The Effect Of Oxidized Oil On Primary Culture Of Hepatocytes From Ctenopharyngodon Idellus

Exp.1Study on Separation and Primary Culture of Hepatocytes fromCt enopharyngodon i dell usIn this study, the utilization of hepatocytes from grass carp after strengthencultured used for experiment, using warm trypsin digestion and red blood cell lysateto separate and purify hepatocytes. Proliferation of hepatocytes was tested by MTTassay, function of hepatocytes were examined using the levels of lactic aciddehydrogenase (LDH), Albumin(ALB), and urea nitrogen(BUN) in supernatant atdifferent times, respectively. The results showed that0.25%concentration of trypsinto dissociate liver tissue for20minutes per time and collect the hepatocytes step bystep, the initial viabilities routinely exceeding99%which detected by trypan blue andblood cell counting chamber. The cells were cultivated in M199medium supplementwith10%foetal bovine serum and10μ g/mL insulin, at1.7×106cell/mL cellingconcentration under the temperature of28℃, concentration of CO2being4.5%for along term. The test observed that lactate dehydrogenase was significantlydecreased(P<0.05), urea nitrogen was significantly increased(P<0.05) and albuminrised during24-72h, at the same time cells have a strong proliferation. This stage wassuited for study metabolism, damage and gene expression of hepatocytes.Exp.2The Effect of Wate rsoluble Matter of Oxidized Soybean Oil on PrimaryCulture of Hepatocytes from Ctenopharyngodon idellusIn this study, the hepatocytes separate from grass carp after cultured24hoursused for experiment. In order to identify the relationship among the accumulation ofconcentration and time of watersoluble matter of oxidized soybean oil and the injurylevel of hepatocytes. Assay the lacticdehydrogenase (LDH)、total antioxidant capacity(T-AOC) and malondialhydries (MDA) in the culture fluid and observe themorphologic changes of hepatocytes by oil red O staining and transmission clectronmicroscope. High concentration of watersoluble matter of oxidized soybean oil in the culture affect3hours could increase LDH leakage、T-AOC capacity and the concentof MDA is higher. There was a significant relationship between the degree of grasscarp hepatocytes damage with dose and time. The degree of injury in highconcentration group was more seroius than control grooup. Moreover, the degree ofinjury increased with the extension of time.Exp.3The Effect of Watersoluble Matter of Oxidized Soybean Oil on GenesExpression of Primary Culture of Hepatocytes from Ctenopharyngodon idellusIn this study, the utilization of hepatocytes from grass carp after cultured24hours were used for experiment, We collected the hepatocytes samples after highconcentration of watersoluble matter of oxidized soybean oil in the culture affect1、1.5、2、2.5and3hours in order to explore the damage mechanism. Using real-timequantitative reverse transcription polymerase chain reaction (qPCR) method to detectthe mRNA abundance of FAS, PGC1-α, SCD1, UCP2, PPAR-α, PPAR-γ,IGF-Ⅰgenes of the hepatocytes so as to discuss the changes of the expression activity.The results showed that, expression of IGF-Ⅰ、UCP2、PGC1-α、PPAR-α genesignificantly decreased, but the expression of upstream genes such as FAS、SCD1、PPAR-γ all significantly increased, meanwhile, expression of all genes significantlychanged in the2.5and3hours compared with1hours in experimental group. Thereason may be due to the water-soluble components such as FFA, MDA increase theexpression of FAS gene, while decrease expression of the PGC1-α、PPAR-α geneswhich may result in increased lipid accumulation in the hepatocytes. Meanwhile, theexpression of PPAR-γ and SCD1increased for self-repair of hepatocytes. Thisinteraction of inhibition and repairtion resulted in the expression changes of all genesat different times.