CN113106154B - Prmt3在诊断或治疗复发性自然流产中的应用 - Google Patents
Prmt3在诊断或治疗复发性自然流产中的应用 Download PDFInfo
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Abstract
本发明涉及PRMT3在诊断或治疗自然流产中的应用。本发明通过分析比较正常妊娠和复发性流产(RSA)患者蜕膜中PRMT3的表达水平，发现RSA患者蜕膜组织及蜕膜巨噬细胞中存在PRMT3表达水平升高的现象；体外抑制PRMT3可降低滋养细胞HTR‑8/SVneo的凋亡；体内动物试验显示，PRMT3抑制剂可降低CBA/J×DBA/2自然流产小鼠模型的胚胎吸收率，从而为临床上自然流产的诊断和治疗提供了新方法。
Description
技术领域
本发明涉及疾病诊断和治疗领域，具体地说，涉及PRMT3在诊断或治疗复发性自然流产中的应用。
背景技术
复发性自然流产(RSA)是一个重要的全球性健康问题，对受影响的夫妇而言，其心理和经济负担并未得到充分重视。RSA与父母染色体异常，血栓性疾病以及结构性子宫异常相关，但是有50％的RSA病因不明，因此阐明RSA的发生机制以及提供相应的防治策略具有重要的理论和临床意义。妊娠是一个复杂的生理过程，在妊娠初期，胎儿滋养细胞侵入子宫粘膜并重塑子宫血管。子宫内膜通过蜕膜化过程变成蜕膜，以维持胚胎生长发育。滋养细胞、蜕膜免疫细胞和蜕膜基质细胞是母胎界面主要的细胞群体，彼此之间的相互对话对成功妊娠至关重要。在母胎界面的免疫细胞中，巨噬细胞约占蜕膜白细胞总数的20％，是仅次于子宫NK细胞的第二大白细胞种群，并且在整个妊娠过程中，蜕膜巨噬细胞的数量保持稳定。尽管胚胎植入是一个炎症过程，但成功的妊娠取决于胚胎植入引起的炎症反应是否及时消失，并在母胎界面转换为免疫耐受的微环境。越来越多的研究显示：蜕膜巨噬细胞在妊娠早期发挥抗炎吞噬作用，并参与调节母胎界面免疫微环境、胎盘血管重塑、胚胎植入以及维持妊娠等生理过程。因此，探讨蜕膜巨噬细胞可能的致病因素能为复发性自然流产的临床诊断和治疗提供一些新思路和新方法。
一氧化氮(Nitric oxide，NO)是一种重要的细胞信使分子和效应分子。它在多种生理和病理过程中发挥重要调控作用。NO主要由一氧化氮合酶(Nitric oxide synthase，NOS)催化L-精氨酸所产生。不对称二甲基精氨酸(Asymmetric dimethylarginine，ADMA)是一种非特异性内源性NOS抑制剂。它由蛋白精氨酸残基经精氨酸甲基转移酶(Argininemethyltransferase，PRMT)甲基化产生，PRMTs的甲基化是一个重要的翻译后修饰。它调节基因转录、DNA修复、信使RNA的加工，和信号转导等多种生理过程。至今已在哺乳动物细胞中发现9种PRMTs。PRMT3包含一个C2H2锌指结构域，该结构域在其他PRMT中不存在，并且该结构域对于底物识别至关重要。PRMT3主要是胞质蛋白。两个先前的研究表明，PRMT3在体外通过锌指结构域与rpS2相互作用使得rpS2甲基化。PRMT3基因敲除小鼠的后代生育力并未受到影响。也有文献报道，PRMT3敲除的新生小鼠发育迟缓。由于确定的底物有限，目前对于PRMT3功能的研究还非常有限。
发明内容
本发明的目的是针对现有技术中的不足，提供PRMT3的新用途。
第一方面，本发明提供了PRMT3作为诊断标志物在制备复发性自然流产的诊断试剂或试剂盒中的应用。
作为本发明的一种优选实施方式，所述诊断试剂或试剂盒用于检测蜕膜组织或蜕膜巨噬细胞中PRMT3基因或蛋白的表达水平。
第二方面，本发明提供了检测PRMT3基因或蛋白表达水平的试剂在制备复发性自然流产的诊断试剂或试剂盒中的应用。
作为本发明的一种优选实施方式，所述诊断试剂或试剂盒用于检测蜕膜组织或蜕膜巨噬细胞中PRMT3基因或蛋白的表达水平。
第三方面，本发明提供了PRMT3基因或蛋白的抑制剂在制备治疗复发性自然流产的药物中的应用。
作为本发明的一种优选实施方式，所述抑制剂为化合物小分子或生物大分子。
第四方面，本发明提供了一种复发性自然流产动物模型的建立方法，包括给予动物物质以降低动物蜕膜组织或蜕膜巨噬细胞中PRMT3基因或蛋白的表达水平的步骤。
作为本发明的一种优选实施方式，所述物质为化合物小分子或生物大分子。
作为本发明的另一种优选实施方式，所述物质通过腹腔注射的方式给予动物。
第五方面，本发明提供了一种筛选治疗复发性自然流产的潜在物质的方法，包括利用如上任一所述的建立方法建立复发性自然流产动物模型的步骤。
作为本发明的一种优选实施方式，所述方法还包括以下步骤：
a)给予复发性自然流产动物模型受试物质；
b)比较复发性自然流产动物模型给药前后动物蜕膜组织或蜕膜巨噬细胞中PRMT3基因或蛋白表达水平的变化，如降低，则表明所述受试物质为治疗复发性自然流产的潜在物质，反之则不是。
本发明优点在于：
1、由于孕早期蜕膜对胚胎发育以及血管形成具有重要作用，因此，我们的假设是某些信号分子对蜕膜的功能有调控作用，我们比较了正常妊娠和RSA患者蜕膜组织中PRMT3蛋白的表达水平，发现RSA患者胚胎蜕膜组织中PRMT3表达水平上调，提示胚胎蜕膜组织中PRMT3的表达水平可作为诊断自然流产的标志物，从而为临床上复发性自然流产的诊断提供了新方法。
2、我们体外实验证实PRMT3下游产物ADMA处理可促进滋养细胞的凋亡，PRMT3抑制剂可抑制滋养细胞的凋亡；同时体内实验证实PRMT3抑制剂SGC707腹腔注射可降低CBA/J×DBA/2自然流产小鼠模型的胚胎吸收率，从而为临床上复发性自然流产的治疗提供了新方法。
3、我们通过腹腔注射SGC707，降低小鼠体内PRMT3，导致小鼠胚胎吸收率降低，从而建立了一种新的、与临床现象相符且机制明确的自然流产小鼠模型。
附图说明
图1：正常妊娠妇女和复发性自然流产患者的蜕膜组织(左)、蜕膜巨噬细胞(右)中PRMT3的表达水平。其中，normal：正常妊娠，RSA：复发性自然流产。*表示P<0.05，具有统计学差异。
图2：抑制PRMT3降低HTR8细胞凋亡。其中，Control：巨噬细胞使用溶剂对照处理，再与HTR-8/SVneo细胞共培养；SGC707 200nM：巨噬细胞使用浓度200nM SGC707处理，再与HTR-8/SVneo细胞共培养。
图3：增加ADMA促进滋养细胞凋亡。其中，Control：巨噬细胞使用溶剂对照处理，再与HTR-8/SVneo细胞共培养；ADMA 60μM：巨噬细胞使用浓度60μM ADMA处理，再与HTR-8/SVneo细胞共培养。
图4：注射PRMT3抑制剂的小鼠胚胎吸收率降低。其中CBA/BALB：阴性对照的小鼠；CBA/DBA：自然流产小鼠；吸收胚胎常伴有缺血、出血、坏死，体积会明显小于正常存活胚胎，并且从胚胎的色泽来看，吸收胚胎呈黑褐色(黑色箭头所示)，存活胚胎则呈粉红色。据此统计存活胚胎数和吸收胚胎数。按照胚胎个数/(存活胚胎个数+死亡胚胎个数)计算小鼠胚胎吸收率。*表示P<0.05，具有统计学差异。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著的《分子克隆实验指南》(科学出版社，2002)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：分析正常妊娠妇女和复发性自然流产患者的蜕膜组织及蜕膜巨噬细胞中PRMT3的表达
蜕膜细胞包含终末分化的子宫基质细胞(也称为蜕膜基质细胞)和大量母体免疫细胞，其主要种群包括子宫自然杀伤细胞和巨噬细胞。通过Western blot分析我们发现蜕膜组织中PRMT3蛋白表达水平显著上调。为了进一步验证哪种类型的蜕膜细胞中PRMT3蛋白表达上调，我们通过Western blot分别在蜕膜组织提取的蜕膜基质细胞，uNK细胞和巨噬细胞中检测了PRMT3的蛋白表达水平。结果表明，与正常孕妇相比，RSA患者蜕膜巨噬细胞中PRMT3蛋白表达(1.358±0.2073vs 0.4888±0.08541,P＝0.0002)显著上调(图1中右图)。
实施例2：抑制PRMT3降低HTR8细胞凋亡
蜕膜巨噬细胞在妊娠期间具有许多功能。先前的研究表明蜕膜巨噬细胞可通过吞噬凋亡的滋养层细胞来执行“清理”功能，从而阻止蜕膜中促炎途径的激活。在本研究中，我们通过Transwell培养板建立由蜕膜巨噬细胞和HTR-8/SVneo细胞的共培养模型，以探究巨噬细胞可能对滋养细胞凋亡的调节作用。RSA患者来源的蜕膜巨噬细胞经CD14磁珠分选提纯后培养在Transwell板的下室，HTR-8/SVneo细胞铺在Transwell上室。下室蜕膜巨噬细胞分成两组，处理组使用200nM的PRMT3特异性抑制剂SGC707刺激蜕膜巨噬细胞，对照组使用与SGC707等体积的药物溶剂DMSO进行处理。当处理48h或72h后，收集上室的HTR-8/SVneo细胞，通过流式细胞术进行凋亡分析。结果表明：与对照组巨噬细胞相比，经SGC707处理48h或72h的巨噬细胞与HTR-8/SVneo共培养，HTR-8/SVneo的细胞凋亡活性受到抑制(图2)。
实施例3：增加ADMA增加滋养细胞凋亡
为了进一步验证PRMT作为滋养层细胞凋亡调节剂的重要性，我们使用其蛋白水解产物ADMA来佐证。正常孕妇来源的蜕膜巨噬细胞经CD14磁珠分选提纯后培养在Transwell板的下室，HTR-8/SVneo细胞铺在Transwell上室。下室蜕膜巨噬细胞分成两组，处理组使用60μM外源性ADMA处理，对照组使用与ADMA等体积的药物溶剂DMSO进行处理。当处理24h，48h或72h后，收集上室的HTR-8/SVneo细胞，通过流式细胞术进行凋亡分析。实验结果显示，与对照组巨噬细胞相比，经ADMA处理48h或72h的巨噬细胞与HTR-8/SVneo共培养，HTR-8/SVneo的细胞凋亡活性受到促进；而经ADMA处理24h的巨噬细胞与HTR-8/SVneo共培养，HTR-8/SVneo的细胞凋亡活性未发生明显变化(图3)。
实施例4：注射PRMT3抑制剂的小鼠胚胎吸收率降低
CBA/J雌性小鼠分别与BALB/c和DBA/2J雄性小鼠交配。CBA/J雌性×DBA/2J雄性交配代表自然流产组，CBA/J雌性×BALB/c雄性交配为正常对照组。图4左上代表妊娠第10.5天CBA/J×DBA/2小鼠的妊娠子宫。其中黑色箭头所指的地方即为胚胎吸收位点。右上侧代表妊娠第10.5天CBA/J×BALB/c小鼠的妊娠子宫。正常组与实验组小鼠于孕0.5天、孕3.5天、孕6.5天经腹腔注射给予SGC707(剂量为：30mg/kg)及等体积溶剂对照DMSO后，于妊娠第10.5天发现SGC707处理可降低CBA/J×DBA/2妊娠小鼠胚胎吸收率(图4)。
因此，本研究通过体内、体外实验证实抑制PRMT3可以降低自然流产的发生，从而为临床上复发性自然流产的风险评估及治疗提供了一种新策略。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (5)
1.PRMT3作为诊断标志物在制备复发性自然流产的诊断试剂或试剂盒中的应用。
2.检测PRMT3基因或蛋白表达水平的试剂在制备复发性自然流产的诊断试剂或试剂盒中的应用。
3.根据权利要求1或2所述的应用，其特征在于，所述诊断试剂或试剂盒用于检测蜕膜组织或蜕膜巨噬细胞中PRMT3基因或蛋白的表达水平。
4.PRMT3基因或蛋白的抑制剂在制备治疗复发性自然流产的药物中的应用。
5.根据权利要求4所述的应用，其特征在于，所述抑制剂为化合物小分子或生物大分子。
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