不同温度下体外大鼠睾丸支持细胞胶质细胞源性神经生长因子的表达

Expression of GDNF in rat Sertoli cells cultured in vitro at different temperatures

目的:通过观察不同温度下体外大鼠睾丸支持细胞(Sertoli cell,SC)胶质细胞源性神经生长因子(GDNF)表达特点,探讨高温致不育的机制。方法:采用组合酶消化法和选择性贴壁法分离雄性Wistar大鼠睾丸SC,将分离的SC分别置于不同温度进行体外培养,观察其贴壁、形态学变化,FasL免疫组化鉴定。实验分为对照组(35℃)、实验组(36℃、37℃、38℃、39℃)。CCK-8法检测SC增殖情况,HE染色观察细胞形态及结构,RTPCR、免疫荧光及Western印迹检测细胞GDNF的表达。结果:本实验条件下,体外分离培养SC纯度=(95.30±2.15)%(n=10),CCK-8实验结果显示,36℃时细胞增殖率最高(P<0.01),>36℃时,随着温度的提高,增殖率逐渐降低,39℃对细胞增殖具有明显抑制作用(P<0.01);免疫荧光结果显示,GDNF表达于SC胞质,36℃时荧光最强,RT-PCR及Western印迹检测结果显示,高于36℃时,GDNF mRNA及蛋白的表达随着温度的增加而降低,36℃、37℃、38℃、39℃4组与对照组比较,差异均有统计学意义(P<0.05)。结论:不同温度下,体外培养SC的增殖能力和GDNF表达明显不同。>36℃时,随着温度的提高,增殖能力受到抑制,GDNF表达水平显著降低。本研究结果证实,高温下大鼠睾丸SC正常功能受到抑制,从而影响生精功能。

Objective： To explore the mechanism of hyperthermia inducing infertility by observing the expression of glial cell line-derived neurotrophic factor （GDNF） in rat Sertoli cells cultured in vitro at different temperatures. Methods： Using combination enzyme digestion and selective adhesion, we isolated Sertoli cells from male Wistar rats and cultured them in vitro at different tempera- tures, followed by observation of the changes in their adhesion and morphology and identification by FasL immunohistochemical stai- ning. We divided the Sertoli cells into a control group （35 ℃ ） and four experimental groups （36 ℃, 37 ℃, 38 ℃, and 39 ℃ ）, measured their proliferation by CCK-8, observed their morphology and structure by HE staining, and determined the expression of GDNF by RT-PCR, immunofluorescence and Western blot. Results： Sertoli cells were successfully isolated and in vitro-cultured, with a purity of （95.30± 2.15 ）% （n = 10 ）. The CCK-8 assay showed that the proliferation of the Sertoli cells was the highest at 36 ℃, gradually decreasing with the temperature above 36 ℃, and significantly inhibited at 39 ℃ （P 〈0. 01 ）. Immunofluorescence revealed the expression of GDNF in the cytoplasm, with the highest fluorescence intensity at 36 %. RT-PCR and Western blot exhibited a decreasing trend of the GDNF expression with the increasing temperature above 36℃. There were statistically significant differ- ences in the expression of GDNF between the control group and the four experimental groups （P 〈 0. 01 ）. Conclusion ： The prolifera- tion and GDNF expression of in vitro-cuhured Sertoli cells differ significantly at different temperatures. At 〉 36℃, the higher the tem- perature is, the lower the Sertoli cell proliferation and GDNF expression are. Our findings suggest that high temperature above 36℃ suppresses the function of Sertoli cells and may also damage spermatogenesis.