Factors affecting morphogenesis of normal gallbladder epithelial cells and those regulationg differentiation in gallbladder cancer cells.

影响正常胆囊上皮细胞形态发生的因素和调节胆囊癌细胞分化的因素。

基本信息

批准号：
09671322
负责人：
MIYAZAKI Kohji
金额：
$ 2.11万
依托单位：
Saga Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Rabbit gallbladder epithelial cells (RGEC) suspended in collagen gels form spherical cysts with morphologic polarity. EGF, HGF, epimorphin, and FCM promoted cyst maturation by accelerating the proliferation and aggregation of clear, polarized vesicles. In contrast, TGF-β1 markedly inhibited DNA synthesisin both monolayer and collagen gel cultures an promoted formation of branching structures in collagen gels. Furthermore, in the presence of EGF, TGF-β1 induced a drastic change in morphogenesis, with the formation of branching networks that showed cell-cell contact only at sites where branches touched. RGEC-forming multicellular cysts did not express vimentin but expressed significant amounts of cytoskeratin and regained junctional complexes. In contrast, TGF-β1 treated cells strongly expressed vimentin along with branching structures and showed decreases in cytokeratin expression and junctional complexes. Thus TGF-β1 induces a mesenchyme-like cell shape accompanied by cytoskeletal mole … More cular changes, with loss of both epithelial polarization and junctional complexes. These results suggest that the morphogenetic program of RGEC is likely tobe determined by the interaction of these peptides and the timing of their presence.To clarify the role of cell adhesion molecules in the morphology of gallbladder cancers, four human gallbladder cancer cell lines (GB-d1, KMG-C, GBK-1 and G-415) were eximined in vitro. They showed noticeably different morphologics in our standard gel cultures (SC). GB-dl and KMG-C formed cystic and spheroid structures, respectively, which seemed to represent well- and moderately-differentiated cancers, respectively. GBK-1 and G-415 showed branching and "pseudo-glandular" structures, respectively, both of which seemed to indicate original dedifferentiated cancers. In floating gel culture (FC), only GB-d1 showed a highly increased tendency toward cyst formation. Expression of E-cadherin and α-catenin was detected in GB-d1 and KMG-C, but not in GBK-1 and G-415 cells. Furthermore, E-cadherin expression in GB-d1 was 1.82 times greater in FC than in SC, while E-cadherin expression levels of KMG-C did not change. Neither GB-d1 nor KMG-C showed any difference in α-catenin expression between SC and FC.Immunostaining of GB-d1 revealed that these proteins were localized to the cell membrane. In contrast, heterogeneous localization of these proteins was detected in the spheroid structures of KMG-C, in both SC and FC.Electromicroscopic examination revealed that reestablishment of junctional complex was occurred only in GB-d1 cells cultured in FC.The formation of cystic structures in GB-d1 was completely inhibited by an antibody against human E-cadherin. Both expression of E-cadherin and its membranous localization are required for well-differentiated-type morphogenesis in gallbladder cancer cells. Less

悬浮在胶原凝胶中的兔胆囊上皮细胞 (RGEC) 形成具有形态极性的球形囊肿，通过加速透明极化囊泡的增殖和聚集来促进囊肿成熟。单层和胶原凝胶培养物中的合成促进胶原凝胶中分支结构的形成此外，在 EGF 存在下， TGF-β1 诱导形态发生的巨大变化，形成分支网络，仅在分支接触的部位显示细胞与细胞的接触，形成 RGEC 的多细胞囊肿不表达波形蛋白，但表达大量的细胞角蛋白并恢复连接复合物。相反，TGF-β1 细胞强烈处理表达波形蛋白和分支结构，并显示细胞角蛋白表达和连接复合物减少，因此 TGF-β1 诱导间充质样细胞形状伴随着细胞骨架痣的细胞变化，以及上皮极化和连接复合物的丧失。这些结果表明，RGEC 的形态发生程序可能是由这些肽的相互作用及其存在的时间决定的。为了阐明细胞粘附分子在胆囊癌形态中的作用，研究了四种人胆囊癌细胞系（GB-d1、KMG-C、 GBK-1 和 G-415) 在我们的标准培养物凝胶 (SC) 中表现出明显不同的形态，分别形成囊状和球状结构，这似乎代表了良好和中等的结构。 GBK-1和G-415分别显示出分支和“假腺”结构，这两者似乎都表明原始的去分化癌症。漂浮凝胶培养（FC）中，仅 GB-d1 在 GB-d1 和 KMG-C 中检测到 E-钙粘蛋白和 α-连环蛋白的表达显着增加，但在 GBK-1 和 G-415 中未检测到。此外，FC 中 GB-d1 中的 E-钙粘蛋白表达量是 SC 中的 1.82 倍，而 KMG-C 中的 E-钙粘蛋白表达水平没有变化。 KMG-C 显示 SC 和 FC 之间的 α-连环蛋白表达存在差异。GB-d1 的免疫染色显示这些蛋白质定位于细胞膜，相反，在 KMG-C 的球状结构中检测到这些蛋白质的异质定位。，在 SC 和 FC 中。电镜检查显示，仅在 FC 中培养的 GB-d1 细胞中发生了连接复合物的重建。GB-d1 中囊状结构的形成被抗人 E-钙粘蛋白的抗体完全抑制。E-钙粘蛋白的表达及其膜定位对于胆囊癌细胞中分化良好的形态发生是必需的。