LaQuaglia M, Shima H, Hudson P, Takahashi M, Donahoe P K
J Urol. 1986 Jul;136(1 Pt 2):219-24. doi: 10.1016/s0022-5347(17)44821-x.
Sertoli cell cultures were monitored for müllerian inhibiting substance with a solid phase radioimmunoassay. Sertoli cells from newborn calf testes were placed in defined media free of serum in monolayer culture after treatment with trypsin-collagenase followed by gravity separation. Immunoreactive müllerian inhibiting substance was detected in the culture media of Sertoli cells but not National Institutes of Health 3T3 cells. To verify that the Sertoli cells were intact, cyclic adenosine monophosphate levels were determined after follicle-stimulating hormone stimulation. Cyclic adenosine monophosphate levels were elevated in Sertoli cells but not National Institutes of Health 3T3 cells. The newborn calf Sertoli cell culture provides a useful system in which to study factors affecting müllerian inhibiting substance production and release, and documents this substance as another reliable marker for the Sertoli cell. Müllerian inhibiting substance levels also could be measured in media beneath testicular fragments in organ culture, and were increased and prolonged in comparison to müllerian inhibiting substance release from Sertoli cell monolayer cultures. Modulation of müllerian inhibiting substance release from monolayer and organ cultures then was attempted. Neither gonadotropin nor steroid additions affected the release of müllerian inhibiting substance during 24 to 72 hours in organ or tissue culture. We are using the müllerian inhibiting substance radioimmunoassay to monitor attempts to immortalize a Sertoli cell line capable of continuous müllerian inhibiting substance production using viral deoxyribonucleic acid transfection techniques.
采用固相放射免疫分析法监测支持细胞培养物中的苗勒管抑制物质。新生小牛睾丸的支持细胞在用胰蛋白酶 - 胶原酶处理后经重力分离，置于无血清的限定培养基中进行单层培养。在支持细胞的培养基中检测到免疫反应性苗勒管抑制物质，而在国立卫生研究院3T3细胞的培养基中未检测到。为了验证支持细胞是否完整，在促卵泡激素刺激后测定环磷酸腺苷水平。支持细胞中环磷酸腺苷水平升高，而国立卫生研究院3T3细胞中未升高。新生小牛支持细胞培养提供了一个有用的系统，可用于研究影响苗勒管抑制物质产生和释放的因素，并将该物质作为支持细胞的另一个可靠标志物。也可以在器官培养的睾丸组织块下方的培养基中测量苗勒管抑制物质水平，与支持细胞单层培养物中苗勒管抑制物质的释放相比，其水平升高且持续时间延长。然后尝试调节单层培养和器官培养中苗勒管抑制物质的释放。在器官或组织培养的24至72小时内，添加促性腺激素或类固醇均未影响苗勒管抑制物质的释放。我们正在使用苗勒管抑制物质放射免疫分析法来监测尝试使用病毒脱氧核糖核酸转染技术使能够持续产生苗勒管抑制物质的支持细胞系永生化的情况。
