改良BIGH3对角膜上皮细胞迁移的实验研究  

Modified BIGH3 and its experimental study on corneal epithelial migration

作　　者：王琛[1] 葛红岩[2] 

机构地区：[1]哈尔滨医科大学第二临床医学院医务科,黑龙江哈尔滨150081 [2]哈尔滨医科大学第一临床医学院眼科,黑龙江哈尔滨150001

出　　处：《哈尔滨医科大学学报》2010年第1期53-55,59,共4页Journal of Harbin Medical University

摘　　要：目的研究比较改良前后重组人BIGH3对人角膜上皮细胞迁移的影响。方法培养人角膜上皮细胞,取同一代细胞进行实验;促细胞迁移实验采用改良的Boyden微孔膜双槽法进行细胞迁移实验,下槽液体用含不同浓度(10,20,30,100μg/mL)的野生或诱变BIGH3蛋白的KSFM培养液,2%BSA作为对照组,上槽加入0.4 mL密度3.5×105cells/mL的细胞悬液(细胞总数为1×105)。置于37℃二氧化碳培养箱,温育4 h后酒精固定、Toludine篮染色、压片固定。用倒置相差显微镜,计算膜上相对固定区域的9个高倍视野(×400)的细胞数,取均值。结果诱变型重组人BIGH3比野生型BIGH3拥有更高的促角膜上皮细胞迁移作用,以剂量依赖式方式促进人角膜上皮细胞的黏附。在10μg/mL时,诱变型重组人角膜上皮素即可促进人角膜上皮细胞黏附,随着浓度的增高,作用增强;浓度达到30μg/mL时,促进作用达到高峰。结论诱变型人BIGH3比野生型BIGH3拥有更高的促角膜上皮细胞迁移作用,为进一步临床应用提供了理论基础。Objective To investigate the effect of native and modified BIGH3 protein containing an Arg-Gly-Asp-Arg-Gly-Asp（RGDRGD） motif on the migration of HCE cells.Methods Human fibroblast cells were cultured and the same cells were studied.HCE cell migration assays were performed in transwellplates（8 μm pore size）.The undersurface of the membrane was coated with various concentrations of recombinant BIGH3 or modified BIGH3 containing an RGDRGD motif（10,20,30,100 μg/mL） at 4℃ and then blocked for 1 h at room temperature with PBS containing 2% BSA.Cells were suspended in medium at a density of 3.5×105 cells/mL,and 0.4 mL of the cell suspension was added to the upper compartment of the filter.Cells were allowed to migrate for 4 h at 37 ℃.Migration was terminated by removing the cells from the upper compartment of the filter with a cotton swab,and the filters were fixed with 8% glutaraldehyde and stained with crystal violet.The extent of cell migration was determined by light microscopy,and within each well,counting was performed in nine randomly selected high-magnitude microscopic fields.Results Modified BIGH3 protein increased HCE cell migration in a concentration-dependent manner.There was a significant enhancement of cell migration when the protein concentration was increased to 10 μg /mL,and it reached a plateau at a concentration of 30 μg /mL.Conclu-sion The BIGH3 with a RGDRGD mutation has a stronger promoting HCE epithelial migration than the wild-type one,and is very important to treatment of corneal epithelial wound healing in clinic.

关 键 词：BIGH3 RGDRGD 角膜上皮细胞 迁移 

分 类 号：R772.2[医药卫生—眼科] R-332[医药卫生—临床医学]