Study On Regulating Of Neuroendocrine Factor And Metabolite On PEPCK,SCD Genes Expression Of Hepatocytes In Vitro Culture And HSL Genes Expression Of Adipocyte In Vitro Culture

The ketosis and adiposis hepatica, which based on the pathobiology agent of perinatal stage energy metabolism disturbance are significant cow frequently encountered diseases. Research indicates that ketosis and adiposis hepatica are caused by negative balance of energy and fat mobilization, hepar lipidosis, hypoglycemia. ketoplasia are the main element.The energy metabolism feature of cow in perinatal stage is decrease in dry matter englobement and negative balance of energy, glyconeogenesis is the main path of produceing glucose in ruminant, PEPCK is the key enzyme of glyconeogenesis, glyconeogenesis and hypoglycemia can be effected through promoting PEPCK gene expression.Fat mobilization is the only path to relievenegative balance of energy,which can retrieve negative balance of energy as well as produce massive free fatty acid to initiate ketosis and adiposis hepatica. Fat mobilization is catalysised by HSL. So the expression of HSL personalizes mobilization level directly and controlling fat mobilization is significant measure to prevent and cure perinatal stage energy metabolism disturbance.Hepar lipidosis is another invasion origin of ketosis and adiposis hepatica.SCD is rate-limiting enzyme of MUFA production, which can promote triglyceride synthesis. So. fat synthesis can be controlled by controlling SCD expression.Neuroendocrine agent and metabolic are main path to control glycometabolism, fat metabolism and relieve ketosis and adiposis hepatica. The purpose of this research is to investigate molecule control mechanism of PEPCK,SCD,HSL expression and HSL activity, illuminate regulation of neuroendocrine agent and metabolic in the course of ketosis. triglyceride production and fat mobilization, establish molecular biological theory fundament for revealling pathogenesy in the genic and proteinaceous level.Health newborn calves were selected for primary monolayer hepatocytes culture in vitro, which was the best time for spotting while adherent cells emerged after 48h.Target genes(β-actin,PEPCK) were amplificated, cloned, identified and sequenced successfully by applying the reference ofβ-actin, the template of same cDNA. optimized reflect condion of RT-PCR. The results were at equal with Genebank's. INS of 0, 1, 10, 100, 1000nmol/mL and GN of 0, 1, 10, 100, 500pg/mL and IGF-I of 0, 5. 10, 15, 20, 30. 40, 50, 60, 100, 150ng/mL (three array in each gradient) were added to culture fluid.The effect of INS, GN, IGF-Ⅰon expression of PEPCKmRNA were detected by RT-PCR method.Target genes(P-actin,SCD) were successfully amplificated through SYBR greenⅠreal-time quantitative PCR and coined. INS of 0, 5, 10, 20, 50IU/mL and GN of 0, 50, 100, 500, 1000pg/mL, and NPY of 0, 50, 100, 500, 1000pg/mL(three array in each gradient) were added to culture fluid. Then the expression of SCD mRNA was detected applying the reference ofβ-actin and fluorescent PCR methods to detect the expression of SCD mRNA.Primary monolayer preadipocyte was cultured for 14d. Observe cells at 8d and 12d after staining with oil red O and trypan blue solution. IGF-1 of 0, 10. 20, 30, 40, 50ug/L. GLP-Ⅰof 0, 100, 250, 500, 750, 1000nmol/L, dexameth of 0, 10, 20, 40, 80,160mg/L, oleic acid of 0, 0.5, 1.0, 1.5, 2.0, 2.5mmol/L, and lactic acid of 0, 10, 20. 30, 40, 50mg/L were added to culture fluid after simple lipids emerged at 14d, then isolated total RNA after 24h. The expression of HSL mRNA was detected by fluorescent quantitation PCR in adipocyte handled with IGF-Ⅰ,GLP-Ⅰ, dexameth. oleic acid and lactic acid. Then total protein was isolated and detected. HSL activity was detected using lipase kit.The results were as follows:1. The PEPCK mRNA expression was notably inhibited by INS and IGF-Ⅰ. and the effect was dose dependent. While the concentration of INS was between 1 and 10nmol/mL or between 100 and 1000nmol/mL.the suppression was gently. The suppression of IGF-Ⅰwas dose dependent. While the concentration of IGF-Ⅰwas between 10 and 15ng/mL, the suppression was gently, and while the concentration was more than 100ng/mL, there was nearly no PEPCK. The PEPCK mRNA expression dose-dependently increased by GN. Liver gluconeogenesis ability was adjusted by INS and IGF-Ⅰthrough inhititing PEPCK mRNA expression and adjusted by GN through increasing PEPCK mRNA expression.2. The expression of SCD mRNA was notably promoted in hepatocytes while the concentration of INS and NPY was more than 5IU/mL and 50pg/mL respectively. This expression was dose dependent. The expression was notably suppressed while the concentration of GN was more than 50pg/mL. This suppression was dose dependent. INS and NPY promoted SCD mRNA expression and liver fat storing. GN inhibited SCDmRNA express and liver fat storing.3. The expression of HSL mRNA and the activity of HSL were suppressed in adipose cell, treated with IGF-Ⅰ, GLP-Ⅰ, oleic acid and lactic acid. This effecion was dose dependent. While the concentration of IGF-Ⅰ, GLP-Ⅰ, oleic acid, lactic acid was more than 20ng/L,1.0mmol/L and 20mg/L respectively, the suppression towards HSL mRNA abundance was notable. While the concentration was more than 30ug/L,0.5mmol/L and 40mg/L respectively, the suppression towards HSL activity was notable. While the concentration of GLP-Ⅰwas more than 100nmol/L, the suppression towards HSL mRNA abundance and HSL activity was notable. While the concentration of dexameth was more than 20mg/L, the expression of HSL mRNA was promoted, and while the concentration was more than 1 0mg/L, the HSL activity was promoted. It was concluded that IGF-Ⅰ, GLP-Ⅰ, oleic acid, lactic acid, dexameth regulated the fat lipoclasis through regulating the abundance of HSL mRNA and the HSL activity. Dexameth promoted lipoclasis and IGF-Ⅰ, GLP-Ⅰ, oleic acid, lactic acid inhibited lipoclasis, and then promote fat storing.