| Pancreas,which can secret amylase,plays a key role in small intestinalstarch digestion.In dairy cows’ duodenum,only about 42%-60% of the starch can be digested and absorbed.Low starch digestibility in small intestine is mainly caused by limited secretion of pancreatic amylase.In present study,we used modified trypsin cold digestion method to isolate pancreatic acinar cells from Holstein calves.Then on this basis,the effects of leucine on amylase secretion as well as the corresponding mechanism were studied by in vitro cell culture of calves’ pancreatic acinar cells to provide a theoretical reference for improving the digestibility of small intestinal starch in ruminants.The main results are as follows:Studies on the isolation of calves’ pancreatic acinar cells The modifiedtrypsin cold digestion method was used to isolate calves’ pancreatic acinar cells.Morphological observation of freshly isolated cells was performed and we set three incubation time gradient of 3,6 and 9 hour.The number of cells obtained and the percentage of living cells were counted.The results showed that the morphological structure of pancreatic acinar cells is similar to that of mouse and rat pancreatic acinar cells,all of them are suspended cells and there isa large number of zymogen granules and endoplasmic reticulums within the cells.The number of cells obtained at6 and 9 incubation hourwas significantly higher than that at 3 hour(P<0.05),and there was no significant difference in cell viability among treatments(P<0.05).Effects of leucine on amylase secretionin calves’ pancreatic acinar cellsFreshly isolated calves’ pancreatic acinar cells were cultured in vitro,and we setleucine treatments of 0,0.5,1,3,9,27 times of normal leucine concentration of DMEM/F12(i.e.,0,0.225,0.45,1.35,4.05 and 12.15 mmol/L)and incubated the cells for 3 hours.The results showed that:(1)As the leucine concentration in the culture medium increased from 0 to 0.5,1 and 3 times,the amylase activity in the supernatant decreased significantly(P<0.05)(2)Compared with the control group,the expression of AF and CCK1 R protein in all the other leucine concentration groups was significantly lower(P<0.05).(3)Compared with the control group,the proteasome activity of the other treatment groups decreased significantly(P<0.05),which are 76%,63%,24%,7% and 9% of that in the control group respectively.Effects of proteasome activity on amylase secretion in calves’ pancreatic acinar cellsBased on the results of experiment 2,we compared the effects of addition without leucine,normal concentration of leucine and high concentration of leucine respectively(i.e.,0,1,27 times of the normal concentration,corresponding to 0,0.45,12.15 mmol/L)with mixed addition of the corresponding concentration of leucine and 5 μmol/L proteasome inhibitor MG-132 in vitro cultured calves’ pancreatic acinar cell.The results showed that: In the 0-fold and 1-fold addition groups,the amount of amylase in the supernatant of culture medium was significantly decreased by MG-132(P<0.05);In the 27-fold addition group,the treatment of leucine and MG-132 did not decrease the secretion of ɑ-amylase(P>0.05).In the 0-fold and 1-fold groups,MG-132 treatments decreased expression of CCK1R(P<0.05),and in the 27-fold group,the addition of MG-132 had a tendency to decrease the expression of CCK1R(P<0.1).In the 0-fold and 1-fold addition groups,the addition of MG-132 significantly reduced the activity of proteasome(P<0.05),accounting for 63% and 72% of the proteasome activity of corresponding concentration of leucine was added alone.Mixed addition of leucine and MG-132 in the 27-fold leucine addition group did not cause significant changes in proteasome activity when compared to addition of leucine alone(P>0.05).In summary,the modified trypsin cold digestion method is suitable for the preparation of pancreatic acinar cells.Leucine can inhibit the secretion of amylase through reducing CCK1 receptor expression and the activity of proteasome. |
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