睾丸FasL^+Sertoli细胞的制备及功能测定

Preparation and functional identification of testicular Sertoli cells

目的简化大鼠睾丸支持细胞分离培养方法,获取高成活率、高数量、高表达的Fas配体阳性支持细胞。方法取2-3周龄大小的Wistar雄性大鼠,应用胶原酶、胰蛋白酶及脱氧核糖核酸酶消化制备大鼠睾丸支持细胞,体外与活性淋巴细胞共同培养,了解其对淋巴细胞的杀伤作用。用不同方法进行形态学观察、鉴定,免疫组化检测Fas配体和卵泡刺激素受体的表达情况,进一步鉴定睾丸支持细胞。结果大鼠睾丸支持细胞的分离培养中,支持细胞存活率在90%以上,并有明显的Fas配体表达。支持细胞可杀伤与之共同培养的活性淋巴细胞。结论本法提高了支持细胞的获取量,获取的对活性淋巴细胞具有的杀伤作用的Fas配体阳性睾丸支持细胞可以作为联合移植的供体,发挥其局部免疫豁免作用。

Objective To simplify the method for separation and cultivation of rat testicular Sertoli cells with high viability, quantity and expression efficiency. Methods Testicular Sertoli cells from 2 to 3-week-old male Wistar rats were prepared by digestion with collagenase, trypsin and DNase and cultured together with active lymphocytes to observe their killing effect against lymphocytes. After cell culture for 72 h, the Sertoli cells were morphologically observed by different means and identified with transmission electron microscope. Fas ligand and follicle-stimulating hormone receptor （FSHR） were examined y to identify testicular Sertoli cells. SABC method was used for labeling the Fas ligand on the testicular Sertoli cells. Results The viability of the isolated and cultured Sertoli cells was more than 90%, and in in vitro culture, Sertoli cells, which expressed the Fas ligand, could kill the active lymphocytes. Conclusion This method improves the efficiency in acquisition of rat testicular Sertoli cells expressing Fas ligand, which are believed to he a potential donor for co-transplantation with parathyroid cells to offer immune privilege.