大鼠睾丸支持细胞的分离、纯化和鉴定

目的 培养高纯度的大鼠睾丸支持细胞,并应用检测ABP mRNA原位杂交方法加以鉴定.方法 选用18～22d龄SD雄性大鼠睾丸,采用0.25%胰蛋白酶、0.1%透明质酸酶、 0.1%胶原酶三酶依次消化法分离支持细胞,置于32℃ 5% CO2的培养箱培养,48h后用20mmol/L Tris-HCl低渗处理培养细胞.培养1周后应用伊红染色、吖啶橙荧光染色、Feulgen染色对所培养的支持细胞进行鉴定,同时应用原位杂交检测ABP mRNA方法鉴定分离培养的支持细胞.结果 培养1周后所获培养的支持细胞纯度达95%以上.分离培养的支持细胞ABP mRNA表达阳性,其形态结构特征与用其他方法鉴定为支持细胞的形态结构特征一致.结论 采用三酶连续消化及低渗处理法分离培养的支持细胞纯度高,而且应用原位杂交方法检测ABP mRNA是一种新的、特异、有效的鉴定支持细胞及其功能的方法.

ISOLATION, PURIFICATION AND IDENTIFICATION OF SERTOLI CELLS FROM RAT TESTIS

Objective To obtain highly pure cultured Sertoli cells from rat testis and identificate cultured cells by in situ hybridization for ABP mRNA. Methods Testes from 18-22 day old SD rats were removed and decapsulated,then chopped and sequentially digested with three enzymes at 37℃: first with 0.25% trypsin for 15 minutes,then with 0.1% hyaluronidase for 30 minutes,at last with 0.1% collagenase V for 2-3 hours.The isolated cells were incubated at 32℃ in a humidified atmosphere of 5% CO2.To increase the purity of the Sertoli cells, the cultured cells were subjected to hypotonic shock(treatment) with 20 mmol Tris-HCl after 48 hours of incubation.After a week’s incubation,the cultured cells were identificed by HE staining,AO fluorescence staining,Feulgen staining,and in situ hybridization with digoxin-labeled rat ABP cDNA. Results Over 95% of the cultured cells were Sertoli cells.Most of the cultured cells expressed ABP mRNA and their structural features were the same as those of the Sertoli cells identified by other methods.Highly pure Setoli cells can be obstained by the sequentiall digestion with three enzymes and hypotonic shock.Conclusion As a specifical secreted protein secreted Sertoli cells in testes,ABP mRNA detected by in situ hybridization is a new, specific,effective identification method of Sertoli cells from testes.Meanwhile as an