EXPRESSION OF A UNIQUE GLYCOCONJUGATE IN PRIMORDIAL GERM CELLS AND CULTURE OF EMBRYO DERIVED CELLS COLLECTED FROM THE GENITAL RIDGE AND ITS PERIPHERAL ORGANS

原始生殖细胞中独特糖缀合物的表达以及从生殖嵴及其周围器官收集的胚胎衍生细胞的培养

• 批准号: 08456142
• 负责人: MIYAKE Masashi
• 金额: $ 2.62万
• 依托单位: GRADUATE SCHOOL OF SCIENCE AND TECHNOLOGY,KOBE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456142/
• 关键词: porcine primordial germ cells; unique glycoconjugate; Lewis^x; migration and proliferation; 増殖パターン

This study was designed to determine a glycoconjugate which was unique to porcine primordial germ cells (PGC) and was employed as their marker and to examine whether marked cells morphologically and ultrastructurally showed typical features of PGC.Intracellular distribution of the glycoconjugates and localization of PGC in fetuses at various developmental stages were also examined in order to select a suitable marker and to establish an effective method for collection of porcine PGC.Three kinds of anti-Lewis^x, anti-Lewis^y and anti-sialyl Lewis^x antibodies were reacted with sections embedded in paraffin and resin (K4M). Anti-Lewis^x showed the strongest positive reaction with PGC,and surrounding cells, i.e.the hindgut, mesenchyme and genital ridge, were negative. Treatments of Paraffin embedded specimens from fetuses at 16 to 24 dpc with anti-Lewis^x antibody revealed that existence of PGC in yolk sack and epithelial regions of the hind gut at 16 dpc, and PGC migrated through the mes … More enchyme from 19 to 22 dpc to the genital ridge. Majority of PGC settled in the genital ridge until 24 dpc. Until 20 dpc, number of anti-Lewis^x positive cells increased and no negative cell with the morphological features of PGC was observed. Anti-Lewis^x negative PGC,however, were observed on 22 dpc fetuses with decrease of number of anti-Lewis^x positive PGC.The most negative cells were in the genital ridge, suggesting that Lewis^x may have a role in migration of PGC.Immunogold method showed that Lewis^x were regularly distributed on the cell surface and heterogeneously distributed in cytoplasm except for nucleus. A lot of spherical mitochondria and rough endoplasmic reticulum were also observed by electronmicrography. Migrating PGC were surrounded by small space (s) rather than contacted with somatic cells, PGC with pseudopod were observed often.From these results, the ventral mesonephros including genital ridge with the dorsal mesentery dissected from 18 to 22 dpc fetuses are optimum for collection of porcine PGC.Embryo derived cells collected by this method and isolated by trypsin treatment were cultured in typical ES medium for the mouse. Proliferating cells formed anti-Lewis^x positive colonies, but there is no evidence that these cells are derived from PGC. Less

本研究旨在确定猪原始生殖细胞（PGC）特有的糖缀合物本研究以PGC为标记物，观察标记细胞的形态学和超微结构是否具有PGC的典型特征，并观察不同发育阶段胎儿PGC的细胞内分布和定位，以筛选合适的标记物，建立有效的PGC检测方法。用抗刘易斯^x、抗刘易斯^y和抗唾液酸刘易斯^x三种抗体与石蜡包埋的K4 M树脂切片反应。抗-Lewis ^x显示与PGC的最强阳性反应，并且周围细胞，即后肠、间充质和生殖嵴为阴性。用抗Lewis ^x抗体处理16 ~ 24 dpc的石蜡包埋标本，发现16 dpc的卵黄囊和后肠上皮区存在PGC，PGC通过肠上皮区迁移， ...更多信息 食糜从19到22 dpc到生殖器脊。大多数PGC在生殖嵴中定居，直到24 dpc。直到20 dpc，抗Lewis ^x阳性细胞数量增加，未观察到具有PGC形态特征的阴性细胞。22例dpc胎鼠抗刘易斯^x抗体阴性，抗刘易斯^x抗体阳性的PGC数量减少，阴性细胞主要位于生殖嵴，提示刘易斯^x抗体可能参与了PGC的迁移，免疫胶体金法显示刘易斯^x抗体在细胞表面呈规则分布，在胞浆中除细胞核外呈不均匀分布。电镜下可见大量球形线粒体和粗面内质网。从18 ~ 22 dpc胎猪腹侧中肾（包括生殖嵴和背侧肠系膜）中分离出PGC是最适宜的。用这种方法收集的胚胎来源细胞，经胰蛋白酶处理后分离，在典型的小鼠ES培养基中培养。增殖细胞形成抗Lewis ^x阳性集落，但没有证据表明这些细胞来源于PGC。少