CAJ | 학술논문

目的：研究游离脂肪酸（ FFA）混合物对肝细胞L-02的脂毒性及脂代谢相关基因表达的影响。方法：正常肝细胞L-02分别用正常培养基和0．5、1、2 mmol/L FFA混合物（油酸和软脂酸的比例为2∶1）培养24 h后，尼罗红染液室温避光染色，激光共聚焦显微镜及流式细胞仪确定细胞内脂质堆积情况。组织细胞酶法测定试剂盒测定细胞内甘油三酯含量。 MTT法分析细胞存活率，Annexin V-PI凋亡检测试剂盒分析肝细胞的凋亡情况，丙氨酸氨基转移酶（ ALT）和天冬氨酸氨基转移酶（ AST）测定试剂盒分别检测培养液中ALT和AST活性。实时定量PCR技术检测脂代谢相关的脂肪分化相关蛋白（ ADRP ）和固醇调节元件结合蛋白1（ SREBP-1）的mRNA表达情况。结果：各浓度FFAs混合物均可剂量依赖性增加肝细胞脂肪堆积和肝细胞甘油三酯含量，且1 mmol/L FFA混合物可增高肝细胞的甘油三酯含量2．6倍，与非酒精性脂肪肝病人的变化基本相同。2 mmol/L FFA混合物可降低肝细胞L-02细胞存活率并诱导细胞凋亡，而0．5 mmol/L和1 mmol/L FFA混合物对细胞无明显影响。与对照组相比，各浓度FFA混合物对细胞上清中ALT和AST活性无明显影响。1 mmol/L FFA混合物作用后可分别上调肝细胞的ADRP和SREBP-1 mRNA的表达2．660和2．758倍。结论： FFA混合物可诱导肝细胞L-02脂肪变性且2 mmol/L FFA混合物可造成轻度细胞损伤。脂代谢相关基因ADRP和SREBP-1表达上调与FFA混合物诱导的脂肪变性相关。
목적：연구유리지방산（ FFA）혼합물대간세포L-02적지독성급지대사상관기인표체적영향。방법：정상간세포L-02분별용정상배양기화0．5、1、2 mmol/L FFA혼합물（유산화연지산적비례위2∶1）배양24 h후，니라홍염액실온피광염색，격광공취초현미경급류식세포의학정세포내지질퇴적정황。조직세포매법측정시제합측정세포내감유삼지함량。 MTT법분석세포존활솔，Annexin V-PI조망검측시제합분석간세포적조망정황，병안산안기전이매（ ALT）화천동안산안기전이매（ AST）측정시제합분별검측배양액중ALT화AST활성。실시정량PCR기술검측지대사상관적지방분화상관단백（ ADRP ）화고순조절원건결합단백1（ SREBP-1）적mRNA표체정황。결과：각농도FFAs혼합물균가제량의뢰성증가간세포지방퇴적화간세포감유삼지함량，차1 mmol/L FFA혼합물가증고간세포적감유삼지함량2．6배，여비주정성지방간병인적변화기본상동。2 mmol/L FFA혼합물가강저간세포L-02세포존활솔병유도세포조망，이0．5 mmol/L화1 mmol/L FFA혼합물대세포무명현영향。여대조조상비，각농도FFA혼합물대세포상청중ALT화AST활성무명현영향。1 mmol/L FFA혼합물작용후가분별상조간세포적ADRP화SREBP-1 mRNA적표체2．660화2．758배。결론： FFA혼합물가유도간세포L-02지방변성차2 mmol/L FFA혼합물가조성경도세포손상。지대사상관기인ADRP화SREBP-1표체상조여FFA혼합물유도적지방변성상관。
AIM: To investigate the lipotoxicity of free fatty acid (FFA) mixture and the effect of the FFA mixture on lipid metabolism-related genes in L-02 cells.METHODS:A normal human hepatocytes-derived cell line L-02 was treated with 0.5, 1 and 2 mmol/L FFA mixture (oleate and palmitate, 2∶1) for 24 h.The cellular total lipid accumu-lation was determined after Nile red staining by confocal laser scanning microscopy and flow cytometry .Intracellular triglyc-eride ( TG) content was measured using an enzymatic kit .The viability of L-02 cells was determined by MTT assay and the apoptosis-inducing effect of FFA mixture was evaluated by annexin V/PI staining .The levels of alanine aminotransferase ( ALT) and aspartate aminotransferase ( AST ) in the culture medium were detected by ALT and AST kits . The mRNA levels of lipid metabolism-related genes, adipose differentiation-related protein (ADRP) and sterol regulatory ele-ment-binding protein 1 (SREBP-1), were examined by quantitative real-time PCR.RESULTS:All the different concen-trations of FFA mixture increased intracellular lipid accumulation and TG content in a dose -dependent manner .FFA mix-ture at concentration of 1 mmol/L increased intracellular TG by 2.6 folds, which matched with the change in non-alcoholic fatty liver disease patients .Treatment for 24 h with 0.5 mmol/L and 1 mmol/L FFA mixture did not trigger apparent cell death and apoptosis , while treatment with 2 mmol/L FFA mixture resulted in a marked decrease in cell viability and in-duced early and late stages of apoptosis in L-02 cells.The levels of ALT and AST in the culture supernatant had no signifi-cant difference between control group and FFA treatment group .Treatment with 1 mmol/L FFA mixture up-regulated the expression of ADRP and SREBP-1 by 2.660 and 2.758 folds, respectively.CONCLUSION:FFA mixture induces the he-patic steatosis and 2 mmol/L FFA mixture causes mild cells damage in L-02 cells.The up-regulation of ADRP and SREBP-1 may be involved in FFA-induced hepatic steatosis .