合物，并沉淀；对沉淀下来的复合物进行清洗，除去一些非特异性结合；洗脱，得到富集的靶蛋白-DNA 复合物；解交联，纯化富集的DNA-片断；qPCR分析DCN启动子区域片断。得到所需cDNA 用于下一步qRT-PCR 分析。
蛋白质免疫印迹（Western blotting）
将变性好的细胞蛋白裂解产物加入至预先制好的10%变性聚丙烯酰氨凝胶(SDS-PAGE)的加样孔中，分离样本中蛋白。随后转至NC膜，并以特异性抗体孵育。最后用ECL发光液压片曝光。GAPDH抗体为对照，DCN抗体购自Proteintech公司。
数据处理
实验数据皆用SPSS17.0软件分析，以三次实验的平均值±标准误表示，组间差异用双尾Student’s T检验、秩和检验和卡方检验。单因素分析中p<0.05的随后再使用多因素分析。
相对于现有技术，本发明具有的技术效果：
我们发现了一个新的lncRNA PDIA3P1并证明其在子痫前期组织中表达下调。下调的PDIA3P1表达与子痫前期发病机制有着密切的关系。敲低PDIA3P1后抑制细胞增殖,血管形成,促进滋养细胞凋亡。此外,我们认为敲低PDIA3P1介导的滋养细胞生长抑制在一定程度上依赖于DCN的表达。在这里,我们首次证实PDIA3P1在人类滋养细胞中行使相关功能，是通过抑制的滋养细胞DCN的表达。lncRNAs通过各种机制调节靶基因的表达,如招募染色质调节酶到靶基因和顺式或反式调节转录,作为脚手架结合相关分子原件或吸附miRNA。在这项研究中,我们发现PDIA3P1招募JMJD2A介导的滋养层DCN上调网络的关键调控因子。抑制JMJD2A后，DCN表达上调，qPCR和免疫印迹显示。总之，这些发现证实，下调的PDIA3P1表达可通过招募JMJD2A介导JDP2的表达损害滋养层的生物学功能。
我们的研究首次发现, 在子痫前期孕妇胎盘组织和细胞PDIA3P1表达下调。在子痫前期孕妇胎盘滋养细胞HTR-8/SVneo敲低PDIA3P1的表达，表现出细胞抑制功能，下调PDIA3P1表现为抑制细胞增殖、血管形成能力以及促进细胞凋亡增加。此外。我们的发现会进一步丰富子痫前期发病机制,并促进lncRNA指导的诊断和治疗。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式描述中所需要使用的附图作简单地介绍。
图1、PDIA3P1 在子痫前期孕妇胎盘组织中表达下调
1A-1BPDIA3P1在子痫前期孕妇胎盘组织（n=30)表达下调。
1C在四种滋养细胞株中检测PDIA3P1 的相对表达情况，发现HTR-8/SVneo 表达最高，而JEG-3中表达最低。
图2、PDIA3P1对HTR-8/SVneo细胞血管形成能力的影响
2A-2D敲低PDIA3P1能够抑制滋养细胞HTR-8/SVneo血管形成能力。
图3、通过测序检测参与PDIA3P1介导滋养细胞生长的潜在下游靶基因
3A 在HTR-8/SVneo滋养细胞干扰PDIA3P1后进行RNA 转录组测序。
3B 对测序结果进行包括 GO 富集分析及COG功能分类在内的多种生物信息学技术分析，下游差异基因功能主要为：细胞迁移及凋亡相关基因、细胞外基质相关基因、增殖相关基因。
3C-3D 敲低PDIA3P1后通过RT-PCR和western blotting方法验证DCN是其潜在的靶基因。
图4、PDIA3P1通过招募JMJD2A调节DCN的表达。
4A-4BFish和核质分离实验在HTR-8/SVneo中检测到PDIA3P1的位置。
4C RIP 实验发现 PDIA3P1 与JMJD2A 蛋白结合关联度及上调倍数最高。
4D-4E干扰JMJD2A后，DCN在基因和蛋白水平表达相应上调。
具体实施方式
以下通过实施例对本发明作进一步的阐述，但不限制本发明。
实施例中末注明具体条件的的实验方法，基本上都按照Sambrook，J等人编著的《分子克隆实验指南(第3版)》(MolecularCloning:ALaboratoryManual,3rded.黄培堂等译，科学出版社.2002.8)中所述的条件及方法或按照材料提供商所建议的条件及方法进行，其它没有详细描述的技术相应于本领域人员来说是熟知的标准方法。
本发明的材料：本申请中提及的细胞株以及培养基均有商品供应或以别的途径能为公众所得，它们仅作举例，对本发明不是唯一的，可分别用其它适合的工具和生物材料来代替。
实施例1
检测PDIA3P1在组织和细胞中的表达情况
取0.1 g组织，液氮研磨充分（成粉末状）或1-5×107细胞弃培养基，预冷的PBS润洗2次。加入1 ml的Trizol裂解液，以无酶枪头吹打混匀，静置5 min，将裂解液移入预先标记好的无酶1.5 ml的离心管中。4℃ 7500 g离心5分钟，取上清加入1/5体积的氯仿，颠倒混匀30 s，静置2 min。4℃，12000 g离心，15 min。溶液分三层（水相-白色沉淀-红色有机物），转移水相层至新的1.5 ml离心管中，尽量不要吸到白色沉淀。加入等体积异丙醇，轻轻颠倒混匀，放置5-10 min。4℃，12000 g离心，10 min。吸弃上清，加入1 ml 75%的乙醇（现配），洗涤RNA沉淀。4℃，7500 g离心，5 min，弃上清。尽量去除75%的酒精，于室温中晾干，约15min。用无RNA酶水(20-25 μl)溶解RNA沉淀。
紫外吸收测定法测定RNA的浓度。使用紫外分光光度计测定RNA浓度和纯度，测量前先用溶解RNA用的DEPC水调零。在260 nm处读值1为表示40 ng/μl，RNA溶液的A260/A280的比值用于RNA纯度的检测，比值范围在1.8到2.1表明符合要求。琼脂糖凝胶电泳鉴定RNA的完整性。配制1% 的琼脂糖胶。加热溶解琼脂糖，冷却，加入1 μl 溴化乙锭(EB, 10 mg/ml)。摇匀后倒胶，待胶冷凝后，置于电泳槽中，浸于1 × TAE缓冲液中平衡10 min，待用。点样。按1:4 (v/v)将5 × 核酸电泳上样缓冲液与样本混合，准确将各样本含有1 μg的RNA加入凝胶孔中。80 V恒压电泳50 min。电泳结束后，在凝胶成像仪上观察结果。
Tris-乙酸(TAE)缓冲液配方(1L) 50 ×：
2 M Tris碱242 g
1 M乙酸57.1 mL冰乙酸(17.4 M)
100 mM EDTA200 mL 0.5 M EDTA (pH8.0)
去离子水至1 L
实时定量PCR
子痫前期孕妇胎盘组织及正常孕妇胎盘组织标本，HTR-8/SVneo细胞的总RNA，逆转录反应应用TaKaRa PrimeScript试剂盒（大连宝生物工程有限公司）。逆转录反应体系如下：
5 × PrimeScript Buffer（for Real Time）4 μl
Total RNA (1 μg/μL)1 μl
Random或Oliga dT2 μl
Primescript RT enzyme Mix1μl
RNase Free dH2O至20 μl
反转录反应条件如下：37°C 15min（反转录反应）；85°C 5sec（反转录酶的失活反应）。根据Genebank提供的基因序列，设计引物序列，
QPCR 应用7300 PCR***（Applied Biosystems，Warrington，UK）。cDNA样品采用三部法PCR扩增标准程序。反应体系：
SYBR Premix Ex Taq2 μl
F primer0.4 μl
R primer0.4 μl
ROX0.4 μl
cDNA1 μl
ddH2O5.8 μl
反应条件：
95℃30 s
95℃5 s
60℃34 s
68℃45 s
结果分析：分析引物的特异性及扩增效率，根据溶解曲线判断引物的反应特异性。根据扩增曲线得到Ct值，采用相对量法与内参GAPDH进行目的基因相对表达量的分析。计算公式为：2^(-△Ct)，△Ct=Ct gene-Ct control。
PDIA3P1的引物如下：
Primer F 5’- ATGGGCCTGTGAAGGTAGTG-3’，SEQ ID NO：2，
Primer R 5’- GTGGCATCCATCTTGGCTAT-3’， SEQ ID NO：3。
结果表明，lncRNA PDIA3P1在子痫前期孕妇胎盘组织表达较正常组织下调。我们利用实时定量PCR检测了30对子痫前期孕妇胎盘组织表达较正常组织中PDIA3P1的表达水平。结果显示与正常孕妇胎盘组织相比，PDIA3P1的表达在60%(12/20)的子痫前期孕妇胎盘组织中较正常组织表达降低（倍数>1.5,P<0.05）(图1A和1B)。提示PDIA3P1可能在子痫前期疾病的诊断，发生发展及治疗中扮演着重要的作用。
实施例2
为了研究PDIA3P1对正常滋养细胞HTR-8/SVneo表型的影响。
首先，选取正常滋养细胞HTR-8/SVneo细胞系作为本实验的研究对象，利用lip2000作为载体，转染PDIA3P1干扰序列以敲低PDIA3P1的表达模拟子痫前期的发病过程，MTT和克隆增殖实验检测发现,在HTR-8/SVneo细胞中转染PDIA3P1的干扰序列，敲低PDIA3P1表达后抑制细胞生长。由此可知，这些数据表明, PDIA3P1可促进HTR-8/SVneo细胞的增殖能力。
实施例3
PDIA3P1对胎盘滋养细胞HTR-8/SVneo凋亡的影响。
为了研究是否PDIA3P1对HTR-8/SVneo细胞的增殖影响了细胞周期转换, 以正常滋养细胞HTR-8/SVneo细胞系作为研究对象，利用lip2000作为载体，转染PDIA3P1干扰序列以敲低PDIA3P1的表达模拟子痫前期的发病过程。我们进行了流式细胞仪分析细胞凋亡是否参与了PDIA3P1敲低后诱导的细胞生长受抑。如图2C所示,早期凋亡(UR)和晚期凋亡率(LR)在PDIA3P1敲低的HTR-8/SVneo细胞中高于对照组细胞。由此可见，PDIA3P1抑制滋养细胞凋亡作用。
实施例4
PDIA3P1参与HTR-8/SVneo细胞血管形成能力。
滋养细胞血管形成能力在子痫前期发病机制中的一个重要方面。HTR-8/SVneo细胞系作为研究对象，利用lip2000作为载体，转染PDIA3P1干扰序列下调PDIA3P1的表达。利用血管形成能力实验研究了PDIA3P1敲低以后对HTR-8/SVneo细胞血管形成能力影响。可见，PDIA3P1的低表达影响着正常滋养细胞的血管形成能力，进一步影响着胎盘的浅着床，诱导子痫前期疾病的发生。
除非另外具体说明，否则在这些实施例中阐述的数值并不限制本发明的范围。在这里示出和描述的所有示例中，除非另有规定，任何具体值应被解释为仅仅是示例性的，而不是作为限制，因此，示例性实施例的其他示例可以具有不同的值。
SEQUENCE LISTING
<110> 江苏省人民医院（南京医科大学第一附属医院）
<120> 一种长非编码RNA及其在诊断／治疗子痫前期中的应用
<130> 2020
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 2099
<212> DNA
<213> Homo sapiens
<400> 1
aaactaaatc aaacttgagt atgaaacttc ttttttttta agtgcctaag tcctgaatga 60
caacaaaaga ctgaagaact gacaacgatt agaggacact aaggagactt aacaactaaa 120
tgcaaaatgg gaccctgaat ctgatcctgg aacacaaaaa ctaaattagt agaaaagtgg 180
taaaataggt ggtcgcgcgc ccgaccgccg cagtcccagt cgagccgcga cccttccggc 240
tgcccccacc ccacctcgcc gccatgtgcc tccgccgccc agcgctgttc ccgggcgtgg 300
cgctgcttct cgccgcggcc cgcctcgcgg ctgcctccga cgtgctagga ctcagggacg 360
acaacttgga gagtcgcatc tccgacacgg gctctgcggg cctcatgctc gtcgagttct 420
tcgccccctg gtgtggacac tgcaagagac ttgctcctga gtatgaagct gcagctacca 480
gattaaaagg aatagtccca ttagcaaagg ctgattgcac tgccaacact aacacctgta 540
ataaatatgg agtcagtgga tatccaaccc tgaatatgtt tagagatggt gaagaagcag 600
gtgcttatga tggacctagg actgctgatg gaattgtcag ccacctgaag aagcaggcag 660
gcccagcttc agtgcctctc aggactgagg aagaatttaa gaaattcatt agtgataaag 720
atgcctctat agtaggtttt ttcgatgatt cattcagtga agctcactcc gagttcctaa 780
aagcagccag caacttgagg gataactacc gatttgcaca tacgaatgtt gagtctctgg 840
tgaacgagta tgatgataac ggagatggta tcatcttatt tcgtccttca catctcacta 900
acaagttgga ggacaagact gtggcatata cagtgcaaaa aatgaccagt ggcaaaatta 960
aaaagtttat ccaggaaaac atttttggta tctgccctca catgacagaa gacaataaag 1020
atttgataca gggcaaggac ttacttattg cttactatga tgtggactat gaaaagaatg 1080
ctaaaggttc caactactgg agaaacaggg taatgatggt ggcaaagaaa ttcctggatg 1140
ctgggcacaa actcaacttt gctgtagcta gccgcaaaac ctttagccat gaactttctg 1200
attttggctt ggagagcact gctggagaga ttcctgttgt tgctatcaga actgctaaag 1260
gagagaagtt tgtcatgcag gaggatttct cgcgtgatgg gaatgctctg gagaggttcc 1320
tgcaggatta ctttgatggc aatctgaaga gatacctgaa gtctgaacct atcccagaga 1380
gcaatgatgg gcctgtgaag gtagtggtag cagagaattt tgatgaaata gtgaataatg 1440
aaaataaaga tgtgctgatt gaattttatg ccccttggtg tggtcactgt aagaacctgg 1500
agcccaagta taaagaactt ggcgagaagc tcagcaaaga cctgaatatc gtcatagcca 1560
agatggatgc cacagccaat gatgtgcctt ctccatatga agtcagagtt ttcctaccat 1620
atacttctct ccagccaaca agaagctaaa tccaaagaaa tatgaaggtg gccatgaatt 1680
aagtgatttt attagctatc tacaacgaga agctacaaac ccccctgtaa ttcaagaaga 1740
aaaacccaag aagaagaagg cacaggagga tctctaaagc agtaggcaaa caccactttg 1800
taaaaggact cttccaccag agatgggaaa accactgggg aggactagga cccatatggg 1860
aattattacg tctcagggcc gagaggacag aatggatata atctgaatcc tgttaaattt 1920
tctctaagcc atttcttagc tgcactgtta tggaaatacc aggaccagtt tatgtttgtg 1980
gttttgggaa aaattattgg tgttggggga aatgttgtgg gagtcgggtt gagttggggg 2040
tattttctaa tttttttgtg catttggaac agtgacaata aataagaccc ctttaaact 2099
<210> 2
<211> 20
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 2
atgggcctgt gaaggtagtg 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 3
gtggcatcca tcttggctat 20
<210> 4
<211> 25
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 4
cattagtgat aaagatgcct ctata 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 5
gataacggag atggtatcat cttat 25
Claims (3)
1.PDIA3P1在制备治疗子痫前期药物中的应用，其特征在于，所述PDIA3P1的核苷酸序列为SEQ ID NO：1。
2.包含权利要求1中所述的PDIA3P1的药物组合物在制备治疗子痫前期药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述组合物中还包括辅料，所述辅料包括lip2000、Opti-mem培养液、PBS磷酸缓冲盐溶液。
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