精氨酸酶调控奶牛乳腺中酪蛋白合成的分子机制

发布时间：2018-05-22 19:16

本文选题：精氨酸 + 精氨酸酶　；参考：《扬州大学》2016年硕士论文

【摘要】：课题组前期试验发现精氨酸对奶牛乳腺上皮细胞中酪蛋白合成具有促进作用,并且对精氨酸代谢相关酶活力也有显著影响。在此基础上,本试验通过向奶牛乳腺上皮细胞培养基中添加不同精氨酸代谢相关酶抑制剂,分别抑制精氨酸的不同代谢通路,并检测细胞中酪蛋白浓度,筛选出精氨酸调控乳腺上皮细胞中酪蛋白表达的关键途径。接着在乳腺上皮细胞培养基中通过补充代谢底物和代谢产物的方法,对筛选出的关键代谢途径进行验证。最后,在泌乳中期的荷斯坦奶牛上,通过颈静脉灌注关键的精氨酸代谢相关酶抑制剂,并测定奶牛乳中酪蛋白浓度,乳成分、产奶量、饲料中养分的消化率以及血液指标等。旨在探讨精氨酸调控奶牛乳腺中酪蛋白合成的关键代谢通路,以期为牛奶中乳蛋白提高,乳品质的改善提供一定的基础数据。试验1：选择Nω-hydroxy-nor-Arginine (Cayman, nor-NOHA,10006861)作为精氨酸酶特异性抑制剂；二氟甲基鸟氨酸2-(difluoromethyl)ornithine (Sigma, DFMO, D193)作为鸟氨酸脱羧酶抑制剂以及S-(2-氨乙基)异硫脲二氢溴酸盐2-(2-Aminoethyl) isothiourea dihydrobromide (Sigma, A5879)作为一氧化氮合酶抑制剂。添加不同抑制剂培养后,分别检测细胞增殖活力,细胞中精氨酸代谢相关酶活力,及细胞中酪蛋白合成的情况。结果发现添加nor-NOHA和DFMO处理后的细胞增殖活力显著降低(P0.05),并且细胞中的酪蛋白浓度也有所降低(P0.05)。而当添加NOS酶抑制剂处理后,细胞增殖活力及细胞中酪蛋白合成并没有显著变化。综合以上试验结果提示,精氨酸可能通过“精氨酸—鸟氨酸—多胺”代谢途径调控乳腺上皮细胞中酪蛋白合成。试验2：在试验1的基础上,试验2分别向培养基中添加nor-NOHA、nor-NOHA+Orn, nor-NOHA+Arg处理奶牛乳腺上皮细胞,并检测细胞增殖,细胞中精氨酸代谢相关酶活力及基因表达量,细胞中酪蛋白浓度及基因表达情况,结果发现添加nor-NOHA处理后,细胞中总的精氨酸酶活力及精氨酸酶的基因表达量显著降低(P0.05),细胞的增殖活力有所下降(P0.05),并且细胞中酪蛋白浓度及基因表达量也显著降低(P0.05)。而当补充精氨酸处理后,细胞的增殖活力,细胞中精氨酸代谢相关酶活力和酪蛋白合成并没有显著变化(P0.05)。但是当向培养基中补充鸟氨酸处理后,细胞的增殖活力有所上升(P0.05),并且上皮细胞中酪蛋白浓度也均有所恢复(P0.05)。体外细胞试验结果初步提示“精氨酸—鸟氨酸”代谢通路是调控乳腺上皮细胞中酪蛋白合成的关键通路。试验3：在泌乳中期的荷斯坦奶牛中,通过颈静脉灌注nor-NOHA, nor-NOHA+Arg后,检测奶牛血液中精氨酸代谢相关酶活力,泌乳奶牛产奶量,乳品质,乳中酪蛋白浓度,乳腺中酪蛋白基因以及精氨酸转运载体蛋白基因表达量。结果发现,颈静脉灌注nor-NOHA后,奶牛血液中总精氨酸酶和鸟氨酸脱羧酶活力显著降低(P0.05),而总的NO合酶的活力却没有显著变化(P0.05),乳中乳蛋白率及乳脂率也有所降低(P0.05),但是产奶量却没有变化(P0.05)；此外乳中酪蛋白浓度及乳腺中酪蛋白基因表达量也有所下降(P0.05),但是乳腺中精氨酸转运载体蛋白的基因表达量却没有显著变化(P0.05)。而当补充灌注精氨酸处理后,奶牛血液中总精氨酸酶和鸟氨酸脱羧酶活力没有显著变化(P0.05),但是总的NO合酶活力显著上升(P0.05),而乳中乳蛋白率,乳脂率和酪蛋白浓度却没有显著变化(P0.05),此外当补充灌注精氨酸后,奶牛乳腺中部分精氨酸转运载体的基因表达量有了显著上升(P0.05),奶牛的产奶量也有所提高(P0.05)。动物在体试验得出与体外细胞试验一致的结论,即精氨酸通过“精氨酸—鸟氨酸”代谢通路调控酪蛋白生成。试验4：本试验主要是研究灌注nor-NOHA和nor-NOHA+Arg处理后,奶牛饲料消化率,奶牛体内氮素循环以及奶牛血液指标的变化。具体试验方法同试验3。本试验发现,灌注nor-NOHA对饲料中养分的表观消化率没有显著影响(P0.05),但是尿氮的含量却有所上升(P0.05),而乳氮却有所下降(P0.05),并且血中尿素氮含量也有所上升(P0.05)。而当补充灌注精氨酸后,饲料中粗脂肪的消化率有所提高(P0.05),而尿氮、乳氮和血中尿素氮浓度均恢复至正常水平(P0.05)。此外,灌注nor-NOHA和nor-NOHA+Arg对奶牛血常规指标和免疫球蛋白水平均没有显著影响(P0.05)。本试验结果提示,不同灌注处理并不影响奶牛机体健康状况,但是当“精氨酸—鸟氨酸”通路抑制后奶牛体内氮素循环受到显著影响。综合以上体内外试验结果提示,精氨酸对奶牛乳腺中酪蛋白合成主要是通过“精氨酸—鸟氨酸”代谢通路实现。
[Abstract]:In the previous study, we found that arginine could promote the synthesis of casein in the mammary epithelial cells of dairy cows and had a significant effect on the activity of arginine metabolism related enzymes. On this basis, the experiment was made to inhibit arginine by adding different arginine metabolism related enzyme inhibitors to the medium of mammary epithelial cells of dairy cows. The key pathway of casein expression in mammary epithelial cells was screened by different metabolic pathways, and the concentration of casein in cells was detected. Then the key metabolic pathways were verified by supplementing the metabolic substrates and metabolites in the mammary epithelial cell culture medium. Finally, in the middle lactation Holstein. In dairy cows, the key arginine metabolism related enzyme inhibitors were perfused through the jugular vein, and the concentration of casein, milk composition, milk production, nutrient digestibility and blood indexes were measured in dairy cow's milk. The aim was to explore the key metabolic pathway of casein synthesis in cow mammary glands by arginine to improve milk protein in milk and dairy products. Quality improvement provides certain basic data. Experiment 1: select N Omega -hydroxy-nor-Arginine (Cayman, nor-NOHA, 10006861) as a specific inhibitor of arginase; two fluoromethyl ornithine 2- (difluoromethyl) ornithine (Sigma, DFMO, D193) as ornithine decarboxylase inhibitor and S- (2- ammonia ethyl) isothiourea two hydrobromate Minoethyl) isothiourea DIHYDROBROMIDE (Sigma, A5879) as a nitric oxide synthase inhibitor. After the addition of different inhibitors, the proliferation activity, the activity of arginine metabolism related enzymes and the synthesis of casein in cells were detected respectively. The results showed that the proliferation activity of cells after adding nor-NOHA and DFMO was significantly reduced. Low (P0.05), and the concentration of casein in cells decreased (P0.05). The proliferation activity and casein synthesis did not change significantly after the addition of NOS enzyme inhibitors. The results suggested that arginine may regulate mammary epithelial cells through the "arginine nianorate polyamine" metabolic pathway. Casein synthesis. Test 2: on the basis of test 1, test 2 added nor-NOHA, nor-NOHA+Orn, and nor-NOHA+Arg to milk cow mammary epithelial cells respectively, and detected cell proliferation, activity of arginine metabolism related enzymes and gene expression, concentration of casein and gene expression in cells, and found the addition of nor-NO. After HA treatment, the total arginase activity and the gene expression of arginase decreased significantly (P0.05), the cell proliferation activity decreased (P0.05), and the concentration of casein and gene expression in the cells also decreased significantly (P0.05). The proliferation activity of cells and the activity of arginine metabolism related enzymes in the cells were supplemented by arginine treatment. There was no significant change in the synthesis of force and casein (P0.05). But when supplementation with ornithine treatment in the medium, the proliferation activity of cells increased (P0.05), and the concentration of casein in epithelial cells was also restored (P0.05). The results of cell test in vitro suggested that the "arginine ornithine" metabolic pathway was the regulation of mammary epithelium. Key pathways in the synthesis of casein in cells. Test 3: in Holstein cows in the mid-term lactation, the activity of arginine metabolism related enzymes in the blood of dairy cows, milk production, milk quality, milk casein concentration, casein gene and arginine transporter protein in milk of dairy cows were detected after nor-NOHA and nor-NOHA+Arg in the jugular vein of Holstein cows. The results showed that the activity of total arginine and ornithine decarboxylase in the blood of dairy cows decreased significantly (P0.05), but the total activity of NO synthase was not significantly changed (P0.05), the milk protein rate and milk fat rate in milk decreased (P0.05), but the milk yield was not changed (P0.05); in addition, milk cheese and eggs were also found. The white concentration and the expression of casein gene in the breast also decreased (P0.05), but the gene expression of the arginine transporter protein in the mammary gland was not significantly changed (P0.05). The total arginase and ornithine decarboxylase activity in the blood of dairy cows did not change significantly (P0.05), but the total NO synthase Activity significantly increased (P0.05), but milk protein rate, milk fat rate and casein concentration in milk were not significantly changed (P0.05). In addition, after supplementation of arginine, the gene expression of partial arginine transporter in dairy cow's mammary gland increased significantly (P0.05) and milk production increased (P0.05) in dairy cows (P0.05). The conclusion of the cell test is that arginine regulates the production of casein through the "arginine aline" metabolic pathway. Test 4: this experiment was mainly to study the digestibility of cow feed, the nitrogen cycle in dairy cows and the change of blood index in dairy cows after the treatment of perfusion nor-NOHA and nor-NOHA+Arg. The specific test method was same as the test of 3. experiments. It was found that the apparent digestibility of nutrients in the feed was not significantly affected by perfusion nor-NOHA (P0.05), but the content of urine nitrogen increased (P0.05), but the milk nitrogen decreased (P0.05), and the content of urea nitrogen in the blood also increased (P0.05). When supplemented with arginine, the digestibility of crude fat in the feed was improved (P0.05) and urine nitrogen was increased. The concentration of milk nitrogen and blood urea nitrogen all recovered to the normal level (P0.05). In addition, perfusion nor-NOHA and nor-NOHA+Arg had no significant effect on the blood routine index and immunoglobulin level of dairy cows (P0.05). The results of this test suggest that different perfusion treatments do not affect the health status of dairy cows, but when the "arginine ornithine" pathway is inhibited. The nitrogen cycle in dairy cows was significantly affected. The results of comprehensive and in vivo experiments showed that the synthesis of casein in dairy cow's mammary gland was mainly through the "arginine ornithine" metabolic pathway.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S823

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