CN113820498A - 一种非梗阻性无精子症的生物标志物组合及其应用 - Google Patents
一种非梗阻性无精子症的生物标志物组合及其应用 Download PDFInfo
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Abstract
本发明提供了一种用于诊断非梗阻性无精子症的生物标志物的组合，所述生物标志物的组合包括FGF1、FGF9和FGF14，可将上述生物标志物的组合用于制备ELISA检测试剂盒，通过检测患者的血样中FGF1、FGF9和FGF14的表达情况来进行非梗阻性无精子症的诊断，可极大的减轻患者的痛苦，具有广阔的应用前景。
Description
技术领域
本发明属于医学诊断领域，更为具体的，本发明涉及一种非梗阻性无精子症的生物标志物的组合及其应用。
背景技术
大约30％-55％的人类不孕不育与男性因素有关，男性不育症最常见的原因是精子发生障碍，临床上称为弱精子症或无精子症。其中10-15％归因于无精子症。近年来，随着人们生殖健康需求的不断提高，男性不育越来越成为社会关注的焦点。WHO不育症防治专题组的研究表明生殖内分泌功能紊乱，特别是精液质量低下是男性不育的重要病因。在过去50年中，男性精液质量，尤其是精子生成数量有了相当程度的下降，我国的研究也呈现出相类似的情况。男性不育受到多方面因素的影响，包括环境因素，遗传因素以及表观遗传因素。由于短期内遗传变异相对有限，因此我们有理由相信，非梗阻性无精子症更可能是受到了某些环境危险因素的影响。
成纤维细胞生长因子(Fibroblast growth factor,FGF)家族具有促进细胞发育、细胞增殖、转移和分化等功能。FGF家族的22个成员通过结合和激活四种受体酪氨酸激酶(RTKs)编码的不同异构体来介导细胞反应。四种RTKs分别为FGFR1、FGFR2、FGFR3和FGFR4。FGF与肝素或硫酸乙酰肝素蛋白聚糖(HSPG)协同作用，以激活FGFR并诱导多效反应，从而导致FGF诱导多种细胞反应。不同的信号蛋白在FGF的刺激下磷酸化，包括Shc、磷脂酶-Cγ(PLCγ)、STAT1、Gab1和对接蛋白成纤维细胞受体底物2α(FRS2α)，从而刺激细胞内的信号通路，控制细胞繁殖、细胞分化、细胞转移、细胞存活和细胞形态。FGF1作为FGF家族中的一员，因其等电点偏酸性，又被称为酸性成纤维细胞生长因子(acid fibroblast growthfactor,aFGF)。FGF1不仅有激活细胞表面特定受体的经典作用方式，也可以独立于受体激活的信号通路来保护细胞免受应激条件的影响。FGF9最初发现于人类神经胶质瘤细胞系，是一种自分泌或旁分泌生长因子,在许多生理过程起着至关重要的作用,包括胚胎发育。主要分布于细胞和组织中,包括成骨细胞、软骨细胞、血管内皮细胞、晶状体上皮细胞,以及皮肤、肝、肾脏、胃、生殖系统、淋巴组织等。研究发现，FGF9具有多种生物学活性,作为胞间信号分子参与血管形成、胚胎发育、损伤修复、细胞凋亡、神经再生、肿瘤生长等多项生理和病理过程,尤其是在卵巢癌发生、骨骼发育、神经再生、性腺分化等过程中起着重要的作用。FGF14起初被发现主要在发育中的脑、脊髓、胸腺中表达，而进一步研究证实FGF14主要在发育中及成熟的神经系统表达，在CNS中高表达。
Elisa生物试验是一种敏感性高，特异性强，重复性好的实验诊断方法。由于其试剂稳定、易保存，操作简便，结果判断较客观等因素，已广泛应用在免疫学检验的各领域中。ELISA的基础是抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性，酶标记的抗原或抗体既保留其免疫学活性，又保留酶的活性。在测定时，受检标本(测定其中的抗体或抗原)与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开。再加入酶标记的抗原或抗体，也通过反应而结合在固相载体上。此时固相上的酶量与标本中受检物质的量呈一定的比例。加入酶反应的底物后，底物被酶催化成为有色产物，产物的量与标本中受检物质的量直接相关，故可根据呈色的深浅进行定性或定量分析。由于酶的催化效率很高，间接地放大了免疫反应的结果，使测定方法达到很高的敏感度。
目前，临床诊断无精子症的方法主要是通过睾丸穿刺活检，给病人带来巨大痛苦。因此，开发无精子症的生物标志物并将其用于无精子症的检测，是广大患者的普遍需求。
发明内容
为了弥补现有技术的缺陷，本发明公开了一种非梗阻性无精子症的生物标志物的组合及其应用，该组合在患者睾丸组织中和血清中均存在显著的差异性表达，因此可通过对患者血液中生物标志物表达量的分析，即可对非梗阻性无精症进行诊断，显著降低患者的痛苦。
本发明的第一个方面，提供一种非梗阻性无精子症的生物标志物的组合及其应用，其中，所述生物标志物的组合为FGF1、FGF9和FGF14。
在一种实施方式中，在非梗阻性无精子症患者血清和睾丸组织中，FGF1和FGF9的表达量上升，FGF14的表达量下调。
本发明的第二个方面，提供上述生物标志物的组合在用于制备无精子症诊断试剂盒中的应用。优选的，所述试剂盒为ELISA试剂盒。
本发明的第三个方面，提供一种用于诊断非梗阻性无精子症的试剂盒，其中，所述试剂盒包括用于检测样品中FGF1、FGF9和FGF14表达量的试剂。
在一种实施方式中，上述试剂盒优选为ELISA法检测试剂盒。
本发明相对于现有技术具有如下显著的进步：
1、本发明提供的生物标志物的组合在非梗阻性无精子症患者的血清中和睾丸组织中均存在相同的差异化表达，特异性强、准确度高；
2、根据上述生物标志物的组合制备的诊断试剂盒可通过抽血检查实现诊断，克服了现有技术中仅能通过睾丸穿刺活检诊断的技术障碍，极大地减轻了患者的痛苦，推动了实现疾病早发现、早诊治的治疗方针，对于无精子症的诊治和研究具有重大的意义。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1NOA造模后，ELISA实验结果：FGF1、FGF9和FGF14在血清中的表达水平；
图2NOA造模后，WB实验结果：FGF1、FGF9和FGF14在组织蛋白中的表达水平；
图3FGF1免疫组织化学染色；
图4FGF9免疫组织化学染色；
图5FGF14免疫荧光染色。
具体实施方式
以下结合附图对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
实施例1小鼠NOA模型的构建
方法：利用性成熟的雄性小鼠，体重为25-35g。取白消安溶解于DMSO，以生理盐水稀释得稀释液，在小鼠下腹部酒精消毒，晾干后，吸取白消安稀释液小鼠腹腔注射，完成首次腹腔注射之后，同一天内再间隔3h注射3次，35-37天后，即得所述无精子症小鼠模型。处死小鼠后取小鼠全血用于ELISA(酶联免疫吸附测定法)，同时取小鼠睾丸组织进行WB、H&E以及免疫荧光染色法检测，从而检验小鼠全血ELISA法的准确性。
实施例2ELISA法检测小鼠NOA模型血液样本的蛋白表达分析
NOA造模后，采用酶联免疫试剂盒(ELISAKit)检测小鼠血清中FGF1、FGF9、FGF14含量。
ELISA检测操作步骤如下：
(1)在已经包被好抗体的96孔板中分别加入50μl的FGF1、FGF9、FGF14标准品、血清样品和对照品，所述对照品为试剂盒中的空白对照品，即样品稀释液，用于去除试剂带来的本底吸光度差异。
所述标准品是试剂盒中给定的不同浓度标准品，用于制备标准曲线。
所述血清样品制备方法为：NOA模型小鼠摘眼球取血后，3000r，4℃离心15min，取上清，-80℃分装保存，然后直接用于试剂盒检测。
(2)每孔加入100μl的1×的STP-HRP液，室温孵育60min。
(3)每孔加入1×的洗涤液300μl，静置1min，弃去液体，拍干，重复洗涤5次，拍干。
(4)用移液器在每孔中加入50μl的底物液，室温避光孵育15min。
(5)每孔加入50μl的终止液，轻轻晃动几秒钟，保证液体混合均匀。
(6)即刻在450nm波长处检测吸光度。
(7)以标准品的浓度为横坐标，吸光度为纵坐标，绘制标准曲线，得出曲线方程，计算样品浓度。
结果显示：
采用Elisa法(酶联免疫吸附测定法)检测小鼠造模前后血清中蛋白表达差异，结 果如图1所示，小鼠NOA模型的睾丸组织及血清中的蛋白水平FGF9、FGF1表达显著上升， FGF14表达水平表达显著下降。
实施例3WB法、H&E法和免疫荧光染色法检测小鼠NOA模型睾丸组织样本的蛋白表达分析
为了进一步验证NOA模型血清中蛋白分析的结果，取血同时取建模小鼠睾丸组织用于WB、H&E法和免疫荧光染色法进行蛋白表达分析。具体操作步骤如下：
1.Westernblot操作步骤如下：
(1)总蛋白抽提：取NOA模型小鼠睾丸组织0.05g，加入0.5ml含PMSF的哺乳动物组织蛋白抽提试剂(抽提试剂：PMSF＝100:1)，用匀浆机匀浆至组织均匀。冰上静置15min后，然后置于4℃，12000rpm离心15min，将上清转移至EP管中。
(2)BCA法测定蛋白浓度：取0，1，2，4，8，12，16，20μl的0.5mg/ml的标准液于96孔板中，并且用0.1mol/LPBS溶液补足至20μl。然后，加2μl样品于96孔板中，加入0.1mol/LPBS溶液补足至20μl。根据样品量的需要，以1:50配制适量BCA工作液。将每孔200μl BCA溶液加到96孔板中，37℃恒温箱中放置30min。在562nm波长处用酶标仪测定各个孔的OD值(包括标准液和实验组)。
(3)固定样品体积及每孔上样量，根据样品浓度计算样品加样量，0.1mol/L PBS溶液补足至样品固定体积。混匀，用沸水煮10min，然后放置于-20℃中冷藏。
(4)配胶：根据蛋白分子量选择合适浓度的分离胶，按说明书配好，冷凝一段时间。再配5％的蛋白浓缩胶。
(5)电泳：80V电压下跑胶30min左右。此时，能在浓缩胶上将蛋白样品压成一条直线，待Marker开始分离，转换为120V电压下跑胶70min至结束。
(6)转膜及注意事项：300mA转膜90min。提前将海绵、滤纸等放入转膜液中浸润，PVDF膜提前用甲醇激活后使用，注意转膜液的温度不能过高，因此需要在外层加冰。
(7)封闭：在转膜操作完成后，将PVDF膜放入5％的脱脂牛奶中，置于摇床上封闭60min。
(8)孵育一抗：将一抗用一抗稀释液稀释至适宜浓度，放入4℃摇床摇过夜。第二天置于室温摇床，0.1％TBST洗涤3次各7min。
(9)孵育二抗：将二抗用0.1％的TBST稀释至适宜浓度，室温下用摇床(90rpm)振荡1h。0.1％TBST洗涤3次,每次各7min。
(10)曝光：在PVDF膜上加入ECL曝光液，用凝胶成像仪对条带进行曝光。对曝光时间进行适当调整，使成像清楚为宜。
(11)数据处理：运用Image Lab 6.0分析系统对结果进行灰度分析。以目的蛋白和相应内参条带的灰度比值表示目的蛋白的表达水平。
2.H&E操作步骤如下：
(1)脱蜡：将切好的石蜡切片放入干净的二甲苯I中30min，然后二甲苯II中30min。
(2)酒精梯度水化：利用100％的无水乙醇配置不同浓度的乙醇溶液。切片从二甲苯中脱蜡后，取出依次置于100％乙醇溶液I中5min，100％乙醇溶液II中5min，95％乙醇溶液I中5min，95％乙醇溶液II中2min，80％乙醇溶液中2min。
(3)干净的自来水中漂洗。
(4)染色和反蓝：用新鲜的苏木素浸泡切片5min，纯化水冲洗除去苏木素。放入10％氨水中返蓝10s，再用自来水清洗。
(5)将切片浸入5％的醋酸-乙醇溶液中分化5～20s。
(6)将切片置于伊红中3min，用纯化水稍洗干净。
(7)将切片放入95％酒精中45s，100％乙醇I中1min，100％乙醇II中2min。
(8)透明：将切片放入干净的二甲苯I中5min；二甲苯II中10min。
(9)将切片从二甲苯中取出，趁二甲苯未干时，用中性树脂快速封片。待切片干燥之后，显微镜下观察。
3.免疫荧光染色法操作步骤如下：
(1)脱蜡和水化：将制备好的NOA小鼠睾丸组织石蜡切片放入切片槽中，置于新鲜的二甲苯中，浸泡3次每次10min；去除多余的液体后置于无水乙醇中，浸泡3次，每次3min；去除多余的液体后置于95％乙醇中浸泡2次，每次3min；去除多余的液体后再置于75％乙醇中浸泡2次，每次3min；然后再蒸馏水冲洗1min，置于PBS缓冲液中。
(2)高压抗原修复：将配制好0.01mol/L枸橼酸钠缓冲液放入高压锅内加热至95℃左右，然后将切片放入高压锅的枸橼酸钠缓冲液槽中，加盖后加热至冒气2min，然后停止加热。当高压锅的蒸汽压力和外面大气压平衡后，打开锅盖。等锅内温度降至室温后，取出各组切片。放于0.1mol/L PBS溶液中冲洗5min。
(3)阻断内源性过氧化物酶：加入适量的内源性过氧化物酶阻断剂。室温孵育10min；PBS缓冲液冲洗3min×3次。
(4)滴加一抗：根据切片上组织的大小，滴加100μl或适量的一抗，于37℃恒温箱中孵育60min，PBS缓冲液冲洗3次，每次3min。
(5)0.1mol/L PBS洗三次，将带有IgG/FITC荧光标签的酶标二抗(稀释比例：1:1000)滴到组织上，然后放入湿盒中37℃避光孵育1h。
(6)0.1mol/L PBS洗三次，用含抗荧光淬灭剂的DAPI试剂避光作用7min染核，盖玻片进行封片。
利用倒置荧光显微镜对细胞进行观察及拍照。
结果如图2-5所示：
通过WB实验(图2)可以明显看出在睾丸组织中，FGF1和FGF9表达升高，FGF14表达下降。
通过免疫组织化学染色法可以看出FGF1(图3)和FGF9(图4)表达升高表达升高。
通过免疫荧光染色法(图5)可以看出FGF14表达下降。
由此可见，小鼠NOA模型中FGF1、FGF9和FGF14在血清中和睾丸组织中的表达结果均一致，相对于未患病对照组具有显著的差异化表达，可作为非梗阻性无精症的生物标志物的组合用于无精症的诊断。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (5)
1.一种非梗阻性无精子症的生物标志物的组合，其特征在于，所述生物标志物的组合为FGF1、FGF9和FGF14。
2.根据权利要求1所述的生物标志物的组合，其特征在于，在非梗阻性无精子症患者血清和睾丸组织中，FGF1和FGF9的表达量上升，FGF14的表达量下调。
3.根据权利要求1所述的生物标志物的组合在用于制备无精子症诊断试剂盒中的应用。
4.一种用于诊断非梗阻性无精子症的试剂盒，其特征在于，所述试剂盒包括用于检测样品中FGF1、FGF9和FGF14表达量的试剂。
5.如权利要求4所述的试剂盒，其特征在于，所述试剂盒为ELISA法检测试剂盒。
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