The Comparative Study Of The Response Pattern Of Glu、NAA、 Dipeptide (Lys-Phe) Transporter Gene And Protein On The Substrate In The Caco-2Cells

Our purpose is to assess whether the absorption of small peptide would have thenutrition significance from the other side, compared with the three transporters（glucose、neutral amino acid and dipeptide）on the mRNA and protein levels.Trial1： The human colon cancer cell lines (Caco-2cells) were selected as the vitromodel of nutrition substrate transporter in the small intestine. It is to observe the expressionof peptide transporter PepT1mRNA at different time points under starvation conditions, thetrial is divided into20treatment groups, each group has six repeat. Set the zero time for thecontrol group, collecting the control group and each treatment group total RNA in Caco-2cells to detect the expression of the PepT1mRNA. The results showed that the quadraticfunction was obtained via linear regression of the time and the PepT1mRNA: y=-0.000382t~2+0.0112t+0.8284（t：time，y：the relative expression levels of PepT1mRNA），the related coefficient r=0.62. t=15h, the expression of PepT1mRNA was highest, theshort-time to hunger can up-regulated the expression of PepT1mRNA (P<0.05); with theextension of time, the expression of PepT1mRNA was gradually decreased. The analysis ofthis phenomenon may be that there is a temporary compensatory changes to balance thepeptide supply shortage in Caco-2cells, increasing the level of PepT1mRNA and ensuringthe number of small-peptide carrier to not reduce the absorption of small peptides, whichmaybe a physiological response of Caco-2cells to adapt to the environment.Trial2： The human colon cancer cell lines (Caco-2cells) were selected as the vitromodel of nutrition substrate transporter in the small intestine too. Three trials:1、Adding25mmol/L Glucose after Caco-2cells were hunger2h,then we detect the relative levels ofSGLT1mRNA by the means of RT-PCR at different time points;2、Adding0.8mmol/Ldipeptide (Lys-Phe) after Caco-2cells were hunger2h,then we detect the relative levels ofPepT1mRNA by the means of RT-PCR at different time points;3、Adding0.8mmol/L neutralamino acid (Ser，Thr，Cys，Ala) after Caco-2cells were hunger2h,then we detect the relativelevels of ASCT2mRNA by the means of RT-PCR at different time points, each trial wasdivided into nine treatment groups and each group has six repeat. The results showed that thelevel of the glucose transporter SGLT1mRNA has significant increase on15min compared with the control group (P<0.05), then the expression of SGLT1mRNA was gradually downregulated and was lower than the control group on24h but not significant (P>0.05); Theexpression of neutral amino acid transporter ASCT2mRNA was up regulated on the15min,30min compared with the control group (P<0.05), the expression of ASCT2mRNA hasno difference on15min and30min,then with the extension of time, it was gradual downwardand was significant lower than the control group (P<0.05).The level of small peptidestransporter PepT1mRNA shows the waves downward trend over time, and it has the highestexpression on the10min,30min,120min,24h and it has significant differences comparedwith the control group (P<0.05).The results showed that: adding substrate after starvation ofCaco-2cells can all promote the expression of the corresponding transporter mRNA in a shortperiod of time, and then by increasing the number of the transporter to transport moresubstrates. With the time extension, the transporter protein may be in a dynamic equilibrium,and then, in turn, adjust the carrier mRNA expression. When the substrate concentration wereconstant，the response pattern of the three transporters mRNA on time were basically thesame.Trial3：The experiment is to study the effects of different concentrations of substrateson the expression of corresponding transporters mRNA and protein and Caco-2cells were thevitro model of nutrient transport. The three experiments were that adding differentconcentrations after Caco-2cells were hungry2h, we collect total RNA and protein from cellsafter incubating24h to detect the expression of the corresponding transporters, each trial wasdivided into nine treatment groups and each group has six repeat. It was found that theexpression of each transporter mRNA in the control groups were higher than those in eachtreatment groups (P<0.05), the effect of the substrate concentration gradient increase on theexpression of the respective transporter mRNA is not significant (P>0.05).The increase ofglucose concentration gradient can up-regulate SGLT1protein levels (P<0.05), the SGLT1protein levels of50mmol/L and100mmol/L had no significant effect ((P>0.05); The increaseof the dipeptide (Lys-Phe) and neutral amino acid（Ser、Thr、Cys、Ala）concentration gradienthas no effect on the expression of the respective transporter protein, but an upward trend(P>0.05). These results indicate that under the concentration range of the test conditions, the increase of the substrate concentration has little effect on the expression of the threetransporter gene and protein, but hunger can significantly promote the expression oftransporter mRNA levels, the analysis may be that the transporter protein would maintain adynamic equilibrium between mRNA synthesis and protein degradation. Compared withglucose, neutral amino acid results, the observed appearance that influence of the substratepeptide on the transporter PepT1were basically the same, thus, we hold the view of thepositive support that small peptide has the nutritional significance in animals.