CAJ | 학술논문

目的建立Hsf4-/-小鼠的晶状体永生化上皮细胞系.方法实验研究.取新生P6天的Hsf4-/-小鼠晶状体原代培养晶状体上皮细胞(MLEC),用SV40,T-抗原转染,C418筛选建立永生化细胞系MLEC/Hsf4-/-,免疫印迹法检测α-A晶状体蛋白表达.结果倒置相差显微镜下观察可见细胞贴壁生长,有伪足伸出,多角形,大小均匀,细胞膜边界清晰,细胞质透亮.消化后的细胞呈圆形透亮,胞质丰富.免疫印迹法检测MLEC/Hsf4-/-细胞(两个克隆)中的α-A晶状体蛋白(α-A-crystallin)有表达.转染Hsf4b蛋白后Hsp25蛋白表达上调.结论采用组织块接种法获得小鼠晶状体上皮细胞后,转染SV40大T-抗原,G418筛选可成功建立永生化细胞系MLEC/Hsf4-/-.
목적 건립Hsf4-/-소서적정상체영생화상피세포계.방법 실험연구.취신생P6천적Hsf4-/-소서정상체원대배양정상체상피세포(MLEC),용SV40,T-항원전염,C418사선건립영생화세포계MLEC/Hsf4-/-,면역인적법검측α-A정상체단백표체.결과 도치상차현미경하관찰가견세포첩벽생장,유위족신출,다각형,대소균균,세포막변계청석,세포질투량.소화후적세포정원형투량,포질봉부.면역인적법검측MLEC/Hsf4-/-세포(량개극륭)중적α-A정상체단백(α-A-crystallin)유표체.전염Hsf4b단백후Hsp25단백표체상조.결론 채용조직괴접충법획득소서정상체상피세포후,전염SV40대T-항원,G418사선가성공건립영생화세포계MLEC/Hsf4-/-.
Objective To establish mouse lens epithelial cell lines with the genotype of Hsf4-/-.Methods The expended mouse lens epithelial cells,which were generated from P6 Hsf4-deficient mouse lens epithelia,were immortalized with SV40-T-antigen and named MLEC/Hsf4-/-cell.The expression of alpha A-crystallin was immunoblotted.Hsf4b cDNA was reconstituted by transiently transfection.Results The SV40-immortalized cells were in adherent growth mode with spindle morphology,pseudopodia,clear nuclear bondary membrane and cytoplasm translucent.Immunoblotting results indicated that the lens biomarker protein alpha A-cryatllin was expressed in MLEC/Hsf4-/-cells.Reconstitution of Hsf4b into MLEC/Hsf4-/-cells upregulated the expression of Hsp25.Conclusions The SV40-immortalized MLEC/ Hsf4-/-cells have the lens epithelial characteristics and could be used as a tool for studying the signal transduction in vitro.