基因突变体的构建及其在张氏肝细胞中表达及功能.docxVIP

O．01)；而突变型、空质粒和正常细胞组两两间比较差异均没有显著性意 O．01)；而突变型、空质粒和正常细胞组两两间比较差异均没有显著性意 义(PO．05)。在排出实验中，孵育至第30 min野生型，突变型及空质粒 组脂肪酸的相对排出率分别为48％士7％，42％士7％，33％士12％，三者并没 有明显的差异(尸0．05)。 结论：本研究首次在细胞水平构建的L—FABP T94A稳定表达细胞模型 可用于L．FABP突变的功能研究；高表达的L．FABP可以促进脂肪酸的摄 入；L．FABP T94A的突变可降低肝脏细胞对游离脂肪酸的摄入。L．FABP 的T94A突变对脂质的排出无显著影响。 关键词： 肝脏型脂肪酸结合蛋白，油酸，基因突变，脂质代谢 ABSTRACTBacl屯round：Liver ABSTRACT Bacl屯round：Liver fatty acid binding protein(L—FABP)is a member of the superfamily of lipid—binding proteins，which is a small cytosolic protein involvedin intracellular fatty acid(FA)transfer and metabolism．The specificity of tissue expression and binding properties prompt L—FABP plays an importent role on lipid metabolism in liver． L-FABP gene exist a threonine to alanine substitution at codon 94 in the exon 3，we hypothesize the genetic variant has alteration of function， which will contribute to the liquid metabolism． Objective：This study is to establish stable cell lines to express L—FABP of wild and mutant，approach the differenc in FFA uptake and effiux between the Wi 1 d and mtxtant． Methods：Establish of the Human L—FABP stable expressing cell lines by PCR，RT-PCR，Construction of Plasmid，Site-directed mutagenesis，transformation，transfections，selection with G4 1 8．Protein determination by Westem Blot．Localization of the recombinated protein in the cell by immunohistochemistry．Radiolabeled oleic acid uptake studies were performed on chang liver in different times．Then cells were lysed and the radioactivity taken up was determined by liquid scintillation counting．Effiux studies were performed on chang liver which loaded with[3H]一oleic acid for 1 5 min，the radioactivity was measured for di fferent intervals of times． Results：Construct the L—FABP stable expressing cell lines successfully．Immunohistochemistry identified the recombinated protein mainly expressed in endochylema which was coincident with the normal physiological expression．The function analysis identified L—FABP correlated with the FFA uptake．Differen