亮氨酸通過蛋白酶體調(diào)控犢牛胰腺腺泡細(xì)胞淀粉酶的分泌
發(fā)布時間：2019-05-16 15:48
【摘要】：胰腺作為重要的消化器官,其分泌的淀粉酶在動物消化利用小腸淀粉的過程中發(fā)揮著關(guān)鍵作用。奶牛十二指腸中的淀粉僅有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芻動物小腸淀粉利用的主要因素。本研究利用改進(jìn)的胰蛋白酶冷消化法分離荷斯坦?fàn)倥Ｒ认傧倥菁?xì)胞,并在此基礎(chǔ)上通過體外細(xì)胞培養(yǎng)試驗,研究亮氨酸對犢牛胰腺腺泡細(xì)胞淀粉酶分泌的影響及相應(yīng)的調(diào)控機(jī)制,以期為通過營養(yǎng)手段改善奶牛等反芻動物小腸淀粉消化提供理論參考。主要結(jié)果如下:犢牛胰腺腺泡細(xì)胞的分離技術(shù)研究使用改進(jìn)后的胰蛋白酶冷消化法分離奶犢牛胰腺腺泡細(xì)胞,對分離得到的細(xì)胞進(jìn)行形態(tài)學(xué)觀察,并設(shè)置3、6、9小時的孵育時間梯度處理,對獲得的細(xì)胞數(shù)目及活率進(jìn)行計數(shù)。結(jié)果表明:犢牛胰腺腺泡細(xì)胞的形態(tài)結(jié)構(gòu)和小鼠及大鼠的胰腺腺泡細(xì)胞相似,均為懸浮細(xì)胞,細(xì)胞內(nèi)可見大量的酶原顆粒。孵育6小時和9小時時獲得的細(xì)胞數(shù)目較3小時明顯升高(P0.05),活率并無顯著差異(P0.05)。亮氨酸對犢牛胰腺腺泡細(xì)胞淀粉酶分泌的影響體外培養(yǎng)新鮮分離的犢牛胰腺腺泡細(xì)胞,培養(yǎng)液使用無亮氨酸的DMEM/F12,分別添加其正常亮氨酸濃度的0、0.5、1、3、9、27倍梯度處理(即0、0.225、0.45、1.35、4.05和12.15 mmol/L)并于正常培養(yǎng)條件下培養(yǎng)3小時。結(jié)果顯示:(1)隨著培養(yǎng)液中亮氨酸濃度由0倍提高至0.5、1、3倍時,上清培養(yǎng)液中的酶活出現(xiàn)了顯著下降(P0.05),當(dāng)亮氨酸添加濃度繼續(xù)升高時,酶活并未繼續(xù)降低(P0.05);(2)相較于0倍對照組,其他幾個亮氨酸濃度處理組的AF及CCK1R的蛋白表達(dá)均出現(xiàn)了顯著下降(P0.05);(3)相較于對照組,其他幾個亮氨酸濃度處理組的蛋白酶體活性出現(xiàn)了顯著下降(P0.05),分別為對照組的76%、63%、24%、7%和9%。蛋白酶體對犢牛胰腺腺泡細(xì)胞淀粉酶分泌調(diào)控在試驗二結(jié)果的基礎(chǔ)上,以犢牛胰腺腺泡細(xì)胞為研究對象,設(shè)置無亮氨酸、正常濃度亮氨酸和高濃度亮氨酸(即0、1、27倍濃度,對應(yīng)0、0.45、12.15 mmol/L)的單獨添加及相應(yīng)濃度的亮氨酸與5μmol/L蛋白酶體抑制劑MG-132的混合添加處理,于正常培養(yǎng)條件下培養(yǎng)3小時。結(jié)果顯示:(1)0倍和1倍添加組中亮氨酸和MG-132的混合添加相較于亮氨酸的單獨添加,培養(yǎng)液上清中的ɑ-淀粉酶量出現(xiàn)了顯著下降(P0.05),27倍添加組中,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倍組中,MG-132的添加均降低了CCK1R的蛋白表達(dá)水平(P0.05),27倍組中,MG-132的添加有降低CCK1R表達(dá)的趨勢(P0.1);(3)0倍和1倍亮氨酸添加組中,亮氨酸和MG-132的混合添加均顯著降低了蛋白酶體的活性(P0.05),分別為單獨添加相應(yīng)濃度亮氨酸時蛋白酶體活性的63%和72%。27倍亮氨酸添加組中亮氨酸與MG-132的混合添加相較于亮氨酸的單獨添加,蛋白酶體的活性并未出現(xiàn)顯著變化(P0.05)。結(jié)論:改進(jìn)的胰蛋白酶冷消化法適用用于分離制備體犢牛胰腺腺泡細(xì)胞;亮氨酸可以通過降低蛋白酶體活性及CCK1R表達(dá),進(jìn)而抑制犢牛胰腺腺泡細(xì)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823
本文編號：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823
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，本文編號：2478384
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