小鼠胚胎干细胞源表皮样细胞β_1整合素的表达与序列分析  

Expression and Sequence Analysis of Integrinβ_1 Gene mRNA in Embryonic Stem Cell Derived Epidermal Like Cells

作　　者：张仁礼[1] 高劲松 李海标[1] 

机构地区：[1]中山大学中山医学院组织胚胎学教研室 [2]达安基因诊断中心,广州510080

出　　处：《中山大学学报（医学科学版）》2004年第5期417-420,共4页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家重点基础研究课题(9.73)基金资助项目(G1999054301-2)

摘　　要：【目的】研究小鼠胚胎干细胞源表皮样细胞分化过程中β1整合素(integrinβ1)表达情况与序列分析,为在分子水平上了解胚胎干细胞分化为表皮样干细胞时相关特异性蛋白表达量的变化打下基础。【方法】用双层培养板将小鼠胚胎干细胞与人羊膜共培养4～5d,显微镜下观察其形态分化,分别用免疫组化法和Actin内参照逆转录聚合酶链反应(reversetranscriptionpolymerasechainreaction,RT-PCR)定量检测β1整合素蛋白和mRNA的表达情况,并对RT-PCR产物进行测序,对照组未加羊膜。【结果】与人羊膜共培养4～5d后,ES细胞分化为表皮细胞样的单层细胞,细胞排列紧密,呈多边形;免疫组化结果显示诱导4d后部分细胞呈β1整合素阳性;RT-PCR结果显示实验组β1整合素mRNA有较强的表达;对照组细胞形态各异,不能形成细胞单层,几乎不表达β1整合素,测序及同源性比对结果表明两条扩增序列分别与β1整合素具有100%的同源性。【结论】小鼠ES细胞源表皮样细胞表达较高水平的β1整合素,而未加羊膜诱导的ES细胞几乎不表达β1整合素。To research the expression and analy ze the sequence of integrinβ 1 gene in embryonic stem(ES)cells derived epidermal like cells,and to lay a base for analyzing the exp ression of relative protein in molecular lev el when ES cells differentrate into e pidermal like cells.ES-BALB /c cells were cocultured wit h human amnion in transwells for 4-5d ays.The morphological differentiation were observed under microscope,and the expression of i ntegrinβ 1 protein and mRNA were measured using immunohistochemistry and actin controlled reverse transcription polymerase chain reaction(RT-PCR)respectively.The sequence of RT-PC R products were analyzed and compared with the published sequence of integrinβ 1 gene by BLAST.Cells in control were c ultured alone without amnion.After 4-5days of coculture,ES cells differentiated into single layer of epidermal like cells,fitted tightly,with polyhedral in shape.Immunoh istochemical results showed that part of the cells were integrinβ 1 positive after 4d of induction.The RT-PCR assay showed that the cells after 4d of induction expresse d high level of integrinβ 1 mRNA,and the sequences of the RT-PCR products were 100%homologou s to that of integrinβ 1 .Cells in control group were diverse in morphological shapes,did not form e pidermal like single layer,and expr essed very low level of integrinβ 1 .[Conclusion]Mouse ES cell derived epidermal like cells express high level of integrinβ 1which is scarcely expressed in contr ol group without amnion.

关 键 词：Β1整合素 表达 表皮 干细胞 人羊膜 对照组 RT-PCR 小鼠胚胎 ES细胞 RNA 

分 类 号：R735[医药卫生—肿瘤] R737[医药卫生—临床医学]