Characterization of the p130cas pathway in Ocular Cell Adhesions

使用SV 40转化的人角膜上皮细胞（sCE）、SV 40转化的人透镜上皮细胞（sLE）、正常人角膜细胞（nK）、稳定表达人端粒酶逆转录酶的人视网膜色素上皮细胞（TERT-RPE）和人A-375黑素瘤细胞。进行了第四个实验。首先，从人角膜文库中筛选Crk相关底物（Cas）的同源物。第二，将细胞贴壁于纤维连接蛋白（FN）或胶原I预包被的培养板上，加入RGB肽，观察细胞形态学变化。第三，在每个细胞中评估添加RGD肽前后FAK和Cas的酪氨酸磷酸化水平。第四，用流式细胞仪分析α2、α3、α5和αv整合素亚基在各细胞上的表达。我们的结论是，该克隆是Cas的人类同源物。加入RGBS肽后，仅人A-375黑素瘤细胞从板上脱落。在A-375黑色素瘤细胞中，RGD肽诱导FAK和Cas的酪氨酸去磷酸化，但在其他眼细胞中不诱导。SCE表达α2和α3整合素，sLE、nK和TERT-RPE表达α3整合素，A-375黑色素瘤细胞表达α3和α5整合素。RGB肽仅对靶向黑色素瘤细胞有效，而对来自人眼的细胞无效。提示RGD肽可用于眼黑色素瘤的临床治疗

SV40-transformed human corneal epithelial cells (sCE), SV40-transformed human lens epithelial cells (sLE), normal human keratocyte (nK), human retinal pigment epithelial cell that stably expresses human telomerase reverse transcriptase (TERT-RPE) and human A-375 melanoma cells were used. Forth experiments were performed. First, A human corneal library was screened to isolate the human homologue of Crk-associated substrate(Cas). Second, after these cells attached on the fibronection (FN) or collagen I precoated culture plates, RGB peptides were added into culture medium and the morphological changes were observed. Third, the levels of tyrosine phosphorylation of FAK and Cas pre and after adding RGD peptides were evaluated in each cell. Forth, The expression of α2, α3, α5 andαv integrin subunit on each cell were analyzed by flow cytometryA clone, (H-2), encoding 3228 nucleotides, with a long open reading frame (870 amino acid residues) was isolated. We concluded that this clone was the human homologue of Cas. After addition of RGBS peptides, only human A-375 melanoma cells detached from the plate. RGD peptides induced tyrosine dephosphorylation of both FAK and Cas in A-375 melanoma cells but not in other ocular cells. While SCE significantly expressedα2 andα3 integrin and sLE, nK, and TERT-RPE doniinantly expressedα3 integrin, A-375 melanoma cells expressedα3 andα5 integrin. RGB peptides were effective in only target melanoma cell but not cells derived from human eye. This suggested that RGD peptides could be clinically applied to ocular melanoma