The Study Of Characteristics Of Trans-membrane Transport Of Long-chain Fatty Acids In Goat Mammary Epithelial Cells

The study was conducted to study the characteristics of trans-membrane transport of four long-chain fatty acids(C16:0,C18:0,C18:1n-9,and C18:2n-6)in mammary epithelial cells of goats.Experiment 1 The primary mammary epithelial cells of goat was isolated and cultured by the isolation method from goat milk.The mammary epithelial cells of goats were identified by cytokeratin 18 fluorescence staining and reverse transcription-polymerase chain reaction(RT-PCR)analysis of beta-casein gene expression Experiment 2:The goat mammary epithelial cells were incubated by the medium with the[1-13C]labeled and unlabeled long-chain fatty acids mixture at the final concentration of 60 ?M for 0,1,2,4,6,12,and 24 h.The uptake-time course of long-chain fatty acids in goat mammary epithelial cells was studied by detecting the ?13C value of the cells.Experiment 3:On the basis of the experiment 2,the goat mammary epithelial cells were incubated for 2 h by the medium with 0,25,50,100,200,400,600,and 800 ?M[1-13C]labeled and unlabeled long-chain fatty acids respectively.The concentration dependency of long-chain fatty acids uptake in goat mammary epithelial cells was studied by detecting the ?13C value of the cells Experiment 4:Based on the results of saturation kinetics,a 2 h incubation time and 50 ?M were used to study the effects of different inhibitors(beta-cyclodextrin,methyl-?-cyclodextrin,diisothiocyanodisulfonic acid DIDS,Sulfo-N-succinimidyl 4-maleimido-oleic acid ester SSO and phloretin)on the trans-membrane transport of long-chain fatty acids in goat mammary epithelial cells.The results showed1.Primary isolation and identification of goat mammary epithelial cellsA small number of mammary epithelial cells can be isolated from goat milk for the primary culture The acquisition time of cells by this method is long.However,the obtained cells have high purity and are not easily contamination.Keratin-18 fluorescence stained cells showed obvious red fluorescence;the gene expression of beta-casein was detected in the cells cultured for 48 h with inducible medium,which showed that the obtained cells were goat mammary epithelial cells with normal growth and biological functions2.Uptake-time course study of long-chain fatty acids trans-membrane transport in goat mammary epithelial cellsThe uptake of C16:0,C18:0,C18:1n-9 and C18:2n-6 in the goat mammary epithelial cells showed a saturation of time dependency.The uptake rate of C16:0,C18:0,C18:1n-9 or C18:2n-6 was the highest around at 2 h in goat mammary epithelial cells.Therefore,this incubation time(2 h)was chosen for the following experiments3.Concentration dependence study of long-chain fatty acids trans-membrane transport in goat mammary epithelial cellsThe uptake of C16:0,C18:0,C18:ln-9 and C18:2n-6 in the goat mammary epithelial cells showed a saturation of concentration dependency.The kinetic parameter(Km)for C16:0 absorption is 88.72 ?M,and the maximum absorption rate(Vmax)is 75.19 ?mol h-1 million cells-1.The Km for C18:0 absorption is 61.61 ?M,and the Vmax is 12.00 ?mol h-1 million cells-1;the Km for C18:ln-9 absorption is 50.82 ?M,and the Vmax is 20.58 ?mol h-1 million cells-1;the Km for C18:2n-6 absorption is 48.29 ?M,and the Vmax is 21.60 ?mol h-1 million cells-1.The average Km value of the four types of long-chain fatty acids is 62.36 ?.Among the tested concentrations,50 ?M long-chain fatty acids was closest to the average Km value and this concentration was chosen for the following experiments4.Effects of different membrane protein inhibitors on long-chain fatty acids trans-membrane transport in goat mammary epithelial cellsThe experiment results showed that the uptake of four long-chain fatty acids in goats mammary epithelial cells were significantly affected by the membrane protein inhibitors,and different inhibitors had different inhibitory effects on uptake of long-chain fatty acids in mammary epithelial cells.Treatment of mammary epithelial cells with methyl-?-cyclodextrin resulted in the highest inhibition of C18:0 uptake by 31.7%(P<0.01),the beta-cyclodextrin resulted in the highest inhibition of C18:1n-9 uptake by 31.2%(P<0.01),or SSO had the highest inhibition of C18:2n-6 uptake by 17.3%(P<0.01)In summary,the cells isolated from milk were goats epithelial cells with normal growth and lactation functions.The trans-membrane transport of long-chain fatty acids in the goat mammary epithelial cells showed a saturation of time and concentration dependency.In this study,the optimal incubation time for long-chain fatty acid absorption is 2 h,and the optimal concentration is 50 ?M.The treatment of membrane protein inhibitors showed that inhibitors inhibited the trans-membrane transport of long-chain fatty acids in goat mammary epithelial cells,which indicated that the membrane proteins-mediated uptake plays an improtant role during the transport process of long-chain fatty acids across epithelial cell membrane.