摘要
本文在国内首次报告了一种勿需组织培养的成人阴茎包皮中５ａ－还原酶Ⅰ型、Ⅱ型活性的测定方法与结果。包皮环切后的组织经匀浆，１２００００Ｘｇ离心得到粗微粒体组分。以氚标记睾酮为底物、还原型辅酶Ⅱ型（ＮＡＤＰＨ）为供氢体进行酶促反应。产物─—氚标记双氢睾酮经乙醚提取后薄层层析分离。并根据其放射性强度进行定量。结果表明，Ⅰ型酶和Ⅱ型酶的底物浓度与酶反应速度关系曲线均符合米氏方程，其Ｋｍ值分别为０．４６和０．０７９μｍｏｌ／Ｌ。２２例正常成年男性包皮的５ａ－还原酶活性（ｘ±ｓ）：Ⅰ型酶为２１．５±１２．５３ＤＨＴｐｍｏｌ·ｍｇｐｒｏｔｅｉｎ－１／３０ｍｉｎ；Ⅱ型酶为５０．８３±３５．７０ＤＨＴｐｍｏｌ·ｍｇｐｒｏｔｅｉｎ－１／３０ｍｉｎ。本方法的平均批内变异系数为９．５０％和５．０２％；平均批间变异系数为１２．７５％和８．９１％；灵敏度为０．０４２ｐｍｏｌ。所以，本方法简便可靠，可用于诊断和鉴别诊断性分化异常病和人类生殖的基础研究。
bjectives :To develop a method for measuring the steroid 5a- eductase isozyme type I and type Il ac-tivities in foreskn of Chinese adult men.Design:A laboratorgy methodology and its clinical application.setting:The endocrinology laboratory of National Research Institute for Family Planning.Subjects: 22 Chinese adult men. Intervention :Foreskin specimens were taken from the patients at the time of circumcision with consentand were quickly frozen in liquid nitrogen and stored at一7O℃. Methods :The procedure of the method includes:homogenization of tissue in Tris/chloride buffer on ice;centrifugation of supernatant at 120 000g to get microsomal peIlets and resuspended by Dounce homogeniza-tion :standard assay of 5a- reductase isozyme activities of microsomal pellet;labelled testosterone used as sub-strate; steroids were extracted and separated with thin layer chromatography;3 H-dihydrotestosteroneformed was counted for radioactivitv. Results :The steroid 5a- reductase isozyme activities were characterized by pH optima 5.5 and a broaderrange of 7～9,and exhibit different kinetics with respect to substrate testosterone. Km values for theisozvme II and I were 0.079 and 0.46μmol/L,respectively.The isozyme I and II activities in foreskin of22 Chinese adult men were 21.15± 1 53 and 50.8335.70(x 士s) pmoles DHT formation/mg protein/30min,respectively. Conclusions :This method of measuring 5a-reductase activities has the advantages of being stable,highlysensitive,easy to perform and more accurate than RIA of DH It can be used in clinical practice for diagno-sis of 5a-reductase deficiencv and other sexual differentiation disorders.It is also valuable in the basic re- search on BPH and hurman reproduction.
出处
《生殖医学杂志》
CAS
1995年第1期11-15,共5页
Journal of Reproductive Medicine
