酶标仪快速测定大鼠、小鼠精子密度的方法研究  

Rapidly detecting the sperm concentration in mice and rats based on enzyme-labeled instrument

作　　者：江伟雯 许艺飞 陈娅琦 蔡泳锋 吴清和[1] 黄萍[1] 操红缨[1] 

机构地区：[1]广州中医药大学中药学院,广东广州510006

出　　处：《中国生化药物杂志》2015年第9期26-29,共4页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的利用酶标仪建立一种快速、准确的测定大、小鼠精子密度的方法。方法 1最适波长确立与回归方程建立:雄性KM小鼠和SD大鼠各6只,脱颈椎处死后分离左侧附睾,于PBS内充分剪碎,水浴充分游离精子,用酶标仪分别在300、380、450、530、600 nm下测定吸光度,拟合吸光度曲线,取相关系数最接近于1及标准差最小的为最佳波长;取KM小鼠、SD大鼠各10只,乙醚过量麻醉致死后用PBS稀释得到4、8、16、36倍的精子悬液,用酶标仪检测吸光度并使用细胞计数板对样品进行计数,以精子密度为横坐标及吸光度为纵坐标建立回归方程。2精子吸光度稳定性检测:KM小鼠和SD大鼠各6只,乙醚过量麻醉致死后制备左侧附睾悬液,样品置于室温(25℃)或继续37℃水浴,并于水浴后的0、20、30、40、50、60 min,取样品测定吸光度,记录样品的吸光度变化。3回归方程验证:使用20%乙醇灌胃30 d造KM小鼠少精症模型,采用吸光度-精子密度曲线方程及细胞计数板计数2种方法测定造模KM小鼠精子密度,验证新方法。结果确立KM小鼠、SD大鼠吸光度-精子密度曲线在380 nm处可以建立最适回归方程,KM小鼠精子密度x1与吸光度y1的关系为线性函数,线性回归方程为y1=2×10-9x1+0.0648,R2=0.9743;SD大鼠精子密度x2与吸光度y2的关系为线性函数,线性回归方程为y2=5×10-9x2+0.0621,R2=0.9940;SD大鼠精子悬液在水浴60 min后,A值与0 min时相比显著减低(P<0.05),但在常温下40 min后显著升高(P<0.05);KM小鼠精子悬液在水浴和常温条件下50 min后,与0 min时相比,A值显著升高(P<0.05);与正常对照组相比,用酶标仪检测和标准曲线计算的乙醇少精组的精子密度均显著下降(P<0.05);与标准曲线计算相比,通过细胞计数板方法检测乙醇少精组的精子密度没有显著变化。吸光度-精子密度曲线方程法可有效检测出少精症动物模型精子密度的减少。结论利用酶标仪建立吸�Objective To establish a rapid and accurate method,and to determine the density of mice and rats sperm with enzyme-labeled instrument. Methods 1 The optimal wavelengths and the regression equation set up： After six Kunming mice and six Sprague-Dawley rats were sacrificed,the left epididymis was separated and fully cut up in phosphate buffer saline. With water bath,the sperm were fully dissociated. Using the enzyme-labeled instrument to detect the wavelength absorbance respectively under different wavelength and fitting absorbance curve. The best wavelength will be the most close to 1 of the correlation coefficient（ R2） and the standard deviation of minimum. After ten Kunming mice and ten Sprague-Dawley rats were sacrificed,the sperm suspension of different concentration gradient were got. The regression equation of the sperm density and absorbance was established by using enzyme-labeled instrument and haemocytometer. 2The test of sperm absorbance stability： Mice and rats,six respectively,were used to make the sperm suspension. Samples were put in room temperature（ 25 ℃） or 37 ℃ water bath continued,and after water bath about 0,20,30,40,50,60 min,the change of absorbance was recorded. 3The regression equation verification： The mice were administrated orally with 20% ethanol solution for30 days to make oligospermia. In order to verify the new method,two different method were used to get the sample sperm. Results The optimal absorbancy-sperm density curve could be established at 380 nm. The means of KM mice sperm count（ x1） and absorbance（ y1） are showed to be the linear function,and the linear regression equation is y1= 2 × 10- 9x1＋ 0. 0648,R2= 0. 9743. The means of SD rat sperm count（ x2） and absorbance（ y2） are showed to be the linear function,and the linear regression equation is y2= 5 × 10- 9x2＋ 0. 0621,R2= 0. 9940. SD rat sperm suspension liquid after 60 min in water bath,absorbance value at 0 min significantly decreased（ P 0. 05）,but at room temperature after 40 min

关 键 词：精子密度 吸光度 SD大鼠 KM小鼠 少精模型 

分 类 号：R965[医药卫生—药理学]