CN101319973B - 一种精子核蛋白裂解液 - Google Patents

一种精子核蛋白裂解液 Download PDF

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CN101319973B
CN101319973B CN2008100165047A CN200810016504A CN101319973B CN 101319973 B CN101319973 B CN 101319973B CN 2008100165047 A CN2008100165047 A CN 2008100165047A CN 200810016504 A CN200810016504 A CN 200810016504A CN 101319973 B CN101319973 B CN 101319973B
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sperm
lysate
grams
halo
dna
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CN101319973A (zh
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张丽红
邱毅
王磊光
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SHANDONG PROV INST OF BIRTH CONTROL SCIENCE
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SHANDONG PROV INST OF BIRTH CONTROL SCIENCE
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Abstract

本发明公开了一种精子核蛋白裂解液，每100毫升蒸馏水中含三羟甲基氨基甲烷4.844克、氯化钠11.7克、二硫苏糖醇1.86克、皂角或桔梗提取物8-15克及乙二胺四乙酸二钠盐12.32克，pH值为中性。本发明裂解液所含的皂角提取物不使蛋白变性，丝毫不破坏精子尾部。本发明所述的裂解液实现了精子DNA检测简单、快速、准确、廉价、分辨率高的目的。

Description

一种精子核蛋白裂解液
技术领域
本发明涉及医学临床上使用的试剂，是一种精子核蛋白裂解液。
背景技术
目前，世界许多国家都在研究精子成熟过程中因受内外环境影响形成DNA碎片出现的DNA完整性异常，从而引起的精子形态和功能异常。由于在男性不育检测中DNA的完整性是精液的常规检测，也是评估男性不育的主要指标，因此，如何检测精子DNA完整性是生殖医学领域研究的重要课题之一。国外比较领先的检测方法为精子染色质扩散试验，其英文名为spermchromatin dispersion，简称SCD，其商品化的试验盒已推广使用。SCD检测方法具有较多优点，例如：简单、快速、准确、分辨率高等，但是，由于试剂盒包含知识产权，因此该试剂盒价格昂贵，受此限制，目前在我国尚未推广应用，并且，这种试剂盒中的精子核蛋白裂解液采用聚乙二醇辛基苯基醚，十二烷基碘酸钠等成份，这些成份均为化学试剂，其异味、刺激性及腐蚀性较大，并具有毒性成份，污染环境等。
发明内容
本发明的目的是，提供一种精子核蛋白裂解液，使它具有造价低、无腐蚀，无毒，易于推广应用的优点。
本发明为实现上述目的，通过以下技术方案实现：一种精子核蛋白裂解液，每100毫升蒸馏水中含三羟甲基氨基甲烷4.844克、氯化钠11.7克、二硫苏糖醇1.86克、皂角或桔梗提取物8-15克及乙二胺四乙酸二钠盐12.32克，PH值为中性。皂角或桔梗提取物为10克。
本发明裂解液的试验成功，为我国应用SCD方法检测精子DNA完整性提供了重要的环节。本发明所述的裂解液中采用皂角或桔梗提取物替代了化学试剂，从而减少了化学试剂的应用，减少污染、无刺激性、无腐蚀、无毒，并使裂解液制造成本大幅降低，易于推广应用。本发明裂解液为精子染色质扩散实验试剂盒的裂解液，试验结果表明：采用本发明所述的裂解液用SCD方法检测精子DNA完整性的效果与国外同类试剂盒的检测效果完整性一致。SCD试验后经染色能够在光学显微镜下清晰的观察保留尾巴的单个精子DNA的完整性，根据晕环的大小，能够准确判定精子DNA是否有碎片，从而对男性生育力进行准确评估。
本发明所述裂解液可由附图1、2、3、4、5、6、7中清楚得出：正常精子没有DNA碎片，具有大大的晕环或中等晕环，如附图1、2所示，存在DNA碎片的精子没有晕环形成或有很小的晕环，或没有晕环且着色很淡，呈退化迹象。如附图3、4、5所示，上述试验进一步表明，本发明裂解液能够准确判定精子DNA是否具有完整性。上述试验结果还可具体描述为：计数500个精子，观察精子晕环大小，根据晕环与精子头部横径的比例，粗分为大、中、小和无晕环4个等级，大和中晕环表示精子DNA完整无碎片，小和无晕环表示精子DNA断裂为碎片。小晕环以≤精子头直径四分之一为判断标准，中晕环＞精子头直径四分之一而≤精子头直径三分之二，大晕环＞精子头直径三分之二。
本发明裂解液所含的皂角提取物不使蛋白变性，丝毫不破坏精子尾部。本发明所述的裂解液实现了精子DNA检测简单、快速、准确、廉价、分辨率高的目的。
本发明裂解液以试剂盒商品推广应用，试剂盒包括：
1、DNA变性染液；
2、精子核蛋白裂解液；
DNA变性染液为公知技术。
附图说明
附图1是呈现大晕环的正常精子；附图2是呈现中晕环的正常精子；附图3是呈现极小晕环的有DNA碎片的异常精子；附图4是没有晕环的有DNA碎片的异常精子；附图5是没有晕环且退化的精子；附图3、4、5所示均为有DNA碎片的异常精子；附图6是有三个正常精子和一个异常精子的图片；附图7是有两个正常精子和三个异常精子的图片。
上述图片均为采用本发明裂解液进行检验的结果。
具体实施方式
本发明所述的精子核蛋白裂解液，每100毫升蒸馏水中含三羟甲基氨基甲烷4.844克、氯化钠11.7克、二硫苏糖醇1.86克、皂角或桔梗提取物8--15克及乙二胺四乙酸二钠盐12.32克，PH值为中性。其中三羟甲基氨基甲烷分子式为[(HOCH2)3CNH2]，PH值为7.5，所述皂角或桔梗提取物选用的最佳值为10克。

Claims (2)

1.一种精子核蛋白裂解液，其特征在于：每100毫升蒸馏水中含三羟甲基氨基甲烷4.844克、氯化钠11.7克、二硫苏糖醇1.86克、皂角或桔梗提取物8-15克及乙二胺四乙酸二钠盐12.32克，PH值为中性。
2.根据权利要求1所述的一种精子核蛋白裂解液，其特征在于：皂角或桔梗提取物为10克。
CN2008100165047A 2008-05-28 2008-05-28 一种精子核蛋白裂解液 Expired - Fee Related CN101319973B (zh)

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CN101907620B (zh) * 2010-08-10 2013-09-11 山东省计划生育科学技术研究所 一种同时检测精子脱氧核糖核酸碎片及膜完整性的方法
CN111595658B (zh) * 2020-06-06 2023-11-14 宾傲 一种提取细胞中蛋白的裂解液及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5919621A (en) * 1991-10-24 1999-07-06 Brown; David B. Methods for diagnosing human male infertility
CN1965881A (zh) * 2006-11-01 2007-05-23 山东省计划生育科学技术研究所 中药桔梗作为体外杀精子药物的应用及其制备药物的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5919621A (en) * 1991-10-24 1999-07-06 Brown; David B. Methods for diagnosing human male infertility
CN1965881A (zh) * 2006-11-01 2007-05-23 山东省计划生育科学技术研究所 中药桔梗作为体外杀精子药物的应用及其制备药物的方法

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