Myoumoto A, Manabe N, Imai Y, Kimura Y, Sugimoto M, Okamura Y, Fukumoto M, Sakamaki K, Niwano Y, Miyamoto H
Department of Animal Sciences, Kyoto University, Japan.
J Vet Med Sci. 1997 Aug;59(8):641-9. doi: 10.1292/jvms.59.641.
Two monoclonal antibodies capable of inducing granulosa cell apoptosis were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were dissected from the ovaries of gilts, and then granulosa cells were isolated. BALB/c female mice were immunized with the isolated granulosa cells. Antibodies against the granulosa cells were detected by immunofluorescent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulosa cells were fused with Sp2/O-Ag 14 mouse myeloma cells by standard hybridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which produced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies specifically recognized cell-membrane proteins with molecular weights of 55 and 70 kD and isoelectric points of 5.9 and 5.4, respectively. The monoclonal antibodies immunohistochemically reacted with granulosa cells of healthy follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 or 10 micrograms/m/PFG-1 or PFG-2 antibodies, respectively, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulosa cells.
利用从猪卵巢窦状卵泡制备的颗粒细胞，制备出了两种能够诱导颗粒细胞凋亡的单克隆抗体。从后备母猪的卵巢中解剖出直径4 - 5毫米的健康卵泡，然后分离出颗粒细胞。用分离出的颗粒细胞对BALB/c雌性小鼠进行免疫。通过使用冷冻卵巢切片的免疫荧光染色检测针对颗粒细胞的抗体。通过标准杂交技术，将从产生针对颗粒细胞抗体的免疫小鼠中分离出的脾细胞与Sp2/O - Ag 14小鼠骨髓瘤细胞进行融合。筛选出了两个产生针对颗粒细胞特异性IgM抗体的杂交瘤克隆，即PFG - 1和PFG - 2。蛋白质印迹分析显示，PFG - 1和PFG - 2抗体分别特异性识别分子量为55和70 kD、等电点分别为5.9和5.4的细胞膜蛋白。单克隆抗体与健康卵泡的颗粒细胞发生免疫组织化学反应。当分别将从健康卵泡制备的分离颗粒细胞在含有0.1或10微克/毫升PFG - 1或PFG - 2抗体的培养基中培养时，通过核形态学、DNA电泳和流式细胞术分析确定细胞发生了凋亡。总之，这两种针对颗粒细胞的单克隆抗体在培养的颗粒细胞中具有细胞杀伤活性。
