CN106885794B - 一种人类精子线粒体功能检测方法 - Google Patents
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Abstract
本发明公开了一种人类精子线粒体功能检测的方法，特点是步骤为：(1)将液化的精液用37℃缓冲液稀释，每个标本处理两管，分别作为对照管和检测管；(2)将500μL稀释过的精子加入到流式细胞仪上样管中，同时加入5μL的JC‑1染液，避光染色15分钟，其中对照管置于85℃条件下2分钟，灭活，检测管置于37℃条件下保持活力；(3)设定流式细胞仪的校准，对照管、检测管按顺序上机，每管样本至少连续测定两次，每管样本至少记录5000个细胞并使用MMP软件统计分析；(4)结果判断依据现已有大量临床数据计算出的正常人范围值设定；该方法优点是操作简单、结果易于判定、检测结果准确、稳定可靠、重复性好、且易于临床推广。
Description
技术领域
本发明涉及医疗卫生部门的应用领域，特别涉及一种人类精子线粒体功能检测方法。
背景技术
随着经济的发展和医疗水平的提高，不孕不育的发病率反而呈增长趋势，已成为当今影响人类生活和健康的一类主要疾病，其中在我国，不孕不育症在育龄期夫妇中发病率达10％以上，并有逐渐增多的趋势。男性因素和女性因素各占30％，双方共同因素占40％。男性因素中精子原因居首位。世界卫生组织(WHO)在25个国家9000对不育夫妇的多中心研究中发现，50％的男性不育症是特发性精子异常。精子活力是衡量精液质量和男性生育力的一个重要指标。男科学上把精子因活力不足而导致的男性生育能力低下，称为弱精子症或精子活力低下。在男性不育患者中弱精子症约占19％，已成为影响男性生育力的常见病因。精子尾部中段主要由线粒体构成，线粒体活性的改变会影响精子的能量供应，精子持续运动能力，而且线粒体还是细胞凋亡的调控中枢。因此，线粒体的功能状态是精子功能质量的一个关键性指标。线粒体活性的评价主要通过检测线粒体的跨膜电位变化来分析。
发明内容
本发明所要解决的技术问题是提供一种操作简单、结果易于判定、检测结果准确、稳定可靠、重复性好、且易于临床推广的人类精子线粒体功能检测方法。
本发明解决上述技术问题所采用的技术方案为：一种人类精子线粒体功能检测方法，包括如下步骤：
(1)将液化的精液用37℃缓冲液稀释精子至1～2×106个/mL，每个标本处理两管，分别作为对照管和检测管；
(2)将500μL稀释过的精子加入到流式细胞仪上样管中，同时加入5μL的JC-1染液，避光染色15分钟，其中对照管置于85℃条件下2分钟，灭活，检测管置于37℃条件下保持活力；
(3)设定流式细胞仪的校准，对照管、检测管按顺序上机，每管样本至少连续测定两次，每管样本至少记录样本主群5000个细胞并使用MMP软件统计分析；
(4)根据MMP软件统计分析得出结果，将37℃正常处理样本百分比减去85℃高温处理后的空白样本百分比，得到的MMP百分比差值即区分出正常人群百分比数值；
(5)测定后，分别用漂白剂、进样管清洁剂和除菌的双蒸水彻底清除残留于流式细胞仪进样线中的细胞碎片和荧光染料。
步骤(1)中所述的缓冲液为氯化钠、磷酸二氢钾、葡萄糖、氯化钾、酸镁、氯化钙、N-2-羟乙基哌嗪-N-2-乙磺酸二钠盐、碳酸氢钠、丙酮酸钠、牛血清白蛋白和乳酸钠的混合物。
步骤(2)中所述的JC-1染色液为JC-1和二甲基亚砜的混合物。
与现有技术相比，本发明的优点在于：本发明一种人类精子线粒体功能检测方法，该方法采用对线粒体膜电位变化敏感的染料JC-1，亲脂性阳离子荧光染料5,5′，6,6′-四氯-1,1′,3，3′-四乙基苯并咪唑基-羰化青碘(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanineiodide，JC-1)，是检测精子线粒体膜电位△ψm最理想的染料。正常精子线粒体膜电位△ψm高，在膜内JC-1以聚合体存在，发橙色光；异常精子线粒体膜电位△ψm降低，JC-1以单体形式存在，发绿色光，其采用流式细胞术进行分析，具有操作简单、结果易于判定、检测结果准确、稳定可靠、重复性好、且易于临床推广的优点。
具体实施方式
以下结合实施例对本发明作进一步详细描述。
具体实施例
一种人类精子线粒体功能检测方法，包括如下步骤：
(1)将液化的精液用37℃缓冲液稀释精子至1～2×106个/mL，每个标本处理两管，分别作为对照管和检测管；
(2)将500μL稀释过的精子加入到流式细胞仪上样管中，同时加入5μL的JC-1染液，避光染色15分钟，其中对照管置于85℃条件下2分钟，灭活，检测管置于37℃条件下保持活力；
(3)设定流式细胞仪的校准，对照管、检测管按顺序上机，每管样本至少连续测定两次，每管样本至少记录样本主群5000个细胞并使用MMP软件统计分析；
(4)根据MMP软件统计分析得出结果，将37℃正常处理样本百分比减去85℃高温处理后的空白样本百分比，得到的MMP百分比差值即区分出正常人群百分比数值；
(5)测定后，分别用漂白剂、进样管清洁剂和除菌的双蒸水彻底清除残留于流式细胞仪进样线中的细胞碎片和荧光染料。
上述漂白剂为50％的双蒸水ddH2O和50％约含5％的次氯酸钠的混合物，进样管清洁剂为50％的乙醇、50％的漂白剂和0.5M NaCl的混合物。
当然，上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内做出的变化、改型、添加或替换，也应属于本发明保护范围。
Claims (1)
1.一种人类精子线粒体功能检测方法，其特征在于包括如下步骤：
(1)将液化的精液用37℃缓冲液稀释精子至1～2×106个/mL，每个标本处理两管，分别作为对照管和检测管，其中所述的缓冲液为氯化钠、磷酸二氢钾、葡萄糖、氯化钾、酸镁、氯化钙、N-2-羟乙基哌嗪-N-2-乙磺酸二钠盐、碳酸氢钠、丙酮酸钠、牛血清白蛋白和乳酸钠的混合物；
(2)将500μL稀释过的精子加入到流式细胞仪上样管中，同时加入5μL的JC-1染色液，避光染色15分钟，其中对照管置于85℃条件下2分钟，灭活，检测管置于37℃条件下保持活力，所述的JC-1染色液为JC-1和二甲基亚砜的混合物；
(3)设定流式细胞仪的校准，对照管、检测管按顺序上机，每管样本至少连续测定两次，每管样本至少记录样本主群5000个细胞并使用MMP软件统计分析；
(4)根据MMP软件统计分析得出结果，将37℃正常处理样本百分比减去85℃高温处理后的空白样本百分比，得到的MMP百分比差值即区分出正常人群百分比数值；
(5)测定后，分别用漂白剂、进样管清洁剂和除菌的双蒸水彻底清除残留于流式细胞仪进样线中的细胞碎片和荧光染料。
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