發(fā)布時(shí)間：2018-09-18 06:58

【摘要】：目的通過(guò)觀察不同溫度條件下體外大鼠睪丸支持細(xì)胞GDNF表達(dá)特點(diǎn),探討高溫致不育的機(jī)制方法采用組合酶消化法和選擇性貼壁法分離雄性Wistar大鼠睪丸支持細(xì)胞(Sertoli cell,SC),將分離的SC分別置于不同溫度進(jìn)行體外培養(yǎng),觀察其貼壁、形態(tài)學(xué)變化,FASL免疫組化鑒定。實(shí)驗(yàn)分為對(duì)照組(35℃)、實(shí)驗(yàn)組(36℃、37℃、38℃、39℃)。 CCK-8法檢測(cè)SC增殖情況,HE染色觀察細(xì)胞形態(tài)及結(jié)構(gòu),RT-PCR\免疫熒光及Western Blotting檢測(cè)細(xì)胞GDNF的表達(dá)。結(jié)果本實(shí)驗(yàn)條件下,體外分離培養(yǎng)SC純度=(95.30±2.15)%(n=10), CCK-8實(shí)驗(yàn)結(jié)果顯示,36℃時(shí)細(xì)胞增殖率最高(P0.01),36℃時(shí),隨著溫度的提高,增殖率逐漸降低,39℃對(duì)細(xì)胞增殖具有明顯抑制作用(P0.01)；免疫熒光結(jié)果顯示,GDNF表達(dá)于支持細(xì)胞胞質(zhì),36℃時(shí)熒光最強(qiáng),RT-PCR及Western Blotting法檢測(cè)結(jié)果顯示,高于36℃時(shí),GDNF mRNA及蛋白的表達(dá)隨著溫度的增加而降低,36℃、37℃、38℃、39℃四組與對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論不同溫度條件下,體外培養(yǎng)SC的增殖能力和GDNF表達(dá)明顯不同。36℃時(shí),隨著溫度的提高,增殖能力受到抑制,GDNF表達(dá)水平顯著降低。本研究結(jié)果證實(shí),高溫條件下大鼠睪丸支持細(xì)胞正常功能受到抑制損害,并可能導(dǎo)致睪丸生精環(huán)境的破壞,而影響生精功能。
[Abstract]:Objective to observe the expression of GDNF in rat testicular Sertoli cells at different temperatures. To explore the mechanism of sterility induced by high temperature the testicular Sertoli cells (Sertoli cell,SC) of male Wistar rats were isolated by combined enzyme digestion and selective adherent method. The isolated SC was cultured at different temperatures in vitro to observe its adherent. Morphological changes were identified by FASL immunohistochemistry. The experiment was divided into control group (35 鈩，

本文編號(hào)：2247138