【摘要】 目的观察蛋白质糖基化改变对大鼠肝脏上皮细胞WB-F344表面微绒毛及在培养中生长的影响。方法构建含正义或反义6A8DNA的重组pAGX(+)质粒,用脂质体法将重组质粒或空载质粒转染WB-F344细胞,作G418筛选,以有限稀释法作细胞克隆,进行PCR检测新霉素抗性(neoR)基因以确定转染DNA在细胞基因组中的整合;用P-nitrophenyl-α-D-mannopyranoside作底物检测细胞匀浆上清中ERα-甘露糖苷酶的活性;采用扫描电镜观察细胞表面的微绒毛,并做生长曲线检测。结果转染反义6A8的各WB-F344克隆细胞株的匀浆上清中,ERα-甘露糖苷酶活性有不同程度降低,其中以克隆AS1与AS2的降低最明显。克隆AS1与ConA结合增强,表面微绒毛减少、变短,细胞增殖减慢。结论大鼠肝脏ERα-甘露糖苷酶表达抑制所致的蛋白质糖基化改变,可导致大鼠肝脏上皮细胞WB-F344微绒毛减少、变短,细胞生长速度减慢。更多还原

【Abstract】 To study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB -F344and the growth of the cells in culture.Methods Recombinant adeno-associated virus(rAAV)expression vector pAGX (+)containing an antisense or a sense fragment of6A8cDNA encoding a humanα-mannosidase was constructed.The recombinant vectors or the mock were transfected into WB -F344cells by means of lipofectAmine.The transfected cells were selected in G418medium and cloned by means of limiting dilution.Integration of the transfected DNA into host DNA was detected by neo R PCR.Rat liver ERα-mannosidase activity in cell supernatant was measured by using P-nitrophenyl-α-D-mannopyranoside as a substrate.Microvilli on cell surface were observed upon a scan electron microscope.The growth curves of the cells in culture were drawn.Results The cell clones transfected with antisense6A8showed reduction of ERα-mannosidase activity with various degrees.Clone AS1and AS2cell showed a pronounced reduction of the enzymatic activity.In the study on AS1cells,Con A binding to the cells was found to be enhanced,cell growth in culture became slow from day5.The microvilli on the cells were reduced and blunted.Conclusions Transfection with antisense6A8resulted in reduction and blunting of microvilli on the surface of growing WB -F344cells,which might be related to N-glycosylation modification.更多还原