亮氨酸通过蛋白酶体调控犊牛胰腺腺泡细胞淀粉酶的分泌
发布时间：2019-05-16 15:48
【摘要】：胰腺作为重要的消化器官,其分泌的淀粉酶在动物消化利用小肠淀粉的过程中发挥着关键作用。奶牛十二指肠中的淀粉仅有42%-60%可被消化吸收,胰腺淀粉酶分泌的不足是限制奶牛等反刍动物小肠淀粉利用的主要因素。本研究利用改进的胰蛋白酶冷消化法分离荷斯坦犊牛胰腺腺泡细胞,并在此基础上通过体外细胞培养试验,研究亮氨酸对犊牛胰腺腺泡细胞淀粉酶分泌的影响及相应的调控机制,以期为通过营养手段改善奶牛等反刍动物小肠淀粉消化提供理论参考。主要结果如下:犊牛胰腺腺泡细胞的分离技术研究使用改进后的胰蛋白酶冷消化法分离奶犊牛胰腺腺泡细胞,对分离得到的细胞进行形态学观察,并设置3、6、9小时的孵育时间梯度处理,对获得的细胞数目及活率进行计数。结果表明:犊牛胰腺腺泡细胞的形态结构和小鼠及大鼠的胰腺腺泡细胞相似,均为悬浮细胞,细胞内可见大量的酶原颗粒。孵育6小时和9小时时获得的细胞数目较3小时明显升高(P0.05),活率并无显著差异(P0.05)。亮氨酸对犊牛胰腺腺泡细胞淀粉酶分泌的影响体外培养新鲜分离的犊牛胰腺腺泡细胞,培养液使用无亮氨酸的DMEM/F12,分别添加其正常亮氨酸浓度的0、0.5、1、3、9、27倍梯度处理(即0、0.225、0.45、1.35、4.05和12.15 mmol/L)并于正常培养条件下培养3小时。结果显示:(1)随着培养液中亮氨酸浓度由0倍提高至0.5、1、3倍时,上清培养液中的酶活出现了显著下降(P0.05),当亮氨酸添加浓度继续升高时,酶活并未继续降低(P0.05);(2)相较于0倍对照组,其他几个亮氨酸浓度处理组的AF及CCK1R的蛋白表达均出现了显著下降(P0.05);(3)相较于对照组,其他几个亮氨酸浓度处理组的蛋白酶体活性出现了显著下降(P0.05),分别为对照组的76%、63%、24%、7%和9%。蛋白酶体对犊牛胰腺腺泡细胞淀粉酶分泌调控在试验二结果的基础上,以犊牛胰腺腺泡细胞为研究对象,设置无亮氨酸、正常浓度亮氨酸和高浓度亮氨酸(即0、1、27倍浓度,对应0、0.45、12.15 mmol/L)的单独添加及相应浓度的亮氨酸与5μmol/L蛋白酶体抑制剂MG-132的混合添加处理,于正常培养条件下培养3小时。结果显示:(1)0倍和1倍添加组中亮氨酸和MG-132的混合添加相较于亮氨酸的单独添加,培养液上清中的ɑ-淀粉酶量出现了显著下降(P0.05),27倍添加组中,亮氨酸和MG-132的混合添加并未降低ɑ-淀粉酶的分泌(P0.05);(2)0倍和1倍组中,MG-132的添加均降低了CCK1R的蛋白表达水平(P0.05),27倍组中,MG-132的添加有降低CCK1R表达的趋势(P0.1);(3)0倍和1倍亮氨酸添加组中,亮氨酸和MG-132的混合添加均显著降低了蛋白酶体的活性(P0.05),分别为单独添加相应浓度亮氨酸时蛋白酶体活性的63%和72%。27倍亮氨酸添加组中亮氨酸与MG-132的混合添加相较于亮氨酸的单独添加,蛋白酶体的活性并未出现显著变化(P0.05)。结论:改进的胰蛋白酶冷消化法适用用于分离制备体犊牛胰腺腺泡细胞;亮氨酸可以通过降低蛋白酶体活性及CCK1R表达,进而抑制犊牛胰腺腺泡细的胞淀粉酶分泌。
[Abstract]:The pancreas, as an important digestive organ, plays a key role in the process of animal digestion and the use of small intestinal starch. Only 42% -60% of the starch in the duodenum of the dairy cow can be digested and absorbed, and the insufficiency of the pancreatic amylase secretion is the main factor to limit the utilization of the small intestinal starch of the ruminant such as the cow. In this study, the pancreatic acinar cells of the calf of Holstein were isolated by an improved trypsin-cold digestion method, and the effect of leucine on the secretion of the pancreatic acinar cells and the corresponding regulation and control mechanism of the calf pancreatic acinar cells were studied by in vitro cell culture test. In ord to provide a theoretical reference for improving the digestion of small intestinal starch in ruminants such as dairy cows by means of a nutritional means. The main results are as follows: The isolation technique of the pancreatic acinar cells of the calf uses the modified trypsin-cold digestion method to separate the pancreatic acinar cells of the calf, perform the morphological observation on the separated cells, and set the incubation time gradient treatment of 3,6 and 9 hours, The number of cells obtained and the number of cells obtained were counted. The results showed that the morphological structure of the pancreatic acinar cells in the calf and the pancreatic acinar cells in the mice and the rats were similar, both of which were suspended cells, and a large amount of zymogen granules were visible in the cells. The number of cells obtained at 6 and 9 hours of incubation was significantly higher than that of 3 hours (P0.05), and there was no significant difference in the number of cells (P0.05). The effect of leucine on the secretion of the amylase of the pancreatic acinar cell of the calf, in-vitro culture of the freshly isolated calf pancreatic acinar cells, the culture solution using the leucine-free DMEM/ F12, The 0, 0.5,1,3,9, and 27-fold gradient treatments of their normal leucine concentrations (i.e.,0, 0.225, 0.45, 1.35, 4.05 and 12.15 mmol/ L) were added, respectively, and cultured for 3 hours under normal culture conditions. The results showed that (1) As the concentration of leucine in the culture medium increased from 0 to 0.5,1 and 3 times, the activity of the enzyme in the supernatant was significantly decreased (P0.05). When the concentration of leucine increased, the activity of the enzyme did not continue to decrease (P0.05). (2) Compared with the control group, The protein expression of AF and CCK1R in other leucine-concentration-treated groups decreased significantly (P0.05); (3) the proteasome activity in other leucine-concentration-treated groups decreased significantly (P <0.05) compared with the control group, and 76%,63% and 24% of the control group, respectively. 7% and 9%. The activity of the proteasome on the secretion of the pancreatic acinar cells of the calf was controlled on the basis of the results of the test, and the calf pancreatic acinar cells were used as the research object, and the leucine, the normal concentration of leucine and the high-concentration leucine (i.e.,0,1,27) were set. The mixture of leucine with the corresponding concentration of 0, 0.45, 12.15 mmol/ L and the corresponding concentration of leucine with 5. m u.mol/ L proteasome inhibitor MG-132 was added and cultured for 3 hours under normal culture conditions. The results showed that (1) the mixture of leucine and MG-132 in the 0-fold and 1-fold addition group was added separately compared with that of leucine, and the amount of amylase-amylase in the supernatant of the culture medium was significantly decreased (P0.05) and the addition of 27-fold. The addition of leucine and MG-132 did not decrease the secretion of L-amylase (P0.05); (2) in the group of 0 and 1, the addition of MG-132 decreased the protein expression level of CCK1R (P0.05). In the 27-fold group, the addition of MG-132 had the tendency to decrease the expression of CCK1R (P0.01); (3)0-fold and 1-fold leucine addition group, the mixed addition of leucine and mg-132 significantly reduced the activity of the proteasome (p0.05), respectively, of 63% and 72% of the proteasome activity when the corresponding concentration of leucine was added separately, and the mixture of leucine and mg-132 in the 27-fold leucine addition group was added separately from the leucine, There was no significant change in the activity of the proteasome (P0.05). Conclusion: The modified trypsin-cold digestion method is suitable for the separation of the pancreatic acinar cells in the body of the calf, and the leucine can be expressed by reducing the proteasome activity and the expression of the CCK1R, thereby inhibiting the secretion of the pancreatic acinar cells of the calf.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823
本文编号：2478384
[Abstract]:The pancreas, as an important digestive organ, plays a key role in the process of animal digestion and the use of small intestinal starch. Only 42% -60% of the starch in the duodenum of the dairy cow can be digested and absorbed, and the insufficiency of the pancreatic amylase secretion is the main factor to limit the utilization of the small intestinal starch of the ruminant such as the cow. In this study, the pancreatic acinar cells of the calf of Holstein were isolated by an improved trypsin-cold digestion method, and the effect of leucine on the secretion of the pancreatic acinar cells and the corresponding regulation and control mechanism of the calf pancreatic acinar cells were studied by in vitro cell culture test. In ord to provide a theoretical reference for improving the digestion of small intestinal starch in ruminants such as dairy cows by means of a nutritional means. The main results are as follows: The isolation technique of the pancreatic acinar cells of the calf uses the modified trypsin-cold digestion method to separate the pancreatic acinar cells of the calf, perform the morphological observation on the separated cells, and set the incubation time gradient treatment of 3,6 and 9 hours, The number of cells obtained and the number of cells obtained were counted. The results showed that the morphological structure of the pancreatic acinar cells in the calf and the pancreatic acinar cells in the mice and the rats were similar, both of which were suspended cells, and a large amount of zymogen granules were visible in the cells. The number of cells obtained at 6 and 9 hours of incubation was significantly higher than that of 3 hours (P0.05), and there was no significant difference in the number of cells (P0.05). The effect of leucine on the secretion of the amylase of the pancreatic acinar cell of the calf, in-vitro culture of the freshly isolated calf pancreatic acinar cells, the culture solution using the leucine-free DMEM/ F12, The 0, 0.5,1,3,9, and 27-fold gradient treatments of their normal leucine concentrations (i.e.,0, 0.225, 0.45, 1.35, 4.05 and 12.15 mmol/ L) were added, respectively, and cultured for 3 hours under normal culture conditions. The results showed that (1) As the concentration of leucine in the culture medium increased from 0 to 0.5,1 and 3 times, the activity of the enzyme in the supernatant was significantly decreased (P0.05). When the concentration of leucine increased, the activity of the enzyme did not continue to decrease (P0.05). (2) Compared with the control group, The protein expression of AF and CCK1R in other leucine-concentration-treated groups decreased significantly (P0.05); (3) the proteasome activity in other leucine-concentration-treated groups decreased significantly (P <0.05) compared with the control group, and 76%,63% and 24% of the control group, respectively. 7% and 9%. The activity of the proteasome on the secretion of the pancreatic acinar cells of the calf was controlled on the basis of the results of the test, and the calf pancreatic acinar cells were used as the research object, and the leucine, the normal concentration of leucine and the high-concentration leucine (i.e.,0,1,27) were set. The mixture of leucine with the corresponding concentration of 0, 0.45, 12.15 mmol/ L and the corresponding concentration of leucine with 5. m u.mol/ L proteasome inhibitor MG-132 was added and cultured for 3 hours under normal culture conditions. The results showed that (1) the mixture of leucine and MG-132 in the 0-fold and 1-fold addition group was added separately compared with that of leucine, and the amount of amylase-amylase in the supernatant of the culture medium was significantly decreased (P0.05) and the addition of 27-fold. The addition of leucine and MG-132 did not decrease the secretion of L-amylase (P0.05); (2) in the group of 0 and 1, the addition of MG-132 decreased the protein expression level of CCK1R (P0.05). In the 27-fold group, the addition of MG-132 had the tendency to decrease the expression of CCK1R (P0.01); (3)0-fold and 1-fold leucine addition group, the mixed addition of leucine and mg-132 significantly reduced the activity of the proteasome (p0.05), respectively, of 63% and 72% of the proteasome activity when the corresponding concentration of leucine was added separately, and the mixture of leucine and mg-132 in the 27-fold leucine addition group was added separately from the leucine, There was no significant change in the activity of the proteasome (P0.05). Conclusion: The modified trypsin-cold digestion method is suitable for the separation of the pancreatic acinar cells in the body of the calf, and the leucine can be expressed by reducing the proteasome activity and the expression of the CCK1R, thereby inhibiting the secretion of the pancreatic acinar cells of the calf.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823
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