Role of beta-citryl-L-glutamate in the differentiation of retina pigmented epithelial cells to lens fiber cells

β-柠檬酰-L-谷氨酸在视网膜色素上皮细胞向晶状体纤维细胞分化中的作用

• 批准号: 07671943
• 负责人: MIYAKE Masaharu
• 金额: $ 1.54万
• 依托单位: Kobe-Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671943/
• 关键词: beta-citryl-L-glutamate; glutamate derivative; retina pigmented epithelial cell; lens fiber cell; cell differentiation

1. When adult bovine lens epithelial cells were cultured in the presence of fetal calf serum, they differentiated into fiber cells and formed lentoid bodies at about 6 weeks in the culture. beta-citryl-L-glutamate (beta-CG) concentration increased from 2 to 4 weeks of the culture, reached to the highest level and then decreased gradually. alpha-crystallin, a marker of differentiation into lense fiber cells, increased extremely from 4 to 5 weeks of the culture and then continued to increase gradually. The cells of lentoid bobies were stained strongly by the anti-alpha-crystallin antibody, while cells around the structures were stained by the anti-beta-CG antibody.2. Retina pigmented epithelial cells were dissociated from eyes of 8-to 9-day-old chick embryos and were cultured in the medium containing phenylthiourea and testicular hyaluronidase. They rapidly lost metanosomes as they proliferated and dedifferentiated in culture, but they could not transdifferentiated into lens fiber cells in the medium containing phenylthiourea, hyaluronidase and ascorbic acid at extremely high cell density. The pigmented epithelial cells were transdifferentiated directly into lens fiber cells in the medium containing hyaluronidase at high cell density according to the method of Itoh et al. The epithelial cells differentiated into fiber cells and formed lentoid bodies efficiently, but neither cells of lentoid bodies nor cells around the lentoidbodies were stained by the anti-beta-CG antibody.Sixteen tryptic-digested peptides from the purified beta-CG-hydrolysing enzyme were prepared and their amino acid sequence were determined. None of their amino acid sequences was corresponded to the known amino acid sequence of proteins, but the sequence of some peptide from 100 and 115 kDa subunits showed 100% homology for cytoskeletal keratin.

1.当在胎牛血清存在下培养成年牛透镜上皮细胞时，它们在培养物中约6周时分化成纤维细胞并形成晶状体样体。β-柠檬酰-L-谷氨酸（β-CG）浓度在培养的第2 ~ 4周逐渐升高，达到最高水平，然后逐渐下降。α-晶状体蛋白是分化成纤维细胞的标志物，在培养的4 - 5周内急剧增加，然后继续逐渐增加。抗α-晶状体蛋白抗体对晶状体样结构的细胞染色较强，而抗β-CG抗体对晶状体样结构周围的细胞染色较强.从8 ~ 9日龄鸡胚眼分离视网膜色素上皮细胞，并在含有苯基硫脲和睾丸透明质酸酶的培养基中培养。它们在培养中增殖和去分化时迅速丢失变性体，但在含有苯基硫脲、透明质酸酶和抗坏血酸的培养基中以极高的细胞密度不能转分化为透镜纤维细胞。根据Itoh等的方法，在含有透明质酸酶的培养基中以高细胞密度将色素上皮细胞直接转分化为透镜纤维细胞。上皮细胞分化为纤维细胞并有效地形成晶状体样体，抗β-CG抗体对晶状体体细胞和晶状体体周围细胞均未染色。制备了水解酶并测定了其氨基酸序列。它们的氨基酸序列均与已知蛋白质的氨基酸序列不一致，但来自100和115 kDa亚基的一些肽的序列与细胞骨架角蛋白显示出100%的同源性。