Histochemical and immunohistochemical study on GSA-II binding glycoprotein of alimentary tract

(1)从系统发育角度来看，基底腺的黏液颈细胞（Mucous neck cells, MNCs）最早出现在两栖动物中。我们研究了爪蟾基底腺中跨国细胞的起源和分化。采用凝集素组织化学方法，利用单纯格里菲尼亚凝集素（GSA -II），在成虫根腺中特异检测到MNCs。在变态幼虫胃的形态发生期间，GSA-II反应细胞随机出现在未发育的底腺的各个部位，然后迅速定位于颈部部位。此时，新出现的MNC型粘膜颗粒用gsa - ii胶体金（CG）标记。在幼虫胃区观察到两种类型的细胞，分别为MNCs和SMCs中间细胞和MNCs和OPCs中间细胞。在成人中也发现了MNCs和OPCs的中间细胞。在这些细胞中，MNC型黏液颗粒被GSA-II- cg标记，而SMC型黏液颗粒和酶样颗粒对GSA-II没有反应。这些观察结果表明，无论物种分化如何，GSA-II都是研究跨国公司及其前体分化的有用标记物。(2)采用溴脱氧尿苷（BrdU）免疫组化方法，研究了成虫及其幼虫基底腺的细胞分化和迁移。在成人的基底腺中，BrdU的累积标记显示，在基底腺的颈部，在SMCs和MNCS之间存在增殖细胞区。到第5周，跨国公司的标记指数迅速上升。OPCs的标记指数在第7周呈延迟上升趋势，与MNCs的标记下降趋势一致。在幼体的未成熟底腺中，标记增殖细胞随机分布于发育中的胃粘膜。在蜕变期，未成熟上皮细胞的标记指数在第63期最高。在63期给药后，标记的上皮细胞在蜕变期没有明显的损失。此外，在给药后7天，上皮细胞（如SMCs/生殖细胞、MNCs和OPCs）之间的标记指数无显著差异。在成人胃粘膜中，生殖细胞可能直接向上分化为SMCs。大多数opc似乎在向下迁移过程中发生了分化。生殖细胞经过一个阶段为MNCS，最后成为OPCS。在幼虫胃区组织发生期，未分化细胞可原位增殖分化为SMCS、MNCs和OPCs。它们可能在64和65阶段迁移。成人胃黏膜的组织发生在66期完成。(3)从大鼠胃黏膜中纯化出gsa - ii结合糖蛋白。在对该糖蛋白进行纯化前，我们建立了GSA-II结合糖蛋白的酶联凝集素测定（ELLA）系统。将大鼠胃黏膜糖蛋白溶于胍-盐酸缓冲液中，涂于Sephacryl S-1000柱上。将含有GSA-II结合糖蛋白的部分应用于rca - i琼脂糖柱。在rca - 1柱中保留的样品随后通过gsa - 2糖糖柱进行亲和纯化。纯化的gsa - ii结合糖蛋白可能有助于开发特异性抗体，并为该项目提供线索。少

(1) Mucous neck cells (MNCs) of the fundic gland are phylogenetically thought to have first appeared in amphibians. We studied the origin and differentiation of MNCs in fundic glands of xenopus laeves. By means of lectin histochemical methods using Griffonia simplicifolia agglutinin(GSA)-II, MNCs were detected specifically in fundic glands of adult X. laeves. During the morphogenetic period of the stomach in metamorphosing larvae, GSA-II reactive cells randomly appeared in various portions of the underdeveloped fundic glands and then rapidly localized in the neck portion. At this time, newly appearing mucous granules of MNC type were labeled by GSA-II-colloidal gold(CG). Two types of cells intermediate to MNCs and SMCs and intermediate to MNCs and OPCs were observed in the larval gastric region. Cellsintermediate to MNCs and OPCs were also found in adults. In these cells, mucous granules of MNC type were labeled by GSA-II-CG, but mucous granules of SMC type and zymogen-like granules di … More d not react to GSA-II. These observations suggest that GSA-II is a useful marker in studying the differentiation of MNCs and their precursors regardless of species differentiation.(2)We have examined cellular differentiation and migration in the fundic glands of adult X. laevis as well as its larvae using bromodeoxyuridine(BrdU)immunohistochemistry. In the adult fundic glands, cumulative labeling with BrdU revealed a proliferative cell zone between the SMCs and MNCS, in what is referred to as in the neck portion of the fundic gland. The labeling index of MNCs rapidly increased by week 5. The labeling index of OPCs showed the delayed-increase by week 7, coincident with the decrease in the labeling of MNCs. In the immature fundic glands of larvae, the labeled proliferating cells randomly distributed throughout the developing gastric mucosa. During the metamorphosing period, the labeling index of immature epithelial cells was the highest at stage 63. After the administration of BrdU to larvae at stage 63, there was no significant loss of the labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling indices among the epithelial cells such as SMCs/generative cells, MNCs, and OPCs at 7 days after administration. In adult gastric mucosa, generative cells might differentiate directly to SMCs in an upward direction. A majority of OPCs seemed to differentiate during a downward migration. Generative cells pass through a stage as MNCS, finally becoming OPCS. In histogenetic period of larval gastric region, undifferentiated cells might proliferate and differentiate to SMCS, MNCs and OPCs in situ. They may have migrated during stages 64 and 65. Histogenesis of adult gastric mucosa completed at stage 66.(3)We have purified GSA-II-binding glycoprotein from rat gastric mucosa. Before trying purification of this glycoprotein, we have established the assay system for GSA-II binding glycoprotein using the enzyme-linked lectin assay(ELLA)method. Glycoprotein from rat gastric mucosa was solubilized in the guanidineHCL buffer, then applied on to Sephacryl S-1000 column. The fraction containing GSA-II binding glycoprotein was applied to RCA-I-agarose column. The sample retained in RCA-I column was subsequently affinity-purified through GSA-IIagarose column. It is likely that purified GSA-II-binding glycoprotein allows to develop specific antibody and to shed light on this project. Less