猪睾丸Sertoli细胞的分离、培养及其免疫豁免相关细胞因子的检测

Isolation and culture of neonatal porcine Sertoli cells and detection of immune privilege-related molecules

目的探讨猪睾丸Sermli细胞分离、培养的方法，并对其体外培养的状态及免疫豁免相关细胞因子的表达进行检测，为Sertoli细胞用于移植提供依据。方法无菌条件下取10～15日龄的湖北白猪睾丸，剥离睾丸白膜及表面血管，剪碎后用2．0g／LV型胶原酶以及2．5g／L胰蛋白酶和0．5g／L DNaseI消化（两步法）分离Sertoli细胞，于37℃、含体积分数为5％CO2的培养箱中培养。采用倒置相差显微镜观察细胞形态，透射电镜进行超微结构鉴定，流式细胞仪检测细胞体外活率，逆转录聚合酶链反应检测Sertoli细胞sox9、FasL、转化生长因子B（TGF-β）及Clusterin的表达，四甲基偶氮唑盐（MTT）检测细胞的活性。结果猪睾丸经分离、培养，所获得的Sertoli细胞纯度达90％以上。培养后的细胞凋亡率为（2．61±0．96）％，死亡率为（2．12±0．74）％；培养的Sertoli细胞稳定表达sox9、TGF-β、Clusterin，而FasL表达较弱。体外培养的Sertoli细胞可以保持良好活性达21d。结论采用V型胶原酶以及胰蛋白酶和DNaseI两步法消化分离可获得大量高纯度的Sertoli细胞，该细胞在培养过程中能够稳定表达FasL、TGF-β和Clusterin分子。

Objective To establish the method for isolation and culture of porcine Sertoli cells and detect the expression of the immune privilege-related molecules in the cultured cells. Methods Testes were aseptically removed from the 10- to 15-days old Large White piglets. Testes were decap-ulated, minced and then digested with 0. 2% （W/V） collagenase type V, 0. 25% （W/V） trypsin and 0. 05% （W/V） DNaseI. In the primary isolation, Sertoli cells were cultured at 37 ℃ with 5 % CO2. Inverted phase contrast microscopy and HE staining were used to observe the morphology of Sertoli cells, and the Sertoli cells were identified under an electron microscope. Viability and apoptosis of cultured cells were measured with the AnnexinV-PI staining by flow cytometry. The expression of sox9, FasL, TGF-β and clusterin in Sertoli cells was detected by RT PCR. The viability of long-term cultured Sertoli cells was assayed by MTT. Results In the cultured total cells, Sertoli cells accounted for more than 90%. The apoptosis rate and mortality of Sertoli cells was （2. 61 ±0. 96）% and （2. 12 ±0. 74）% respectively. RT-PCR revealed that the expression of sox9, TGF-β and clusterin in the Sertoli cells was strongly positive, but FasL was weakly positive. Viability of cultured cells measured by MTT conformed that the sertloli cells could survive more than 21 days. Conclusion The isolation and culture methods of the neonatal porcine Sertoli cells was established. Under the cultured ,conditions, the Sertoli cells can express the immune privilege molecules and successfully survive at least 21 days.