改良法分离睾丸支持细胞体外诱导异种淋巴细胞凋亡

目的改进睾丸支持细胞（Sertoli细胞）的分离方法及培养条件,探讨Sertoli细胞体外诱导异种淋巴细胞凋亡的作用机制。方法取2～4周龄的SD大鼠睾丸,采用Ⅴ型胶原酶、胰蛋白酶及脱氧核糖核酸酶二步消化法制备Sertoli细胞。用免疫组织化学法（SABC法）检测Sertoli细胞中Fas配体（FasL）、转化生长因子p,（TGF-β1）、clusterin的表达;流式细胞仪检测Sertoli细胞诱导异种淋巴细胞凋亡情况。结果在分离培养纯化过程中,Sertoli细胞占培养细胞总数的90％以上,生长状况良好,能稳定表达FasL、TGF-β1和clusterin,并且能够诱导异种淋巴细胞发生凋亡。结论应用本实验方法能够稳定获得大量高纯度的Sertoli细胞,并且能够发挥其免疫豁免的功能,从而用于细胞联合移植。

The research of xeno-lymphocytes apoptosis induced by Sertoli cells isolated by modified methods in vitro

OBJECTIVE: To modify the isolation and culture method of Sertoli cells and investigate its' effects on xeno-lymphocytes apoptosis. METHODS: Sertoli cells which was isolated from 2 - 4 week-old Sprague Dawley (SD) rats, were successfully prepared by collagenase type V, trypsin and DNase I and then identified by electron microscope. Viability and apoptosis of cultured cells were measured by flow cytometry. The apoptosis rates of Balb/c mouse lymphocytes were examined which were co-cultured with Sertoli cells of SD rats by flow cytometry, too. The expression of FasL, TGF-beta(1) and clusterin on Sertoli cells were detected by immunocytochemistry. RESULTS: In the co-cultured system, Sertoli cells accounted for more than 90%. The viability of Sertoli cells was above 95% and the apoptosis rate was 10.87% +/- 3.87% in this study. The lymphocytes apoptosis ratio was 15.52% +/- 0.17% (P < 0.01). Streptavidin-biotin-peroxidase-complex immunochemistry staining showed that the Sertoli cells could express FasL, TGF-beta(1) and clusterin, respectively. CONCLUSIONS: It indicates that the expression of FasL, TGF-beta(1) on the Sertoli cells might relate to the immune privilege, and it supposed to be benefit for xenotransplantation.