Terasawa E, Quanbeck C D, Schulz C A, Burich A J, Luchansky L L, Claude P
Wisconsin Regional Primate Research Center, University of Wisconsin-Madison 53715-1299.
Endocrinology. 1993 Nov;133(5):2379-90. doi: 10.1210/endo.133.5.8404690.
The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH cells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro. Six monkey fetuses at the ages of E34-E36 were delivered surgically and the area including the olfactory placode (PL) and the areas that encompass the migratory pathway (MP) were dissected out. The tissues were cut into small pieces and plated on collagen- or poly-L-lysine-coated glass coverslips in medium M199. Cultures were maintained for up to 33 days and immunostained for LHRH, GnRH-associated peptide, neurofilament protein, neuron-specific enolase, and glial fibrillary acidic protein. LHRH positive cells were also positive for neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein. In the first week of culture, LHRH cells remained within the explants of PL, were rounded (average dimensions: 13.0 x 11.3 microns) and stained lightly. By the second week a number of LHRH cells (15.7 x 13.6 microns) with neurites started to migrate out from PL explants, whereas some still remained in the PL. By the third week a large number of LHRH cells (19.3 x 9.4 microns) had migrated out from the PL. They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 microns in length. A few "pioneer" LHRH cells appeared to lead the migration of 100-400 LHRH cells forming 1-3 major migratory paths. In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants. LHRH cells from the ventral part of the MP, which is close to the PL, migrated out by 1-2 weeks and formed several migratory paths, whereas LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were extended tortuously. Cultured LHRH cells released LHRH into the media and responded to challenge with high K+. The results indicate that 1) primary LHRH neurons can be obtained from the embryonic PL and their migratory pathway, 2) these neurons migrate and mature in culture and 3) they are accessible for cellular and molecular studies.
本研究的目的是利用胚胎嗅基板建立原发性促性腺激素释放激素（LHRH）细胞培养系统，并检测源自嗅基板的LHRH细胞以及LHRH神经元的迁移途径在体外是否成熟。通过手术取出6只处于E34 - E36期的猴胎儿，解剖出包括嗅基板（PL）以及包含迁移途径（MP）的区域。将组织切成小块，接种于涂有胶原蛋白或聚-L-赖氨酸的玻璃盖玻片上，置于M199培养基中。培养长达33天，并对LHRH、GnRH相关肽、神经丝蛋白、神经元特异性烯醇化酶和胶质纤维酸性蛋白进行免疫染色。LHRH阳性细胞对神经丝蛋白、神经元特异性烯醇化酶和GnRH相关肽也呈阳性，但对胶质纤维酸性蛋白呈阴性。在培养的第一周，LHRH细胞仍留在PL外植体中，呈圆形（平均尺寸：13.0×11.3微米），染色较浅。到第二周，一些带有神经突的LHRH细胞（15.7×13.6微米）开始从PL外植体中迁移出来，而一些仍留在PL中。到第三周，大量LHRH细胞（19.3×9.4微米）从PL迁移出来。它们呈梭形，细胞核清晰，伸出长达500微米的长的曲张神经突。一些“先驱”LHRH细胞似乎引领着100 - 400个LHRH细胞的迁移，形成1 - 3条主要的迁移路径。相比之下，来自MP外植体的LHRH细胞比来自PL外植体的LHRH细胞更早迁移出来。来自MP靠近PL的腹侧部分的LHRH细胞在1 - 2周内迁移出来并形成几条迁移路径，而来自MP远离PL的背侧部分的LHRH细胞广泛散布在外植体周围，其神经突呈曲折状延伸。培养的LHRH细胞将LHRH释放到培养基中，并对高钾刺激作出反应。结果表明：1）原发性LHRH神经元可从胚胎PL及其迁移途径获得；2）这些神经元在培养中迁移并成熟；3）它们可用于细胞和分子研究。
