Effect Of NEFA,BHBA And GLU On CPT-Ⅰ In Vitro Culture Bovine Hepatocytes

In this study, in vitro primary cell culture, quantitive RT-PCR, and ELISE were applied to research the effect of NEFA,BHBA and GLU on the expression and transcription of the key enzyme CPT-â… for hepatic fatty acid oxidation of dairy cow. The focal point of this study is to probe the regulation mechanism of NEFA,BHBA and GLU. on the above mentioned metabolism. and then identify the characteristics of hepatic fatty acid oxidation of ketosis cow,major restricted factor and key joints ,which may provide rationale and systematic data for the further.According to the publishing sequence in GenBank, primers were designed and RT-PCR was employed to amplify the partial mRNA of CPT-â… and the target sequences were recombinated into cloning vector, The method of fluorescent quantitation RT-PCR was established to detect the mRNA expression of CPT-â… .In the experiment of the effect of NEFA,BHBA and GLU on the transcriptional level of the key enzyme CPT-â… that catalysis fatty acid oxidation in hepatocyte of dairy cow.The effects of NEFA ( 0,0.3,0.6,1.2,2.4,4.8 mM/L ) ,BHBA(0,0.3,0.6,1.2,2.4,4.8 mM/L)and GLU(0,5,10,15,20,25 mM/L)on mRNA expression of CPT-â… in hepatocyte were detected by fluorescent quantitation PCR respectively. The result showed that:â‘ the mRNA expression of CPT-â… in hepatocyte was increased with increasing concentration of NEFA in the limted range in culture media.â‘¡The level of CPT-â… RNA in hepatocyte was decreased markedly with increasing concentration of BHBA in culture media,â‘¢The level of CPT-â… mRNA in hepatocyte was decreased moderately with increasing concentration of GLU in culture media.In the experiment of the effect of NEFA,BHBA and GLU on the translational level of the key enzyme CPT-â… that catalysis fatty acid oxidation in hepatocyte of dairy cow.The effects of NEFA ( 0,0.3,0.6,1.2,2.4,4.8 mM/L ) BHBA(0,0.3,0.6,1.2,2.4,4.8 mM/L)and GLU(0,5,10,15,20,25 mM/L)on mRNA expression of CPT-â… in hepatocyte were detected by fluorescent quantitation PCR. respectively The result showed that:â‘ the expression of CPT-â… in hepatocyte was increased with increasing concentration of NEFA in culture media.in a certain low concentration range ,but when beyond a certain concentration,the promotive effect become c downâ‘¡The translational level of CPT-â… in hepatocyte was decreased markedly with increasing concentration of BHBA in culture media,and showing dose dependentâ‘¢The translational level of CPT-â… in hepatocyte has nothing to do with increasing concentration of GLU in culture media.In conclusion: Firstly The transcriptional and translational level of CPT-â… in hepatocyte was increased with increasing concentration of NEFA in the limted range in culture media. Showing dose dependent effect But exceeding concentration of NEFA restrains the transcriptional and translational level.Secondly The transcriptional and translational level of CPT-â… in hepatocyte was decreased markedly with increasing concentration of BHBA in culture media,Showing dose dependent effect.Thirdly,The level of CPT-â… mRNA in hepatocyte was decreased moderately with increasing concentration of GLU in culture media. Showing little dose dependent.But the translational level of CPT-â… has nothing to with the GLU.Finally,NEFA(in the low concentration range) would decrease its concentration in hepatocyte probably by means of promoting the transcriptional and translational level.to release the TG accumulation in liver.However,when in ketosis and fatty liver condition,because of excessive fat mobilization, NEFA accumulation in both blood and liver promote, which restrains the transcriptional and translational level of CPT-â… ,resulting in fatty acid oxidation disturbance;BHBA would cause NEFA concentration in hepatocyte probably by means of restraining the transcriptional and translational reaction of CPT-â… ,resulting in NEFA concentration.There might be little respondence to GLU for fatty acid oxidation in vitro culture bovine hepatocyte...