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目的:通过观察不同温度对原代大鼠睾丸支持细胞增殖作用与紧密连接蛋白occludin(OCLN)表达的影响,探讨温度因素在男性生殖功能降低或男性不育中的作用机制。方法:体外分离培养雄性Wistar大鼠睾丸支持细胞(Sertoli cell,SC),油红O染色及免疫组化FasL鉴定。实验分为对照组(34℃)和实验组(35℃、36℃、37℃、38℃、39℃)。在相应的温度下培养4 d后,CCK-8法检测SC增殖作用,HE染色观察细胞形态及结构,Western印迹法和免疫荧光法检测OCLN表达水平。结果:体外培养SC的纯度为(96.20±1.95)%,CCK-8结果显示,细胞增殖活性在34～36℃时逐渐增强,36～39℃时逐渐降低,并在镜下观察发现细胞核固缩、裂解明显;免疫荧光及Western印迹显示,36℃时OCLN表达量最高;高于36℃时,OCLN表达量随着温度的增加而降低(P<0.01)。结论:温度升高(>36℃)抑制SC的增殖活性,并降低SC紧密连接蛋白的表达量。因此,高温对SC活性和紧密连接完整性的影响,可能是导致男性生育能力下降或不育的机制之一。
OBJECTIVE: To investigate the effect of temperature on the proliferation of primary cultured rat sertoli cells and the expression of tight junction protein Occludin (OCLN), and to explore the mechanism of the effect of temperature on the reduction of male reproductive function or male infertility. Methods: Sertoli cell (SC), oil red O staining and immunohistochemistry were used to identify and culture male Wistar rats in vitro. The experiment was divided into control group (34 ℃) and experimental group (35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃). After cultured for 4 days at the corresponding temperature, the proliferation of SC was detected by CCK-8 assay, the morphology and structure of cells were observed by HE staining, and the expression of OCLN was detected by Western blotting and immunofluorescence. Results: The purity of SC cultured in vitro was (96.20 ± 1.95)%. The results of CCK-8 showed that the cell proliferative activity increased gradually from 34 ℃ to 36 ℃ and gradually decreased from 36 ℃ to 39 ℃. , And the cleavage was obvious. Immunofluorescence and Western blot showed that the expression level of OCLN was the highest at 36 ℃. When the temperature was above 36 ℃, the expression of OCLN decreased with the increase of temperature (P <0.01). Conclusion: The increase of temperature (> 36 ℃) inhibits the proliferation activity of SC and decreases the expression of SC tight junction protein. Therefore, the effect of high temperature on SC activity and tight junction integrity may be one of the mechanisms leading to decreased fertility or infertility in men.