CN112945924A - 一种高通量筛选KSper缺陷不育人精子样本的方法 - Google Patents
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Abstract
本发明公开了一种高通量筛选KSper缺陷不育人精子样本的方法，属于医学技术领域。本发明基于精子膜电位主要由KSper决定、而膜电位可通过荧光染料以荧光信号形式呈现间接由多功能酶标仪测定，建立基于多功能酶标仪的KSper异常不育样本的高通量筛选方法，为进一步深入探讨人精子KSper通道的生理作用及在评价男性不育病因中的临床意义，同时为寻找治疗不育症的有效途径提供新思路。
Description
技术领域
本发明属于医学技术领域，具体涉及一种高通量筛选KSper缺陷不育人精子样本的方法。
背景技术
精子特异性KSper通道是哺乳动物精子上主要K+通道，与精子运动、膜超极化及顶体反应等息息相关。在体KSper电流研究表明，在小鼠精子中，KSper是由Slo3为主亚基与LRRC52作为辅助亚基共同构成的一种强pH敏感、电压依赖的钾离子通道。Slo3敲除小鼠完全失去生育能力，Slo3-/-精子的前向运动、顶体反应明显下降，并丧失穿卵能力，通过膜片钳也几乎记录不到KSper电流，胞内碱化也不会引发膜的超极化。另外，将小鼠Slo3与LRRC52体外表达，LRRC52能够显著改善KSper通道开放程度，使Slo3更易开放。在LRRC52敲除小鼠中，LRRC52-/-生育能力下降，KSper通道开放程度降低，通道激活所需时间更长，表明在生理条件下小鼠精子KSper通道在各方面调控精子功能影响精子生育能力。而人精子KSper呈现弱pH敏感性而具有强的钙依赖性，且Slo3与LRRC52在人精子中均存在表达，但是关于人精子KSper通道的生理作用、其异常是否是导致男性不育的重要原因尚未清楚的研究。
KSper通道作为精子上唯一介导Iksper的K+通道，其异常是否是导致男性不育的重要原因，也为研究特发性男性不育的诊断与治疗提供了新方向。现有利用人精子膜片钳方法测定KSper电流响应的筛选方法，该方法虽然准确，但步骤繁琐，耗时较长，并存在着技术上挑战，未能高效筛选KSper响应缺陷不育样本，另外存在一种以流式细胞仪通过荧光方法检测膜电位信号，判断膜电位信号的去极化与超极化，但因人精子形态结构的特殊性，该方法对于检测人精子膜电位信号具有较高的仪器设备要求，而且无法达到高通量大样本的碱性刺激后迅速、高效的检测膜电位前后变化，因而无法用于高通量筛选KSper响应缺陷不育样本。
发明内容
本发明的目的是提供一种高通量筛选KSper缺陷不育人精子样本的方法，基于精子膜电位主要由KSper决定、而膜电位可通过荧光染料以荧光信号形式呈现间接由多功能酶标仪测定，建立基于多功能酶标仪的KSper异常不育样本的高通量筛选方法。
为了实现上述发明目的，本发明采用以下技术手段：
一种高通量筛选KSper缺陷不育人精子样本的方法，包括以下步骤：
步骤1，精液样本的处理：将收集到的精液样本水浴液化；
步骤2，梯度离心分离纯化人精子：采用Percoll梯度分离液对液化好的精液样本进行纯化；
步骤3，精子孵育染色：用荧光探针3,3'-二丙基硫杂羰花青碘化物对纯化后的精子样本进行染色；
步骤4，高通量测定精子膜电位信号：向染色后的精子样本中加入刺激物，通过测定加入刺激物前后精子样本膜电位的荧光信号变化，从而筛选出KSper响应缺陷不育人精子样本。
进一步地，步骤1中的水浴液化是在37℃水浴中液化60min。
进一步地，步骤2中采用Percoll梯度分离液对液化好的精液样本进行纯化的具体过程为：将体积浓度为80％的Percoll液加至离心管底部，然后用沿管壁依次加入体积浓度为40％的Percoll液和液化好的精液样本，80％Percoll液、40％Percoll液、液化的精液样本的体积比为1∶1∶1，2000rpm离心分离，弃去上层精浆层、40％Percoll液和部分80％Percoll液，留取底层沉淀，加入37℃预热的HS溶液重悬，1800rpm离心，弃去上清，加入HS溶液重悬，将精子浓度调整至3～8×107/mL，即可。
进一步地，步骤3中用荧光探针对精子样本染色的具体过程为：用2μM荧光探针3,3'-二丙基硫杂羰花青碘化物与纯化后的精子样本重悬混匀，置于37℃、5％CO2培养箱中避光染色25min，取出后1800rpm离心5min，吸去上清液以去除精子细胞外残留的染料，加入HS溶液重悬精子沉淀，1800rpm离心5min，去除上清，重复一次，即得染色后的精子溶液。
进一步地，步骤4中，所述刺激物为：250mM KCl、pH8.5的HS溶液、50μM钾离子载体Valinomycin。
进一步地，步骤4中测定荧光信号变化的条件为：激发波长为620nm，发散波长为680nm，温度37℃。
本发明利用了高通量酶标仪自动加药避免时间差、快速、结果稳定、可重复、大样本量大数据等特点，以及Disc3(5)荧光探针具有很高淬灭系数，强偏光依赖性、很短的激发寿命、稳定和灵敏等优点，高效筛选KSper响应缺陷的人精子不育样本。
相对于膜片钳筛选方法而言，这种高通量膜电位筛选方法能够简便，快捷，高效的监测人精子样本KSper离子通道的缺陷情况。为进一步深入探讨人精子KSper通道的生理作用及在评价男性不育病因中的临床意义，同时为寻找治疗不育症的有效途径提供新思路。
附图说明
图1为实施例1的样本1中人精子膜电位对各种刺激响应信号的测量结果。
图2为实施例1的样本2中人精子膜电位对各种刺激响应信号的测量结果。
图3为人精子检测膜电位信号方法的校准结果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
目前通过人精子膜片钳技术、流式细胞仪筛选KSper电流缺失样本技术难度高，步骤繁琐，耗时较长，未能高效筛选样本得到数据。本发明基于精子膜电位主要由KSper决定、而膜电位通过荧光染料染色可由多功能酶标仪测定，建立基于多功能酶标仪的KSper异常不育样本的高通量筛选方法。以健康生育男性作为正常对照，正常人精子的KSper将响应胞内pH升高(由胞外碱化实现)而开放，其开放将导致精子膜超极化；以特发性不育男性患者作为研究对象，KSper电流低下的精子其超极化程度将显著降低，KSper缺失的精子其不发生超极化。观察KSper对响应胞内碱化人精子膜超极化为指标，连续高效快速筛选KSper是否引起细胞膜超极化的人精子样本，为进一步深入探讨人精子KSper通道的生理作用及评价其异常在男性不育症中的临床意义。
本发明中，所述HS溶液的配方为：135mM NaCl，5mM KCl，1mM MgSO4，2mM CaCl2，20mM HEPES，5mM蔗糖，10mM乳酸，1mM丙酮酸钠，通过使用NaOH调节pH至7.4。
实施例1
1.精液样本的处理
将当天收集到的精液样本置于37℃恒温水浴中液化60min。轻吹打混匀，取10μL液化后的精液，添加入洁净的伟力计算机辅助精液分析仪(CASA)分析板，将分析板放于CASA中，调整焦距，检测人精子的各项参数，对比符合《世界卫生组织人类精液检查与处理实验室手册》第5版(WHO5)精液标准参数(精子活力≥40％、前向运动≥32％、精子浓度≥15％)，初筛精子样本，选取精液常规正常且较好的样本。结果如表1所示。
表1.计算机辅助精液分析仪(CASA)分析人精子样本各项功能参数
| 总活力 | 前向运动 | 精子浓度 | |
| 标准 | ≥40％ | ≥32％ | ≥15×10<sup>6</sup>/mL |
| 样本1 | 56.34％ | 50.23％ | 26.17×10<sup>6</sup>/mL |
| 样本2 | 59.49％ | 50.75％ | 28.81×10<sup>6</sup>/mL |
2.Percoll法梯度离心分离纯化人精子
(1)分别配制40％、80％Percoll梯度分离液，具体配方为40％Percoll液(3％BSA∶10×EBSS培养基∶Percoll液∶ddH2O＝1∶1∶4∶4)、80％Percoll液(3％BSA∶10×EBSS培养基∶Percoll液＝1∶1∶8)。
(2)取15mL无菌离心管，将80％Percoll液加至离心管底部，而用移液器沿管壁依次缓慢的且匀速的分别加入40％Percoll液和液化好的精液样本，80％Percoll液∶40％Percoll液∶液化的精液样本＝1∶1∶1。
(3)2000rpm离心15min，观察有无沉淀，若无沉淀追加离心5min。
(4)弃去上层精浆层、40％Percoll液、部分80％Percoll液，留取底层700μL左右沉淀，加入2ml 37℃预热HS重悬。
(5)1800rpm离心6min。弃去上清，加入适量HS重悬。将精子浓度调整至3～8×107/mL，取10μL置于CASA检测，保证精子活力参数正常。
3.精子孵育染色
用2μM荧光探针3,3'-二丙基硫杂羰花青碘化物(Disc3(5))与纯化样本重悬轻轻混匀，置于37℃，5％CO2培养箱中避光染色25min，1800rpm离心5min，吸去上清液以去除精子细胞外残留的染料，加入HS溶液重悬精子沉淀，1800rpm离心5min，去除上清，重复一次，37℃避光备用既得染色后的精子溶液。
4.高通量测定精子膜电位信号
将多功能酶标仪(Flexstation 3,Molecular Devices)提前开机预热30min，同时配制：pH7.4 HS溶液(对照)、膜电位去极化对照250mM KCl、超极化对照50μM Valinomycin、实验组1M NaOH调节HS pH至碱性pH:8.5，于37℃预热(胞外高浓度K+抑制胞内K+外流，使细胞膜电位去极化；Valinomycin：钾离子载体，能特异性的协助钾离子从高浓度一侧往低浓度一侧扩散，可使K+扩散速率提高10万倍，使胞内K+外流，精子细胞超极化)。
取180μL孵育后的精子样本置于黑色底板透明的96孔板中，添加200μL刺激物至试剂透明96孔板，设置仪器的激发波长为620nm，发散波长为680nm，使仪器温度保持在37℃，设定加样时间为第120s，自动吸取加样的体积为20μL，加样前检测的120s膜电位信号的荧光基底值为F0，加样后检测的膜电位信号荧光基底值为F，酶标仪检测10min内精子膜电位各指标的荧光变化情况，确定胞内pH升高(胞外碱化)对人精子KSper通道的影响，进一步筛选KSper响应缺陷不育人精子样本。根据以下公式计算不同精子样本的KSper离子通道响应精子胞内碱化而引起的超极化的影响情况。公式如下：膜电位荧光信号变化百分数(％)＝ΔF/F0×100％(F0,刺激前平均荧光；ΔF，每个测量点荧光值减去F0)。
比较图1和图2中可知，样本1受到胞外碱化刺激后，精子发生超极化，而样本2受胞外刺激后，精子基本上未出现超极化现象。同时结合表1中CASA测试数据也可初步判断样本1、2均为常规检测参数为正常的正常精子样本，因此，提示样本2精子膜上KSper离子通道可能存在功能障碍。
相对于膜片钳筛选方法而言，本发明的高通量膜电位筛选方法能够简便，快捷，高效的监测人精子样本KSper离子通道的缺陷情况。同时，如图3所示，本发明还对人精子检测膜电位方法进行了校准，可为今后可临床大样本量应用，也为人精子膜电位指标异常检测与筛选提供了前期思路与依据。
人精子检测膜电位方法的校准：
利用Valinomycin(缬氨霉素：K+载体)结合于精子细胞膜，特异性将K+由胞内高浓度释放至胞外低浓度，使K+排空，精子发生超极化，进一步在胞外通过多次加入不同体积的250mM KCl溶液(8μL、13.2μL、13.2μL、13.2μL)，改变胞外K+浓度，形成精子膜内外K+浓度差以及荧光信号变化。
再利用以下方程得到校准曲线：
其中R为气体常数R＝8.314J/(mol.k)，T为绝对温度，F为法拉第常数F＝eNA。精子胞内K+溶度为120mM[K+]i＝120mM。
Claims (6)
1.一种高通量筛选KSper缺陷不育人精子样本的方法，其特征在于：包括以下步骤：
步骤1，精液样本的处理：将收集到的精液样本水浴液化；
步骤2，梯度离心分离纯化人精子：采用Percoll 梯度分离液对液化好的精液样本进行纯化；
步骤3，精子孵育染色：用荧光探针3,3'-二丙基硫杂羰花青碘化物对纯化后的精子样本进行染色；
步骤4，高通量测定精子膜电位信号：向染色后的精子样本中加入刺激物，通过测定加入刺激物前后精子样本膜电位的荧光信号变化，从而筛选出KSper响应缺陷不育人精子样本。
2.根据权利要求1所述的方法，其特征在于：步骤1中的水浴液化是在37℃水浴中液化60 min。
3.根据权利要求1所述的方法，其特征在于：步骤2中采用Percoll 梯度分离液对液化好的精液样本进行纯化的具体过程为：将体积浓度为80% 的Percoll液加至离心管底部，然后用沿管壁依次加入体积浓度为40% 的Percoll液和液化好的精液样本，80% Percoll液、40% Percoll液、液化的精液样本的体积比为1∶1∶1，2000rpm离心分离，弃去上层精浆层、40% Percoll液和部分80% Percoll液，留取底层沉淀，加入37℃预热的HS溶液重悬，1800rpm离心，弃去上清，加入HS溶液重悬，将精子浓度调整至3~8×107/mL，即可。
4.根据权利要求1所述的方法，其特征在于：步骤3中用荧光探针对精子样本染色的具体过程为：用2 μM 荧光探针3,3'-二丙基硫杂羰花青碘化物与纯化后的精子样本重悬混匀，置于37℃、5 % CO2培养箱中避光染色25 min，取出后1800 rpm 离心 5 min，吸去上清液以去除精子细胞外残留的染料，加入 HS溶液重悬精子沉淀，1800 rpm 离心 5 min，去除上清，重复一次，即得染色后的精子溶液。
5.根据权利要求1所述的方法，其特征在于：步骤4中，所述刺激物为：250 mM KCl、pH8.5的HS溶液、50 μM钾离子载体Valinomycin。
6.根据权利要求1所述的方法，其特征在于：步骤4中测定荧光信号变化的条件为：激发波长为 620 nm，发散波长为 680 nm，温度37℃。
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