【摘要】：目的:研究抗雄激素受體氟他胺(Flutamide,Flu)孕中、晚期誘導(dǎo)子代雄鼠發(fā)生隱睪,及其病理組織學(xué)損害,探討其不育的病理學(xué)基礎(chǔ),為臨床治療隱睪所致非梗阻性不育提供依據(jù)。方法:將20只SD(sprague dawley)孕鼠隨機(jī)分為Flu隱睪組(n=10)、正常對(duì)照組(n=10),Flu隱睪組于妊娠12~21 d給予Flu 25 mg/(kg·d)皮下注射,正常對(duì)照組不予任何處理,取各組出生后第60天(postnatal day 60,PND60)的雄性SD大鼠睪丸組織(Flu隱睪組只取隱睪睪丸),HE染色觀察睪丸組織形態(tài)學(xué)的差異,透射電鏡觀察睪丸支持細(xì)胞間的緊密連接結(jié)構(gòu),TUNEL凋亡染色檢測(cè)生精細(xì)胞凋亡情況,取附睪尾進(jìn)行精子計(jì)數(shù)及形態(tài)觀測(cè),免疫組織化學(xué)和Western blot檢測(cè)睪丸組織中生殖細(xì)胞增殖分化指標(biāo)視黃酸刺激因子8(stimulated by retinoic acid gene 8,Stra8)、聯(lián)會(huì)復(fù)合體蛋白3(synaptonemal complex protein 3,SCP3)的表達(dá),采用Q-PCR檢測(cè)Stra8基因水平。結(jié)果:收集正常對(duì)照組正常睪丸30只與Flu隱睪組隱睪睪丸22只,HE染色顯示隱睪睪丸管腔明顯縮窄、生精細(xì)胞發(fā)育遲緩且排列紊亂、管腔中央無(wú)精子形成;TUNEL凋亡檢測(cè)證實(shí)隱睪組有大量生精細(xì)胞發(fā)生凋亡,精子計(jì)數(shù)結(jié)果為隱睪組(1.99±0.13)×108個(gè)/m L,遠(yuǎn)低于正常對(duì)照組[(5.53±0.17)×108個(gè)/m L,P=0.000];透射電鏡(transmission electron microscope,TEM)可觀察到SD大鼠隱睪支持細(xì)胞間的緊密連接結(jié)構(gòu)疏松;免疫組織化學(xué)顯示Stra8在隱睪組織中表達(dá)較正常對(duì)照組少,SCP 3在正常對(duì)照組睪丸生精小管的各級(jí)生精細(xì)胞胞核中均有表達(dá),而在隱睪組睪丸中表達(dá)很弱;Western blot結(jié)果顯示,Flu隱睪組Stra8蛋白表達(dá)量(0.34±0.05)明顯低于正常對(duì)照組(0.96±0.09),P=0.002;Flu隱睪組SCP3蛋白表達(dá)量(0.39±0.03)也明顯低于正常對(duì)照組(0.97±0.07),P=0.001。Q-PCR結(jié)果顯示,Flu組SD大鼠睪丸組織內(nèi)Stra8 m RNA的表達(dá)(0.765±0.015)較正常對(duì)照組(1.00±0.01)低,P=0.01。結(jié)論:Flu誘導(dǎo)的SD大鼠隱睪模型中,睪丸呈明顯病理形態(tài)學(xué)改變,支持細(xì)胞間緊密連接結(jié)構(gòu)破壞,Stra8及SCP3表達(dá)明顯下調(diào),生精細(xì)胞凋亡增多,精子數(shù)量及質(zhì)量下降,這可能是導(dǎo)致生精細(xì)胞發(fā)育障礙的重要原因。
[Abstract]:Objective: to study the histopathological damage of cryptorchidism in male offspring induced by antiandrogen receptor flutamide (Flutamide,Flu) in pregnancy and to explore the pathological basis of sterility in order to provide evidence for clinical treatment of non-obstructive sterility induced by cryptorchidism. Methods: twenty pregnant rats with SD (sprague dawley) were randomly divided into Flu cryptorchidism group (n = 10). The normal control group (n = 10), Flu) received subcutaneous injection of Flu 25 mg/ (kg d) on the 21st day of gestation, while the normal control group did not receive any treatment. The testicular tissues of male SD rats (Flu cryptorchidism group) were collected on the 60th day after birth (Flu cryptorchidism group) to observe the difference of testicular morphology with), HE staining, and the tight junctional structure of testicular Sertoli cells was observed by transmission electron microscope. The apoptosis of spermatogenic cells was detected by TUNEL apoptosis staining, sperm count and morphology were observed from epididymal tail, and Retinoic acid stimulating factor (8 (stimulated by retinoic acid gene 8 Stra8) was detected by immunohistochemistry and Western blot. The expression of synaptophysin 3 (synaptonemal complex protein 3 was detected by Q-PCR. Results: 30 normal testis in normal control group and 22 cryptorchidism testis in Flu cryptorchidism group were collected. HE staining showed that testicular lumen of cryptorchidism was obviously constricted, spermatogenic cells were slow and disordered, and azoospermia was formed in the central lumen. TUNEL apoptosis assay confirmed that a large number of spermatogenic cells were apoptotic in cryptorchidism group. The sperm count in cryptorchidism group was (1.99 鹵0.13) 脳 108 / mL, which was much lower than that in normal control group [(5.53 鹵0.17) 脳 108 / mL]. Transmission electron microscopy (transmission electron microscope,TEM) showed that the tight junctional structure of cryptorchidism Sertoli cells in SD rats was loose. Immunohistochemistry showed that the expression of Stra8 in cryptorchidism was less than that in control group. The expression of SCP 3 was found in the nucleus of spermatogenic cells of testicular seminiferous tubule in normal control group, but was weak in cryptorchidism testis. Western blot results showed that the expression of Stra8 protein in Flu cryptorchidism group (0.34 鹵0. 05) was significantly lower than that in normal control group (0. 96 鹵0. 09). The expression of SCP3 protein in Flu cryptorchidism group (0.39 鹵0.03) was also significantly lower than that in normal control group (0.97 鹵0.07). The expression of Stra8 m RNA in testis of SD rats in Flu group (0.765 鹵0.015) was lower than that in normal control group (1.00 鹵0.01). Conclusion: in the SD rat model of cryptorchidism induced by Flu, the testis showed obvious pathomorphological changes, the structure of supporting cell tight junction was destroyed, the expression of Stra8 and SCP3 was down-regulated, the apoptosis of spermatogenic cells was increased, and the quantity and quality of spermatozoa were decreased. This may be an important factor in the development of spermatogenic cells.
【作者單位】： 重慶醫(yī)科大學(xué)附屬兒童醫(yī)院泌尿外科兒童發(fā)育疾病研究教育部重點(diǎn)實(shí)驗(yàn)室兒科學(xué)重慶市重點(diǎn)實(shí)驗(yàn)室重慶市兒童發(fā)育重大疾病診治與預(yù)防國(guó)際科技合作基地;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(編號(hào):81100415) 教育部博士點(diǎn)基金資助項(xiàng)目(編號(hào):20115503120009)
【分類號(hào)】：R-332;R726.9

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