大鼠睾丸支持细胞的分离、鉴定与培养

Isolation,Identification and Culture of Sertoli Cells from Rat Testis

背景与目的：分离培养高纯度的大鼠睾丸支持细胞。材料与方法：选用SD雄性大鼠（18—21d），处死取出睾丸，采用0．25％胰蛋白酶、0．05％胶原酶二步酶消化法结合低速离心分离支持细胞，置于35℃，5％CO2培养箱培养，48h后用20mmol／LTfis-HCl低渗处理培养细胞。0．25％胰蛋白酶消化后二次贴壁培养，用油红O染色和透射电镜观察对所培养的支持细胞进行鉴定，并用MTT法绘制细胞生长曲线。结果：分离培养的支持细胞纯度达95％，体外培养的对数生长期为4—9d。结论：采用二步酶消化法与低速离心及低渗处理法分离培养的支持细胞纯度高，用油红O染色鉴定支持细胞是一种简便快速的方法。

BACKGROUND AND AIM： To obtain highly purified cultured Sertoli cells from rat testis. MATERIALS AND METHODS： Testes from 18-22 day old SD rats were removed and decapsulated, then chopped and sequentially digested with two enzymes at 37 ℃： first with 0.25% trypsin for 20-40 minutes, then with 0.05% collagenase V for 40-60 minutes. The speed of centrifugation was 800 r/ min. The isolated cells were incubated at 35℃ in a humidified atmosphere of 5% CO2.To increase the purity of Sertoli cells,cultured cells were subjected to hypotonic shock（treatment） with 20 mmol Tris-HC1 after 48 h of incubation. After 0.25% trypsin digestion and adherent culture, the cultured cells were identified by HE staining,oil red 0 staining and transmission electron microscope. The growth curve of Sertoli cells was evaluated as well. RESULTS： Over 95% of the cultured ceils were Sertoli cells.The exponential phase of growth was 4- 9 days. CONCLUSION： Highly purified Sertoli ceils could be obtained by two step enzymes digestion and hypotonic shock, and identification of Sertoli cells by oil red 0 was a convenient method.