猪Sertoli细胞在异种移植模型中对人免疫细胞功能的影响

Effects of Porcine Sertoli Cells on Human Lymphocyte Proliferation and Apoptosis in vitro in A Xenotransplantation Model

目的通过体外实验,研究猪睾丸支持细胞Sertoli细胞对猪-人异种移植模型中人外周血单个核细胞(PB-MCs)功能的影响,以探讨Sertoli细胞在诱导异种移植免疫耐受中的潜在作用。方法采用改良酶消化法获取新生猪睾丸Sertoli细胞,纯化后培养,检测分离的原代猪Sertoli细胞纯度,并在光学显微镜和电子显微镜下对猪Sertoli细胞进行形态学测定。采集新鲜人血液,分离外周血单个核细胞(PBMCs),将原代猪Sertoli细胞与人PBMCs共同孵育,观察Sertoli细胞对人PBMCs凋亡率的影响,并与人PBMCs自身凋亡率进行比较。在体外,用3H-TdR摄入法检测人PB-MCs的增殖,对照组用刀豆蛋白A(ConA)刺激活化人PBMCs,实验组则将猪原代Sertoli细胞与ConA活化的人PB-MCs共同培养,连续4d检测人PBMCs的增殖变化。结果采用该实验改良法分离的新生猪Sertoli细胞,经检测纯度超过93%。光学显微镜及电子显微镜下均可见Sertoli细胞胞体多突起、胞核内多核仁、胞质内多脂滴等典型特征。经Annexin-Ⅴ/PI染色,流式细胞仪检测,猪Sertoli细胞可诱导人PBMCs凋亡率升高(8.92±1.75)%,高于人PBMCs的自身凋亡率(3.09±0.75)%(P<0.01)。并且猪Sertoli细胞能够延迟ConA刺激的人PBMCs体外增殖峰的出现(对照组增殖峰在第2天出现,实验组增殖峰在第3天出现),但是两组增殖峰峰值间的差异没有统计学意义。结论采用该法可以获得高纯度的猪Sertoli细胞。在猪-人异种移植体外实验中,新生猪Sertoli细胞能够诱导人PBMCs凋亡,并且能够延迟ConA活化的人PBMCs增殖反应,对人PBMCs的活化增殖有一定程度的抑制作用。

Objective To investigate whether xenogeneic neonatal porcine Sertoli cells could induce human lymphocyte apoptosis as well as inhibit xenospeeific cellular responses in vitro. Methods Testis was isolated from 10-day-old Yorkshire neonatal pigs. Sertoii cells were successfully prepared by digestion with collagenase type V, trypsin, and DNase I, and additionally identified by morphology under light and electron microscopy. Human peripheral blood mononuclear cells （PBMCss） were non- specifically stimulated and activated by concanavalin A （ConA）. And then they were co cultured with porcine Sertoli cells. Cell proliferation of human PBMCss was evaluated by using ^3H-thymidine incorporation in 4 days. The apoptosis ratio of human PBMCss co-cultured with porcine Sertoli cells was examined by flow cytometry （FCM）. Results The purity of porcine Sertoli cells was more than 930/oo in the culture. In vitro, porcine Sertoli cells delayed the proliferation of human PBMCss, while the peak value showed no significant differences between the co-cultured group and the control group （human PBMCss without Sertoll cells）. The apoptosis ratio of human PBMCss induced by porcine Sertoli cells was 8.92%± 1.75%, significantly higher than in human PBMCss alone （3.09%±0.75%, P〈0.01）. Conclusion Porcine Sertoli cells could delay the proliferation of activated human PBMCss. And they could also induce human PBMCs apoptosis in vitro.