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

1.成年牛晶状体上皮细胞在含胎牛血清的条件下培养，6周左右分化为纤维细胞并形成类豆状小体。β-柠檬基-L-谷氨酸(β-CG)浓度在培养2~4周逐渐升高，达到最高值后逐渐下降。α-晶状体蛋白是分化为晶状体纤维细胞的标志，在培养4~5周后急剧增加，之后逐渐增加。结果1.抗α-晶状体蛋白抗体对晶状体样核细胞染色较强，而抗β-CG抗体对结构周围细胞染色较深。从8日龄至9日龄鸡胚眼分离视网膜色素上皮细胞，在含有苯硫脲和睾丸透明质酸酶的培养液中培养。在细胞密度极高的情况下，它们在含有苯硫脲、透明质酸酶和抗坏血酸的培养液中不能转分化为晶状体纤维细胞。根据Itoh等人的方法，在含有高密度透明质酸酶的培养液中，将着色的上皮细胞直接转分化为晶状体纤维细胞。从纯化的β-CG水解酶中分离纯化的β-CG水解酶制备了16个胰酶解肽，并测定了它们的氨基酸序列。它们的氨基酸序列都不与已知的蛋白质氨基酸序列相对应，但100和115 kDa亚基的某些肽序列与细胞骨架角蛋白有100%的同源性。

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.