Phosphorus Absorption And Gene Expressions Of Related Phosphate Transporters In Primary Cultured Duodenal Epithelial Cells Of Broiler Embryos

On basis of phosphorus?P?nutrition results in early days of the lab,three experiments for this thesis were carried out to investigate the absorption of P by using chicken embryo duodenal epithelial cell model,so that to study the absorptions and mechanisms of P in chicken embryo duodenal epithelial cells in vitro.The first experiment was conducted to evaluate the effect of different density and incubation times of duodenal epithelial cells on close connectivity and cell viability for building absorption model of primary cultured duodenal epithelial cells of broiler embryos in vitro.The primary cultured duodenal epithelial cells of chick embryos were randomly divided into 3 treatments with 6 replicates per treatment.The inoculation densities were 2.9×10⁶ cells/mL?T1?,6.2×10⁶ cells/mL?T2?and 8.8×10⁶cells/mL?T3?.The experiment lasted for 4 days.The results were as follows:1)The isolated duodenal epithelial cells were uniform spherical shape,suspended in the medium.After adhered on the wall,the cells began to spread out and attached uniform and had clear boundaries,showing the growth of"paving stones".2)Duodenal epithelial cells of the positive group were stained blue-black,and the cells of the negative group were not colored after alkaline phosphatase staining.3)Transepithelial electrical resistance?TEER?values in T1 and T2 were greater than 300?·cm²,and the phenol red penetration rate was less than 5%after 24,48 or 72 h cultivations.The TEER value was greater?P<0.03?while the phenol red transmittance of cells was less?P<0.002?in T1 than in T2.4)The lactate dehydrogenase?LDH?enzyme activity in T1 was lower?P<0.0001?than those in T2 or T3 after 24 or 48 h cultivations.It is indicated that the cells with inoculation density of 2.9×10⁶ cells/mL have clear boundary,strong growth,close linkage and the best viability after 48 h cultivations.Therefore,the absorption model of primary cultured duodenal epithelial cells in vitro of broiler embryos is established,which provide a model for studying the absorption and molecular mechanism of phosphorus in primary cultured duodenal epithelial cells.The second experiment was conducted to investigate the absorption characteristics of P in duodenal epithelial cells of broiler embryos at different incubation time.We investigated the kinetic of P absorption in primary cultured duodenal epithelial cells as changing of time.The primary cultured duodenal epithelial cells of chick embryos were randomly divided into 8 treatments with 6 replicates per treatment.The incubation time of 8 treatments were 0,10,20,40,60,80,100 and 120 min,respectively.The results showed that with the increase of incubation time from 0 to 80 min,the P absorption increased linearly?P<0.0001?.Otherwise,we chose the middle time?40 min?as the optimal incubation time to investigate the kinetics of P absorption in embryo duodenal epithelial cells under different concentrations of P.We found that asymptote fitting curve is the best model to fitting the P absorption rate,after using three curve fitting methods of quadratic curve,asymptote and break model.After using asymptote fitting curve model,the optimal incubation time to investigate the molecular mechanism of P absorption in embryo duodenal epithelial cells with different concentrations of P is 87 min.The third experiment was conducted to investigate the absorption kinetics according to the change of P absorption in embryo duodenal epithelial cells with different concentrations of P?0.0,0.75,1.5,3.0,6.0,12.0,24.0 and 48.0 mmol/L P as KH₂PO₄?,and the mRNA and protein expressions of NaP-?b,PiT-1 and PiT-2 of treatments?added 6 or 48 mmol/L?and control group?P-unsupplemented?were determined in order to study the molecular mechanisms of P absorption in duodenal epithelial cells.The primary cultured duodenal epithelial cells of chick embryos were randomly divided into 8 treatments with 6 replicates per treatment.The samples were collected at 40 or 87 min,respectively.The results were as follows:1)The P absorption rate of embryo duodenal epithelial cells linearly or curvely?P<0.0001?increased as added P levels increased.Unsaturated diffusion and saturated carrier transfer mixed model was the optimal model to fit the absorption kinetics of P in primary cultured embryo duodenal epithelial cells with different concentrations of incubation P.2)The mRNA expressions of NaP-?b of primary cultured duodenal epithelial cells were decreased?P<0.04?,the mRNA expressions of PiT-2 were increased?P<0.0001?by added 6.0 mmol/L P.However,the mRNA expressions of PiT-1 and the protein expressions of NaP-?b,PiT-1 and PiT-2 were not affected?P>0.15?by the added P.3)The mRNA and protein expressions of NaP-?b and PiT-1 of primary cultured duodenal epithelial cells were decreased?P<0.04?,the mRNA expressions of PiT-2 were increased?P<0.0001?by added 48.0 mmol/L P.However,the protein expressions of PiT-2 were not affected?P>0.15?by the added P.The The results indicated that the absorption of inorganic phosphorus in the primary cultured duodenal epithelial cells was dominated by unsaturated diffusion and saturated carrier transport.The P absorption was restricted by reducing mRNA and protein expressions of NaP-IIb and PiT-1,when P is overadded in the incubation solution.In conclusion,absorption model of primary cultured duodenal epithelial cells in vitro of broiler embryos was established at cultivated 48 h.The absorption of P in the primary cultured duodenal epithelial cells was dominated by unsaturated diffusion and saturated carrier transport.The P absorption was restricted by reducing mRNA and protein expressions of NaP-IIb and PiT-1,when P is overadded in the incubation solution,which may be a self-feedback regulation mechanism avoiding excessive absorption of P.