| Cholesterol metabolism is closely related to the hepatic steatosis.At present,there is few report about the effect and mechanism of exogenous cholesterol on the metabolism of cholesterol and fatty acids in liver.In this study,in order to research the role of cholesterol in hepatocytes steatosis,we isolated and cultured the primary hepatocytes of Landes goose and Sichuan white goose as material,and investigated the effect of exogenous cholesterol on TG and TC content in hepatocytes,and studied the mRNA expression change of key genes involved in the metabolism of cholesterol and fatty acids,including sterol regulatory element-binding proteins-1 and 2(SREBP-1,SREBP-2),fatty acid synthase(FAS) and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase(HMGR) using the method of Real-time quantitative PCR.The results were as follows:(1) Hepatocytes were successed harvested from Lande goose and Sichuan White goose by using a modified method of the seglen's,and about 6×10~7 hepatocytes could get from each goose.The livability achieved about 90%.(2) The results showed that the TG content was increased markedly induced by 10,20,30μg/ml cholesterol(P＜0.05),and the effect of 20μg/ml cholesterol was especially evident;The TG content increased as the cultured time with cholesterol was prolonged. Compared to 0h,12h,24h groups,TG content in goose primary hepatocytes cells after cultured 48h was the highest.But compared to 72h group,there was no evident difference. Comparing the two different breeds of goose in different cholesterol treatment,the TG content was markedly higher in Langde goose hepatocytes than in Sichuan White goose hepatocytes. (3) The transcription concentration of SREBP-1 was increased by 10～30μg/ml cholesterol(P＜0.05),and the effert of 20μg/ml cholesterol was especially evident.The expression of SREBP-1 was increased as the cultured time with cholesterol was prolonged, Compared to 0h,12h,24h groups,The expression of SREBP-1 mRNA in goose primary hepatocytes cells after cultured 48h was the highest.But compared to 72h group,there is no evident difference.Comparing the two different breeds of goose in different cholesterol treatments,the expression of SREBP-1 was markedly higher in langde goose hepatocytes than in Sichuan goose hepatocytes.(4) The transcription concentration of FAS was increased by 10～0μg/ml cholesterol(P＜0.05),and the effert of 20μg/ml cholesterol was especially evident.The expression of FAS was increased as the cultured time with cholesterol was prolonged. Compared to 0h,12h,24h groups,The expression of FAS mRNA in goose primary hepatocytes cells after cultured 48h was the highest.Compared to 72h group,there was no evident difference.Comparing two different breeds of goose in different cholesterol treatments,the expression of FAS mRNA was markedly higher in langde goose hepatocytes than in Sichuan goose hepatocytes.(5) The results showed that the TC content was not significantly increased by 10,20,30μg/ml cholesterol(P＞0.05).The TC concent was significantly increased as the cultured time with cholesterol was prolonged at first,and it reached the highest point and significantly different from the control group as the cultured time with cholesterol for 24h, after 24h,the TC content began to drop down.When cultured with cholesterol for 72h, there was no significantly difference compared to the control group.Comparing the two different breeds of goose in different cholesterol treatments,the TC concent was markedly higher in Langde goose hepatocytes than in Sichuan goose hepatocytes.(6) The transcription concentration of SREBP-2 was decreased by 10～30μg/ml cholesterol(P＜0.05),and the effert of 20μg/ml cholesterol was especially evident.The expression of SREBP-2 was decreased as the cultured time with cholesterol was prolonged. Compared to 0h,12h,24h groups,The expression of SREBP-2 mRNA in goose primary hepatocytes cells after cultured 48h was the lowest.Compared to the 72h group,there was no evident difference.The expression of SREBP-2 mRNA had no significantly difference between Sichuan goose and Langde goose in all treatments.(7) The transcription concentration of HMGR was decreased by 10～30μg/ml cholesterol(P＜0.05),and the effect of 20μg/ml cholesterol was especially evident.The expression of HMGR was decreased as the cultured time with cholesterol was prolonged. Compared to Oh,12h,24h groups,the expression of HMGR mRNA in goose primary hepatocytes cells after cultured 48h was the highest.Compared to the 72h group,there was no evident difference.The expression of HMGR mRNA has no significantly difference between Sichuan goose and Langde goose hepatocytes in all trentments.(8) The correlation analysis showed that the TG content of two kinds of goose did not have the significant positive correlation with the concentration of cholesterol(P＞0.05),and it had the significant positive correlation with the culture time of adding cholesterol (P＜0.05);SREBP-1 mRNA expression did not have the significant positive correlation with the concentration of cholesterol(P＞0.05),but it had the significant positive correlation with the culture time of adding cholesterol;FAS mRNA expression had the significant positive correlation with the concentration of cholesterol and the culture time of adding cholesterol(P＜0.05);FAS mRNA expression had the higher significant positive correlation with the SREBP-1 mRNA expression(P＜0.01).(9) The correlation analysis showed that the TC content of two kinds of goose did not have the significant correlation with the concentration of cholesterol and the culture time of adding cholesterol;SREBP-2 and HMGR did not have the significant negative correlation with the concentration of cholesterol(P＞0.05),but it had the significant negative correlation with the culture time of adding cholesterol;The TC content did not have the significant negative correlation with SREBP-2 and HMGRmRNA expression. HMGR mRNA expression had the higher significant positive correlation with the SREBP-2 mRNA expression(P＜0.01). |
