CN113151451B - 一种卵子发育成熟诊断生物标志物及其应用 - Google Patents
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Abstract
本发明公开了一种卵子发育成熟过程中生物标志物，所述生物标志物为NSUN5。本发明检测NSUN5的表达水平对胚胎干细胞分化的影响，发现在NSUN5被敲降后，胚胎干细胞向原始生殖细胞分化比例明显下降。同时，进一步构建NSUN5敲除小鼠，发现NSUN5敲除小鼠的卵子发育成熟明显落后于野生型小鼠。可见，NSUN5可以作为卵子发育成熟的生物标志物，为卵巢发育和卵子发生相关疾病的预测、诊断及预后提供理论基础。
Description
技术领域
本发明属于卵巢功能诊断技术领域，具体涉及一种卵子发育成熟诊断生物标志物及其在原发性卵巢功能不全诊断和预后中的应用。
背景技术
原发性卵巢功能不全(Primary ovarian insufficiency，POI)，是一种妇科内分泌疾病，主要表现为40岁前促性腺激素异常增高和雌激素减低，发病率为3.7％。当前，随着中国二胎政策放开及女性受孕年龄的延迟，高龄生育女性数量逐渐增多，有生育需求女性中POI患者比例也相应增加。POI可能由遗传因素、放化疗手术相关医源性因素、代谢因素、线粒体功能紊乱、免疫因素和环境因素等造成，进而导致卵子数量和质量下降，但仍有75％以上POI患者原因不明。POI不仅导致女性生育力下降、妊娠不良结局增多、生殖系统疾病发生，也诱发多种心血管、骨科、乳腺疾病，严重危害女性身心健康和家庭幸福。
5-甲基胞嘧啶(m5C)RNA甲基化修饰是真核生物常见的RNA修饰方式，由甲基转移酶、脱甲基酶和结合蛋白分子调控甲基化水平的动态变化，影响RNA剪切、输出、稳定、翻译和定位，并通过以上方式维持生物稳态或其异常引发疾病。研究表明生物体中m5C水平降低可能引起线粒体功能障碍、配子发生和胚胎发育缺陷、神经和脑发育异常，且与细胞迁移和肿瘤相关。
通常POI患者在出现闭经及围绝经期症状的晚期阶段即POF才能被识别，此时卵巢功能恢复的希望渺茫，识别POF的意义十分有限，如能在卵巢功能衰竭前发现卵巢损伤并给予合理的管理可延缓甚至逆转卵巢损伤，减轻POF患者的远期并发症，因此，早期识别并及时诊断POI在临床工作中显得尤为重要。
目前，对POI的诊断主要从临床表现、实验室检查、超声检查及病理组织活检进行。实验室检查主要包括检测血清促卵泡刺激素FSH、黄体生成素LH和抗苗勒管激素AMH等。有研究表明，只有当卵巢功能损害达到一定程度时才会出现血清FSH水平的明显改变，所以，检测FSH并不能筛查出极早期的卵巢功能减退。当卵巢功能受损时，LH的表现远不如FSH敏感，往往要在FSH水平发生改变几年后才发生改变。AMH是近年来新兴的一项检测指标，是目前外周血中可检测到的最早的卵泡产生的物质，其水平随年龄增加而显著降低，且与窦状卵泡数(AFC)具有强烈相关性，可更直接、真实地反映原始卵泡储备情况。NICE(英国)指南建议，将AMH<(0.1～0.35)ng/mL作为预测生殖能力受损的界值，而AMH>8pmol/L可排除POI，但特异性较低。此外，还可通过超声检查判断卵巢功能储备，AFC是指卵泡早期经阴道超声检测的直径为2～5mm的窦状卵泡数，AFC再发育就会成为成熟卵泡，其可精确地反映卵巢剩余卵泡数。卵巢体积反映卵巢状态，在FSH上升前即有所改变。监测卵巢血流动力学指标，也可了解卵巢功能储备情况。卵巢活检是确诊POI的“金标准”，但受取材位置的影响，诊断POF的意义有限；同时，具有一定危险性，且术后有粘连和感染的可能，患者不易接受，不主张采用卵巢活检对POI进行病因诊断和病情评估。因此，早期识别及诊断POI目前仍然存在较大困难。
因此，急需一种能够准确诊断卵子发育成熟的生物标志物，能够为卵子发育成熟的诊断、预后及诊断提供理论基础。
发明内容
针对上述问题，本申请经过多角度研究发现，NSUN5表达水平降低引起卵子发育及成熟障碍，NSUN5可作为生物标志物检测卵子发育及成熟障碍。
第一方面，本发明提供了用于检测NSUN5的转录水平的物质在以下任一中的应用，
(A)诊断或辅助诊断卵子发育不成熟；
(B)诊断或辅助诊断原发性卵巢功能不全；
(C)比较待测卵巢组织中卵子发育状况；
(D)制备用于诊断或辅助诊断卵子发育不成熟的产品；
(F)制备用于诊断或辅助诊断原发性卵巢功能不全的产品；
(G)制备用于比较待测卵巢组织中卵子发育状况的产品。
进一步地，所述检测NSUN5的转录水平的物质为如下1)或2)：
1)转录测序所需试剂及仪器；
2)扩增所述NSUN5的引物对或含有所述引物对的试剂或试剂盒。
第二方面，本发明提供了一种比较待测卵巢组织中卵子发育成熟度的方法，其特征在于，用于非疾病诊断和治疗目的，包括如下步骤(A1)或(A2)：
(A1)对所述NSUN5直接测序；
(A2)用本发明第一方面所述的试剂或试剂盒对待测卵巢组织中所述NSUN5的转录水平进行测定：
若所述待测卵巢组织中所述NSUN5的转录水平高于其他所述待测待测卵巢组织中所述NSUN5的转录水平，则所述待测卵巢组织中卵子发育水平相对成熟；
若所述待测待测卵巢组织中所述NSUN5的转录水平低于其他所述待测待测卵巢组织中所述NSUN5的转录水平，则所述待测卵巢组织中卵子发育相对不成熟。
第三方面，本发明提供了上述的方法或上述的物质在筛选具有如下性状中至少一种的卵巢组织中卵子发育成熟度的应用：
(a)卵子发育相对成熟；
(b)卵子发育相对不成熟；
(c)原发性卵巢功能相对不全。
第四方面，本发明提供了一种用于诊断或辅助诊断卵子发育成熟或原发性卵巢功能不全的系统，包括：
(A1)用于检测NSUN5的转录水平的物质；
(A2)装置，所述装置包括数据输入模块、数据比较模块和结论输出模块。
进一步地，所述数据输入模块用于输入(A1)检测得到的待测者的样本中NSUN5的转录水平值。
进一步地，所述数据比较模块用于将待测者的样本中NSUN5的转录水平值与对照数值进行比较；所述对照数值为未患有卵子发育成熟或原发性卵巢功能不全的患者的NSUN5的转录水平值。
进一步地，所述结论输出模块用于按照如下标准输出结论：如果待测者的样本中NSUN5的转录水平值小于对照数值，则待测者为卵子发育不成熟或候选为卵子发育不成熟或原发性卵巢功能不全患者。
本发明与现有技术相比，本发明发现在NSUN5被敲降后，胚胎干细胞向原始生殖细胞分化比例明显下降，进一步构建NSUN5敲除小鼠，发现NSUN5敲除小鼠的卵子发育成熟明显落后于野生型小鼠。NSUN5作为卵子发育成熟的生物标志物，为卵巢发育和卵子发生相关疾病的预测、诊断及预后提供理论基础。
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1NSUN5 KD和WT胚胎干细胞流式细胞术检测结果；
图2NSUN5 KO小鼠模型构建方法；
图3NSUN5 KO和WT小鼠卵巢组织免疫化学染色照片；
图4NSUN5 KO和WT小鼠GV期卵母细胞免疫荧光染色照片；
图5NSUN5 KO和WT小鼠MII期卵母细胞免疫荧光染色照片；
图6NSUN5 KO和WT小鼠卵巢组织流式细胞术检测结果。
具体实施方式
下面将结合附图对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只是作为示例，而不能以此来限制本发明的保护范围。需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
实施例1
1.1在饲养层细胞上培养小鼠胚胎干细胞；
1.2转入siRNA对NSUN5进行敲降，将小鼠胚胎干细胞向原始生殖细胞方向诱导，诱导后进行PGC迁移后期标志基因VASA和PGC减数分裂期标志基因SCP3鉴定；
1.3制备单细胞悬液，经特异性荧光染料标记的VASA和SCP3抗体染色，流式细胞仪测定诱导分化比例，如图1所示，发现NSUN5 KD胚胎干细胞向原始生殖细胞转化的比例显著下降。
实施例2
2.1确定编码NSUN5的基因位点，如图2所示，构建sgRNA序列为sgRNA1：CCGATGGAAGGCTCTGCTCTGCTGGGCC(SEQ ID NO.1)、sgRNA2：GGCTGAGCTGGCCCGACTCAAGG(SEQID NO.2)，与Cas9 mRNA一起原核显微注射获得测序鉴定阳性的F0代阳性小鼠；
2.2在F0代小鼠出生后5-7天进行剪趾编号，并将剪取组织用设计好的对应引物进行荧光定量PCR验证，如图2所示，证明NSUN5 KO小鼠模型构建成功。
实施例3
3.1选择成年期NSUN5 KO小鼠和WT小鼠，颈椎脱臼处死后干净分离出卵巢，4％多聚甲醛溶液(PFA)固定24h,梯度乙醇溶液进行脱水，二甲苯透明，浸蜡，包埋切片；
3.2石蜡切片免疫组织化学染色：选择PGC迁移后期标志基因VASA和PGC减数分裂期标志基因SCP3抗体作为一抗，按使用说明比例稀释后4℃孵育过夜，洗去一抗，加入二抗室温孵育2h，封片后显微镜下观察；
3.3采集图像，如图3所示，对窦状卵泡及各级卵泡进行计数，并进行统计学分析，发现NSUN5 KO小鼠窦状卵泡数和各级卵泡总数均明显低于WT小鼠，差异存在统计学意义。
实施例4
4.1选择成年期NSUN5 KO小鼠和WT小鼠，颈椎脱臼处死后干净分离出卵巢，体视显微镜下分离GV期及MII期卵母细胞，对其进行固定；
4.2细胞免疫荧光染色：选择γH2AX和NSUN5抗体，按使用说明比例稀释后4℃孵育过夜，洗去一抗，加入二抗室温孵育2h，封片后显微镜下观察；
4.3采集图像，如图4-5所示，对γH2AX焦点数进行统计，观察MII期卵母细胞纺锤体及染色质状态，发现NSUN5 KO小鼠GV期卵母细胞γH2AX焦点数量明显低于WT小鼠，表明减数分裂不活跃，而MII期卵母细胞纺锤体形成异常，染色体排列紊乱，这些可能是NSUN5水平降低后卵子发育成熟障碍的重要机制。
实施例5
5.1选择成年期NSUN5 KO小鼠和WT小鼠，颈椎脱臼处死后干净分离出卵巢，彻底破碎卵巢组织，联用酶消化法，机械打散法和化学分散法来获得足够数量的单细胞悬液；
5.2所得单细胞悬液，经特异性荧光染料标记的PCNA和KI67抗体染色，流式细胞仪测定增殖活跃细胞比例，如图6所示，发现与WT小鼠相比，NSUN5 KO小鼠的细胞增殖活力明显降低。
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> 苏州市立医院
<120> 一种卵子发育成熟诊断生物标志物及其应用
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Claims (3)
1.用于检测NSUN5的转录水平的物质在制备用于比较待测卵巢组织中卵子发育状况的产品中的应用，其特征在于，若所述待测卵巢组织中所述NSUN5的转录水平高于其他所述待测卵巢组织中所述NSUN5的转录水平，则所述待测卵巢组织中卵子发育水平相对成熟；若所述待测卵巢组织中所述NSUN5的转录水平低于其他所述待测卵巢组织中所述NSUN5的转录水平，则所述待测卵巢组织中卵子发育相对不成熟。
2.根据权利要求1所述的应用，其特征在于，所述检测NSUN5的转录水平的物质为如下1)或2)：
1)转录测序所需试剂及仪器；
2）扩增所述NSUN5的引物对或含有所述引物对的试剂。
3.根据权利要求1所示的应用，其特征在于，所述比较待测卵巢组织中卵子发育状况包括如下步骤(A1)或(A2)：
(A1)对所述NSUN5直接测序；
(A2)用权利要求2中所述的试剂对待测卵巢组织中所述NSUN5的转录水平进行测定。
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