糖尿病對大鼠睪丸病理變化及睪酮合成、17β-HSD3和3β-HSD1 mRNA表達(dá)的影響

1、糖尿病對大鼠睪丸病理變化及睪酮合成、17-HSD3和3-HSD1 mRNA表達(dá)的影響         08-11-03 09:52:00     編輯：studa20               作者：林刻智，王文艷，肖艷，王蓉蓉，方周溪，陳國榮  【摘要】  目的: 研究大鼠糖尿病時睪丸的主要病理變化及對間

2、質(zhì)細(xì)胞（Leydig細(xì)胞）睪酮合成、17-HSD3和3-HSD1 mRNA表達(dá)的影響。方法: 雄性SD大鼠19只，隨機(jī)分為正常對照(NC)組10只，糖尿病(DM)組9只。DM組予高脂飲食加小劑量（25 mg/kg）鏈脲佐菌素（STZ）誘導(dǎo)2型糖尿病大鼠模型，12周后處死。用光鏡及電鏡觀察大鼠睪丸的組織形態(tài)學(xué)改變；RT-PCR法檢測睪丸組織17-羥基類固醇脫氫酶型 (17-HSD3)和3-羥基類固醇脫氫酶型(3-HSD1) mRNA含量；酶聯(lián)免疫吸附法測定血清中睪酮含量。結(jié)果: 糖尿病大鼠光鏡下主要表現(xiàn)為睪丸曲細(xì)精管生精細(xì)胞數(shù)量減少及生精阻滯，Leydig細(xì)胞縮小，細(xì)胞核皺縮；透射電鏡下主要見到

3、Leydig細(xì)胞萎縮，線粒體、內(nèi)質(zhì)網(wǎng)等細(xì)胞器空泡樣變及擴(kuò)張，細(xì)胞核皺縮，染色質(zhì)聚集；睪丸組織的17-HSD3和3-HSD1 mRNA含量和血清中睪酮含量糖尿病組均低于正常對照組。結(jié)論: 糖尿病可使大鼠睪丸Leydig細(xì)胞變性，睪酮合成降低，其機(jī)制可能與降低17-HSD3和3-HSD1 mRNA表達(dá)有關(guān)。 【關(guān)鍵詞】  糖尿?。徊G丸；間質(zhì)細(xì)胞；睪酮；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    間質(zhì)細(xì)胞(Leydig細(xì)胞)產(chǎn)生睪酮和曲細(xì)精管生精是睪丸的兩大主要生理功能，前者制約后者。Leydig細(xì)胞是成年雄性動物睪酮的主要來源細(xì)胞，其功能狀態(tài)影響動物的精子發(fā)生和性功能1。睪丸合成過程中，17-HSD3、3-HSD1催化脫氫異雄酮形成睪酮2，3。有研究表明，糖尿病大鼠睪丸間質(zhì)細(xì)胞功能障礙，睪

10、酮的合成及分泌降低4-6，但其機(jī)制尚未明了。本實驗通過光鏡和電鏡觀察糖尿病大鼠睪丸間質(zhì)細(xì)胞的病理改變，同時測定并分析睪丸組織17-HSD3及 3-HSD1 mRNA含量及血清中睪酮含量變化，以對其機(jī)制進(jìn)行探討。    1  材料和方法    1.1  動物分組及處理  雄性SD大鼠19只(由溫州醫(yī)學(xué)院實驗動物中心提供)，體重180220 g，鼠齡2 個月。隨機(jī)分組：2型糖尿病 (DM) 組9只，予高糖高脂飼料（飼料組成：10.0%豬油，20.0%蔗糖，2.5%膽固醇，1.0%膽酸鹽，66.5%常規(guī)飼料）飼

11、養(yǎng)1個月后，按25 mg/kg劑量腹腔注射鏈脲佐菌素（STZ）(廈門星隆達(dá)化學(xué)試劑有限公司產(chǎn)品，溶于0.1 mmol/L枸櫞酸緩沖液內(nèi)，pH=4.0)，注射前禁食12 h，注射后72 h空腹血糖8.0 mmol/L為模型成功。正常對照 (NC)組10只，喂以普通飼料，腹腔內(nèi)注射等容積枸櫞酸緩沖液。此后，DM組繼續(xù)給以高糖高脂飲食，NC組給以普通飲食，12周后處死。    1.2  血清睪酮測定  股動脈放血處死大鼠，各取血3 ml，2000 r/min離心15 min，吸取上清液置-30 冰箱保存，用酶聯(lián)免疫吸附法測定睪酮含量。 &#

12、160;  1.3  光鏡標(biāo)本的制備  剪開陰囊，暴露睪丸，每只大鼠左睪丸白膜下用2.5%戊二醛原位固定0.5 h后，橫切面剖開睪丸切成3 mm薄片，置于10%中性福爾馬林液中固定，經(jīng)常規(guī)乙醇脫水，二甲苯透明，石蠟包埋，制作切片，厚3m，HE染色，中性樹膠封固。每個睪丸觀察10個低倍視野。    1.4  電鏡標(biāo)本的制備  上述白膜下原位固定0.5 h后的睪丸，切成1 mm×1 mm×1 mm小塊，迅速置2.5%戊二醛4 固定l h，鋨酸后固定，常規(guī)PBS漂洗、丙酮梯度脫水，Epon812包埋，RMC-XL型超薄切片機(jī)切片，鉛鈾雙重染色，于H-7500型透射電鏡下觀察。    1.5  RT-PCR  取右側(cè)睪丸新鮮組織100 mg加1ml Triol(美國Invitrogen公司)，提取各組大鼠睪丸組織的總RNA。取2g總RNA，用Promega逆轉(zhuǎn)錄試劑盒合成cDNA。取1l cDNA為模板，在Taq DNA 聚合酶(ProbeGene公司)催化下應(yīng)用特異性引物行PCR擴(kuò)增。引物設(shè)計參照Gene Bank中17-HSD3，3-HSD1 mRNA，RPS16 mRNA（作為內(nèi)參照）序