CN114062688A - Ccl8蛋白作为非梗阻性无精子症的生物标志物及其应用 - Google Patents
Ccl8蛋白作为非梗阻性无精子症的生物标志物及其应用 Download PDFInfo
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Abstract
本发明公开了CCL8蛋白作为非梗阻性无精子症的生物标志物及其应用。所述生物标志物包括CCL8蛋白。本发明首次发现NOA患者精液中CCL8蛋白的浓度显著低于正常男性,并提出CCL8蛋白可作为评估NOA患者生精功能障碍的潜在预警分子,有助于扩充诊断NOA指标,以扩大诊断范围,对于NOA诊断领域具有重要意义。
Description
技术领域
本发明属于生殖医学技术领域,涉及CCL8蛋白作为非梗阻性无精子症的生物标志物及其应用。
背景技术
无精子症的发生率占男性不育症的10%-20%。根据睾丸中有无精子可分为两类:一类是睾丸具有正常精子发生的阻塞性无精子症(obstructive azoospermia,OA),另一类是睾丸精子发生障碍的非阻塞性无精子症(non-obstructive azoospermia,NOA)。对非阻塞性无精症的准确诊断可以为临床医生提供有价值的指导。
睾丸体积,血清卵泡刺激素(FSH)水平等可辅助诊断NOA患者,亦有报道以RNA作为NOA的标志物,如CN113215246A公开了一种非梗阻性无精子症诊断的circRNA标志物及其应用,通过实验证实与生精功能正常的对照组相比,非梗阻性无精子症患者血清中的hsa_circMAN1A2表达明显升高,且血清中hsa_circMAN1A2的相对表达量对于非梗阻性无极精子症的预测效能较大。因此,hsa_circMAN1A2能够作为非梗阻性无精子症诊断的circRNA标志物;CN112176052A公开了一种与非梗阻性无精子症诊断相关的血浆外泌体tsRNA标志物及其应用,该标志物为tRF-Gly-GCC-002,其在非梗阻性无精子症和梗阻性无精子症患者血浆中差异表达,能够较好地诊断非梗阻性无精子症,可见,目前已开发出多种诊断NOA指标,但是这些指标主要集中于非编码RNA,其定量检测较为困难,仍有一部分患者无法利用这些指标进行正确的诊断。
综上所述,开发新的与非梗阻性无精子症相关的生物标志物,尤其是蛋白质分子标志物,以扩充诊断NOA指标,对于NOA诊断领域具有重要意义。
发明内容
针对现有技术的不足和实际需求,本发明提供CCL8蛋白作为非梗阻性无精子症的生物标志物及其应用,本发明首次发现精液中CCL8蛋白的浓度与非阻塞性无精子症相关,提示CCL8蛋白可作为评估非阻塞性无精子症(NOA)患者生精功能障碍的潜在预警分子。
为达上述目的,本发明采用以下技术方案:
第一方面,本发明提供一种与非梗阻性无精子症相关的生物标志物,所述标志物包括CCL8蛋白。
本发明中,结合测序数据与临床样本,对NOA睾丸巨噬细胞相关的差异性分泌蛋白进行筛选和验证,首次发现NOA患者精液中CCL8蛋白的浓度显著低于正常男性,并提出CCL8蛋白可作为评估NOA患者生精功能障碍的潜在预警分子,有助于扩充诊断NOA指标,以扩大诊断范围,对于NOA诊断领域具有重要意义。
优选地,所述CCL8蛋白的氨基酸序列包括SEQ ID NO.1所示的序列。
SEQ ID NO.1:
PDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLKP。
第二方面,本发明提供第一方面所述的非梗阻性无精子症相关的生物标志物在制备非梗阻性无精子症诊断或监测产品中的应用。
本发明首次将CCL8蛋白作为生物标志物应用于制备非梗阻性无精子症诊断或监测产品中,为开发新的非梗阻性无精子症诊断或监测产品提供思路。
第三方面,本发明提供一种非梗阻性无精子症诊断或监测试剂盒,所述试剂盒包括第一方面中所述CCL8蛋白的抗体。
本发明中,首次提出一种基于检测CCL8蛋白的非梗阻性无精子症诊断或监测试剂盒,所述试剂盒通过检测精液中CCL8蛋白的浓度,能够快速、准确的表征异常样本,从而辅助非梗阻性无精子症诊断或监测。
根据本发明,能够特异性识别CCL8蛋白且可应用于免疫检测的抗体均适用于本发明。
优选地,所述试剂盒还包括CCL8蛋白标准品、多孔板、洗涤液、稀释液、显色剂或终止液中的任意一种或至少两种的组合。
优选地,所述多孔板包括96孔板。
优选地,所述洗涤液包括磷酸盐缓冲液。
优选地,所述显色剂包括TMB底物溶液。
优选地,所述终止液包括硫酸。
本发明的试剂盒,采用常规酶联免疫吸附法(ELISA)测定CCL8蛋白浓度,商品化ELISA试剂均适用于本发明。
优选地,所述试剂盒的使用方法包括:
收集精液样本,以ELISA检测方法检测精液样本中CCL8蛋白的浓度,根据所述CCL8蛋白的浓度判断是否为非梗阻性无精子症阳性。
优选地,所述试剂盒的使用方法包括以下步骤:
(1)收集精液样本进行预处理;
(2)使用CCL8蛋白的抗体包被多孔板;
(3)按ELISA检测方法,检测所述精液样本中CCL8蛋白的浓度,根据所述CCL8蛋白的浓度判断是否为非梗阻性无精子症阳性。
优选地,步骤(1)所述预处理包括将所述精液样本置于35~38℃液化25~35min,离心并收集上清液并进行过滤。
优选地,步骤(2)所述包被多孔板包括使用磷酸盐缓冲液稀释CCL8蛋白的抗体,将稀释后溶液加入多孔板,于4~8℃放置12~16h,使用牛血清白蛋白封闭。
作为优选的技术方案,所述试剂盒的使用方法包括以下步骤:
(1)收集手淫取精的精液样本,在35~38℃水浴锅中放置25~35min进行液化,液化完毕后离心去除精液内精子和不溶物杂质,收集上清液,使用30~45μm滤网进一步去除杂质,得到精浆;
(2)使用磷酸盐缓冲液稀释CCL8蛋白的抗体,稀释至终浓度浓度为100ng/mL~20μg/mL,将稀释液加入多孔板,置于4~8℃冰箱中放置12~16h,使用牛血清白蛋白封闭,然后烘干装袋;
(3)取所述精浆,按照常规ELISA检测方法,检测其中CCL8蛋白的浓度,根据所述CCL8蛋白的浓度判断是否为非梗阻性无精子症阳性。
优选地,所述非梗阻性无精子症阳性的判断标准为精液样本中CCL8蛋白的浓度低于54.42ng/mL。
与现有技术相比,本发明具有以下有益效果:
本发明首次检测到非阻塞性无精子症精液样本中CCL8蛋白浓度显著低于正常精液样本,将精液中CCL8蛋白作为非阻塞性无精子症生物标志物,并提供一种非梗阻性无精子症诊断或监测试剂盒,通过检测精液中CCL8蛋白浓度能够辅助非梗阻性无精子症诊断或监测,具有安全、无创(不用抽血或者睾丸活检)和快速(全部检测流程在4h以内)等特点。
附图说明
图1为正常人与非梗阻性无精子症精浆内CCL8蛋白浓度结果图,****表示p<0.0001;
图2为为标准品中CCL8蛋白浓度与样本OD450关系曲线图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例提供一种非梗阻性无精子症诊断试剂盒,所述试剂盒中有CCL8蛋白的抗体(R&D Systems,MAB281-100)、HRP二抗(R&D Systems,HAF008)、CCL8蛋白标准品(R&DSystems,281-CP-010/CF)96孔板、稀释液、显色剂和终止液,其中稀释液为PBS,显色剂(底物显色A液:取醋酸钠13.6g、柠檬酸1.6g和30%双氧水0.3mL,加蒸馏水至500mL;底物显色B液:取乙二胺四乙酸二钠0.2g、柠檬酸0.95g、甘油50mL和0.15g TMB(溶于3mL DMSO中),加蒸馏水至500mL,现配现用),终止液为硫酸。
此外,还需对96孔板进行包被,包被方法为:使用磷酸盐缓冲液(pH 7.2)稀释CCL8蛋白抗体,稀释抗体至浓度至500ng/mL,将稀释后抗体加入96孔板,于4℃冰箱中放置过夜,第二天使用0.5%的牛血清白蛋白封闭,然后烘干装袋备用。
实施例2
本实施例利用实施例1中的试剂盒对精液样本进行检测。
分别取10名NOA患者的精液和10名正常男性精液,使用实施例1中的试剂盒检测各精液样本中CCL8蛋白的浓度,包括以下步骤:
(1)将80ng/mL的CCL8蛋白标准品通过对比稀释,得到80ng/mL、40ng/mL、20ng/mL、10ng/mL、5ng/mL和0ng/mL标准品液用于制作标准曲线;
(2)患者手淫获取的新型精液样本,放置于37℃水浴锅内30min进行液化;
(3)精液完全液化后,使用3000g/5min离心,取上清精浆;
(4)取100mL精浆和各浓度标准品放置于包被了抗体的96孔板中,37℃孵育30min;
(5)孵育结束后使用洗涤液洗涤3次,加入二抗室温孵育3min;
(6)再次洗涤3次,加入显色剂避光室温孵育10min,加入终止液;
(7)使用酶标仪检测各组吸光度,根据标准曲线(图2)确定精浆样本目标蛋白浓度。
结果如图1所示,NOA样本中CCL8蛋白的浓度显著低于正常样本中CCL8蛋白的浓度(p<0.001),表明CCL8蛋白可作为评估NOA患者生精功能障碍的生物标志物,且本发明分析正常样本中CCL8蛋白的浓度范围为54.42~67.75ng/mL,因此,当检测样本中CCL8蛋白的浓度低于54.42ng/mL时,可判断受检者睾丸生精微环境已经发生损伤,具有高非梗阻性无精子症阳性风险。
综上所述,本发明首次检测到非阻塞性无精子症精液样本中CCL8蛋白浓度显著低于正常精液样本,将精液中CCL8蛋白作为非阻塞性无精子症生物标志物,并提供一种非梗阻性无精子症诊断或监测试剂盒,通过检测精液中CCL8蛋白浓度能够辅助非梗阻性无精子症诊断或监测,具有安全、无创(不用抽血或者睾丸活检)和快速(全部检测流程在4h以内)等特点。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 中山大学附属第五医院
<120> CCL8蛋白作为非梗阻性无精子症的生物标志物及其应用
<130> 20211206
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 75
<212> PRT
<213> 人工序列
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Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr Asn
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Glu Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met Lys
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His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro
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Claims (10)
1.一种与非梗阻性无精子症相关的生物标志物,其特征在于,所述生物标志物包括CCL8蛋白。
2.根据权利要求1所述的生物标志物,其特征在于,所述CCL8蛋白的氨基酸序列包括SEQ ID NO.1所示的序列。
3.权利要求1或2所述的非梗阻性无精子症相关的生物标志物在制备非梗阻性无精子症诊断或监测产品中的应用。
4.一种非梗阻性无精子症诊断或监测试剂盒,其特征在于,所述试剂盒包括CCL8蛋白的抗体。
5.根据权利要求4所述的试剂盒,其特征在于,所述试剂盒还包括CCL8蛋白标准品、多孔板、洗涤液、稀释液、显色剂和终止液。
6.根据权利要求5所述的试剂盒,其特征在于,所述多孔板包括96孔板;
优选地,所述洗涤液包括磷酸盐缓冲液;
优选地,所述显色剂包括TMB底物溶液;
优选地,所述终止液包括硫酸。
7.根据权利要求4-6任一项所述的试剂盒,其特征在于,所述试剂盒的使用方法包括:
收集精液样本,以酶联免疫吸附法检测精液样本中CCL8蛋白的浓度,根据所述CCL8蛋白的浓度判断是否为非梗阻性无精子症阳性。
8.根据权利要求7所述的试剂盒,其特征在于,所述试剂盒的使用方法包括以下步骤:
(1)收集精液样本并进行预处理;
(2)使用CCL8蛋白的抗体包被多孔板;
(3)按酶联免疫吸附法,检测所述精液样本中CCL8蛋白的浓度,根据所述CCL8蛋白的浓度判断是否为非梗阻性无精子症阳性。
9.根据权利要求8所述的试剂盒,其特征在于,步骤(1)所述预处理包括将所述精液样本置于35~38℃液化25~35min,离心收集上清液并进行过滤;
优选地,步骤(2)所述包被多孔板包括使用磷酸盐缓冲液稀释CCL8蛋白的抗体,将稀释后CCL8蛋白的抗体加入多孔板,于4~8℃放置12~16h,使用牛血清白蛋白封闭。
10.根据所述权利要求4-9任一项所述的试剂盒,其特征在于,所述非梗阻性无精子症阳性的判断标准为精液样本中CCL8蛋白的浓度低于54.42ng/mL。
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