中国生化药物杂志
中國生化藥物雜誌
중국생화약물잡지
Chinese Journal of Biochemical Pharmaceutics
2015年
9期
26-29
,共4页
江伟雯%许艺飞%陈娅琦%蔡泳锋%吴清和%黄萍%操红缨
江偉雯%許藝飛%陳婭琦%蔡泳鋒%吳清和%黃萍%操紅纓
강위문%허예비%진아기%채영봉%오청화%황평%조홍영
精子密度%吸光度%SD大鼠%KM小鼠%少精模型
精子密度%吸光度%SD大鼠%KM小鼠%少精模型
정자밀도%흡광도%SD대서%KM소서%소정모형
sperm density%absorbance%SD rat%KM mice%oligospermia model
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＝2×10－9 x1＋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＝5×10－9 x2＋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光度-精子密度曲线方程法可以快速、准确的测定KM小鼠和SD大鼠的精子密度。
目的：利用酶標儀建立一種快速、準確的測定大、小鼠精子密度的方法。方法①最適波長確立與迴歸方程建立：雄性KM小鼠和SD大鼠各6隻，脫頸椎處死後分離左側附睪，于PBS內充分剪碎，水浴充分遊離精子，用酶標儀分彆在300、380、450、530、600 nm下測定吸光度，擬閤吸光度麯線，取相關繫數最接近于1及標準差最小的為最佳波長；取KM小鼠、SD大鼠各10隻，乙醚過量痳醉緻死後用PBS稀釋得到4、8、16、36倍的精子懸液，用酶標儀檢測吸光度併使用細胞計數闆對樣品進行計數，以精子密度為橫坐標及吸光度為縱坐標建立迴歸方程。②精子吸光度穩定性檢測：KM小鼠和SD大鼠各6隻，乙醚過量痳醉緻死後製備左側附睪懸液，樣品置于室溫（25℃）或繼續37℃水浴，併于水浴後的0、20、30、40、50、60 min，取樣品測定吸光度，記錄樣品的吸光度變化。③迴歸方程驗證：使用20％乙醇灌胃30 d造KM小鼠少精癥模型，採用吸光度-精子密度麯線方程及細胞計數闆計數2種方法測定造模KM小鼠精子密度，驗證新方法。結果確立KM小鼠、SD大鼠吸光度-精子密度麯線在380 nm處可以建立最適迴歸方程，KM小鼠精子密度x1與吸光度y1的關繫為線性函數，線性迴歸方程為y1＝2×10－9 x1＋0.0648，R2＝0.9743；SD大鼠精子密度x2與吸光度y2的關繫為線性函數，線性迴歸方程為y2＝5×10－9 x2＋0.0621，R2＝0.9940；SD大鼠精子懸液在水浴60 min後，A值與0 min時相比顯著減低（P＜0.05），但在常溫下40 min後顯著升高（P＜0.05）；KM小鼠精子懸液在水浴和常溫條件下50 min後，與0 min時相比，A值顯著升高（P＜0.05）；與正常對照組相比，用酶標儀檢測和標準麯線計算的乙醇少精組的精子密度均顯著下降（P＜0.05）；與標準麯線計算相比，通過細胞計數闆方法檢測乙醇少精組的精子密度沒有顯著變化。吸光度-精子密度麯線方程法可有效檢測齣少精癥動物模型精子密度的減少。結論利用酶標儀建立吸光度-精子密度麯線方程法可以快速、準確的測定KM小鼠和SD大鼠的精子密度。
목적：이용매표의건립일충쾌속、준학적측정대、소서정자밀도적방법。방법①최괄파장학립여회귀방정건립：웅성KM소서화SD대서각6지，탈경추처사후분리좌측부고，우PBS내충분전쇄，수욕충분유리정자，용매표의분별재300、380、450、530、600 nm하측정흡광도，의합흡광도곡선，취상관계수최접근우1급표준차최소적위최가파장；취KM소서、SD대서각10지，을미과량마취치사후용PBS희석득도4、8、16、36배적정자현액，용매표의검측흡광도병사용세포계수판대양품진행계수，이정자밀도위횡좌표급흡광도위종좌표건립회귀방정。②정자흡광도은정성검측：KM소서화SD대서각6지，을미과량마취치사후제비좌측부고현액，양품치우실온（25℃）혹계속37℃수욕，병우수욕후적0、20、30、40、50、60 min，취양품측정흡광도，기록양품적흡광도변화。③회귀방정험증：사용20％을순관위30 d조KM소서소정증모형，채용흡광도-정자밀도곡선방정급세포계수판계수2충방법측정조모KM소서정자밀도，험증신방법。결과학립KM소서、SD대서흡광도-정자밀도곡선재380 nm처가이건립최괄회귀방정，KM소서정자밀도x1여흡광도y1적관계위선성함수，선성회귀방정위y1＝2×10－9 x1＋0.0648，R2＝0.9743；SD대서정자밀도x2여흡광도y2적관계위선성함수，선성회귀방정위y2＝5×10－9 x2＋0.0621，R2＝0.9940；SD대서정자현액재수욕60 min후，A치여0 min시상비현저감저（P＜0.05），단재상온하40 min후현저승고（P＜0.05）；KM소서정자현액재수욕화상온조건하50 min후，여0 min시상비，A치현저승고（P＜0.05）；여정상대조조상비，용매표의검측화표준곡선계산적을순소정조적정자밀도균현저하강（P＜0.05）；여표준곡선계산상비，통과세포계수판방법검측을순소정조적정자밀도몰유현저변화。흡광도-정자밀도곡선방정법가유효검측출소정증동물모형정자밀도적감소。결론이용매표의건립흡광도-정자밀도곡선방정법가이쾌속、준학적측정KM소서화SD대서적정자밀도。
Objective To establish a rapid and accurate method, and to determine the density of mice and rats sperm with enzyme-labeled instrument.Methods ①The 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 for 30 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 -9 x1 +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 significantly increased ( P<0.05); KM mice sperm suspension in the water bath and under the condition of normal temperature after 50 min, compared with the 0 min, absorbance value increased significantly(P<0.05).Compared with control group, sperm density of ethanol oligozoospermia group by enzyme standard detector and standard curve calculation were significantly decreased ( P <0.05 );compared with absorbancy-sperm density equation, determination of ethanol oligozoospermia group of sperm density by cell counting plate method had not significant difference.The results suggested absorbancy-sperm density equation could effectively detect the reduction of the mice sperm in oligospermia.Conclusion Using enzyme-labeled instrument to set up the curve of absorbancy-sperm density equation can estimate the sperm density of mice and rats rapidly and exactly.
