糖尿病对大鼠睾丸病理变化及睾酮合成、17β-HSD3和3β-HSD1 mRNA表达的影响

1、糖尿病对大鼠睾丸病理变化及睾酮合成、17-HSD3和3-HSD1 mRNA表达的影响         08-11-03 09:52:00     编辑：studa20               作者：林刻智，王文艳，肖艳，王蓉蓉，方周溪，陈国荣  【摘要】  目的: 研究大鼠糖尿病时睾丸的主要病理变化及对间

2、质细胞（Leydig细胞）睾酮合成、17-HSD3和3-HSD1 mRNA表达的影响。方法: 雄性SD大鼠19只，随机分为正常对照(NC)组10只，糖尿病(DM)组9只。DM组予高脂饮食加小剂量（25 mg/kg）链脲佐菌素（STZ）诱导2型糖尿病大鼠模型，12周后处死。用光镜及电镜观察大鼠睾丸的组织形态学改变；RT-PCR法检测睾丸组织17-羟基类固醇脱氢酶型 (17-HSD3)和3-羟基类固醇脱氢酶型(3-HSD1) mRNA含量；酶联免疫吸附法测定血清中睾酮含量。结果: 糖尿病大鼠光镜下主要表现为睾丸曲细精管生精细胞数量减少及生精阻滞，Leydig细胞缩小，细胞核皱缩；透射电镜下主要见到

3、Leydig细胞萎缩，线粒体、内质网等细胞器空泡样变及扩张，细胞核皱缩，染色质聚集；睾丸组织的17-HSD3和3-HSD1 mRNA含量和血清中睾酮含量糖尿病组均低于正常对照组。结论: 糖尿病可使大鼠睾丸Leydig细胞变性，睾酮合成降低，其机制可能与降低17-HSD3和3-HSD1 mRNA表达有关。 【关键词】  糖尿病；睾丸；间质细胞；睾酮；17-HSD3；3-HSD1    Abstract:  Objective: To study the  major pathological change of testis，the c

4、hange of testosterone synthesis function of interstitial cell （Leydig cell) and the expression of 17-HSD3 mRNA and 3-HSD1 mRNA in diabetic rat. Methods: Nineteen male Sprague-Dauley rats were divided randomly into two groups: normal control group and diabetic group. The diabetic group was fed with h

5、igh-fat diet and injected with strepozotocin intraperitoneally to induce type 2 diabetes mellitus. Twelve weeks later，the rats were sacrificed. The morphologic change of testicular tissue was observed under light microscopy (LM) and transmission electron microscopy (TEM)，respectively. The mRNA expre

6、ssion level of 17-hydroxysteroid dehydrogenase type 3（17-HSD3）and 3-hydroxysteroid dehydrogenase type 1（3-HSD1）of testicular tissue were detected by RT-PCR，the serum testosterone (T) concentration was measured by ELISA. Results: The number of  spermatogenic cell in seminiferous tubule was decre

7、ased and germinal arrest. Under LM， the Leydig cells were diminished and the nuclear was shrinked in diabetic group. Ultrastructural analysis of Leydig cells by TEM showed dilatation of the endoplasmic reticulum and mitochondrial swelling，and the chromatin was highly condensed in diabetic group. The

8、 mRNA levels of 17-HSD3 and 3-HSD1 of testicular tissue and the serum testosterone concentrations decreased in diabetic group compared with normal control group. Conclusion: Diabetes can cause morphologic change of Leydig cells and decrease T production. The decreasing mRNA expression of 17-HSD3 and

9、 3-HSD1 may be involved in it.    Key words:  diabetes mellitus；testis；Leydig cells；testosterone；17-HSD3；3-HSD1    间质细胞(Leydig细胞)产生睾酮和曲细精管生精是睾丸的两大主要生理功能，前者制约后者。Leydig细胞是成年雄性动物睾酮的主要来源细胞，其功能状态影响动物的精子发生和性功能1。睾丸合成过程中，17-HSD3、3-HSD1催化脱氢异雄酮形成睾酮2，3。有研究表明，糖尿病大鼠睾丸间质细胞功能障碍，睾

10、酮的合成及分泌降低4-6，但其机制尚未明了。本实验通过光镜和电镜观察糖尿病大鼠睾丸间质细胞的病理改变，同时测定并分析睾丸组织17-HSD3及 3-HSD1 mRNA含量及血清中睾酮含量变化，以对其机制进行探讨。    1  材料和方法    1.1  动物分组及处理  雄性SD大鼠19只(由温州医学院实验动物中心提供)，体重180220 g，鼠龄2 个月。随机分组：2型糖尿病 (DM) 组9只，予高糖高脂饲料（饲料组成：10.0%猪油，20.0%蔗糖，2.5%胆固醇，1.0%胆酸盐，66.5%常规饲料）饲

11、养1个月后，按25 mg/kg剂量腹腔注射链脲佐菌素（STZ）(厦门星隆达化学试剂有限公司产品，溶于0.1 mmol/L枸橼酸缓冲液内，pH=4.0)，注射前禁食12 h，注射后72 h空腹血糖8.0 mmol/L为模型成功。正常对照 (NC)组10只，喂以普通饲料，腹腔内注射等容积枸橼酸缓冲液。此后，DM组继续给以高糖高脂饮食，NC组给以普通饮食，12周后处死。    1.2  血清睾酮测定  股动脉放血处死大鼠，各取血3 ml，2000 r/min离心15 min，吸取上清液置-30 冰箱保存，用酶联免疫吸附法测定睾酮含量。 &#

12、160;  1.3  光镜标本的制备  剪开阴囊，暴露睾丸，每只大鼠左睾丸白膜下用2.5%戊二醛原位固定0.5 h后，横切面剖开睾丸切成3 mm薄片，置于10%中性福尔马林液中固定，经常规乙醇脱水，二甲苯透明，石蜡包埋，制作切片，厚3m，HE染色，中性树胶封固。每个睾丸观察10个低倍视野。    1.4  电镜标本的制备  上述白膜下原位固定0.5 h后的睾丸，切成1 mm×1 mm×1 mm小块，迅速置2.5%戊二醛4 固定l h，锇酸后固定，常规PBS漂洗、丙酮梯度脱水，Epon812包埋，RMC-XL型超薄切片机切片，铅铀双重染色，于H-7500型透射电镜下观察。    1.5  RT-PCR  取右侧睾丸新鲜组织100 mg加1ml Triol(美国Invitrogen公司)，提取各组大鼠睾丸组织的总RNA。取2g总RNA，用Promega逆转录试剂盒合成cDNA。取1l cDNA为模板，在Taq DNA 聚合酶(ProbeGene公司)催化下应用特异性引物行PCR扩增。引物设计参照Gene Bank中17-HSD3，3-HSD1 mRNA，RPS16 mRNA（作为内参照）序