本文選題：精氨酸 + 精氨酸酶�。� 參考：《揚(yáng)州大學(xué)》2016年碩士論文

【摘要】：課題組前期試驗(yàn)發(fā)現(xiàn)精氨酸對奶牛乳腺上皮細(xì)胞中酪蛋白合成具有促進(jìn)作用,并且對精氨酸代謝相關(guān)酶活力也有顯著影響。在此基礎(chǔ)上,本試驗(yàn)通過向奶牛乳腺上皮細(xì)胞培養(yǎng)基中添加不同精氨酸代謝相關(guān)酶抑制劑,分別抑制精氨酸的不同代謝通路,并檢測細(xì)胞中酪蛋白濃度,篩選出精氨酸調(diào)控乳腺上皮細(xì)胞中酪蛋白表達(dá)的關(guān)鍵途徑。接著在乳腺上皮細(xì)胞培養(yǎng)基中通過補(bǔ)充代謝底物和代謝產(chǎn)物的方法,對篩選出的關(guān)鍵代謝途徑進(jìn)行驗(yàn)證。最后,在泌乳中期的荷斯坦奶牛上,通過頸靜脈灌注關(guān)鍵的精氨酸代謝相關(guān)酶抑制劑,并測定奶牛乳中酪蛋白濃度,乳成分、產(chǎn)奶量、飼料中養(yǎng)分的消化率以及血液指標(biāo)等。旨在探討精氨酸調(diào)控奶牛乳腺中酪蛋白合成的關(guān)鍵代謝通路,以期為牛奶中乳蛋白提高,乳品質(zhì)的改善提供一定的基礎(chǔ)數(shù)據(jù)。試驗(yàn)1：選擇Nω-hydroxy-nor-Arginine (Cayman, nor-NOHA,10006861)作為精氨酸酶特異性抑制劑；二氟甲基鳥氨酸2-(difluoromethyl)ornithine (Sigma, DFMO, D193)作為鳥氨酸脫羧酶抑制劑以及S-(2-氨乙基)異硫脲二氫溴酸鹽2-(2-Aminoethyl) isothiourea dihydrobromide (Sigma, A5879)作為一氧化氮合酶抑制劑。添加不同抑制劑培養(yǎng)后,分別檢測細(xì)胞增殖活力,細(xì)胞中精氨酸代謝相關(guān)酶活力,及細(xì)胞中酪蛋白合成的情況。結(jié)果發(fā)現(xiàn)添加nor-NOHA和DFMO處理后的細(xì)胞增殖活力顯著降低(P0.05),并且細(xì)胞中的酪蛋白濃度也有所降低(P0.05)。而當(dāng)添加NOS酶抑制劑處理后,細(xì)胞增殖活力及細(xì)胞中酪蛋白合成并沒有顯著變化。綜合以上試驗(yàn)結(jié)果提示,精氨酸可能通過“精氨酸—鳥氨酸—多胺”代謝途徑調(diào)控乳腺上皮細(xì)胞中酪蛋白合成。試驗(yàn)2：在試驗(yàn)1的基礎(chǔ)上,試驗(yàn)2分別向培養(yǎng)基中添加nor-NOHA、nor-NOHA+Orn, nor-NOHA+Arg處理奶牛乳腺上皮細(xì)胞,并檢測細(xì)胞增殖,細(xì)胞中精氨酸代謝相關(guān)酶活力及基因表達(dá)量,細(xì)胞中酪蛋白濃度及基因表達(dá)情況,結(jié)果發(fā)現(xiàn)添加nor-NOHA處理后,細(xì)胞中總的精氨酸酶活力及精氨酸酶的基因表達(dá)量顯著降低(P0.05),細(xì)胞的增殖活力有所下降(P0.05),并且細(xì)胞中酪蛋白濃度及基因表達(dá)量也顯著降低(P0.05)。而當(dāng)補(bǔ)充精氨酸處理后,細(xì)胞的增殖活力,細(xì)胞中精氨酸代謝相關(guān)酶活力和酪蛋白合成并沒有顯著變化(P0.05)。但是當(dāng)向培養(yǎng)基中補(bǔ)充鳥氨酸處理后,細(xì)胞的增殖活力有所上升(P0.05),并且上皮細(xì)胞中酪蛋白濃度也均有所恢復(fù)(P0.05)。體外細(xì)胞試驗(yàn)結(jié)果初步提示“精氨酸—鳥氨酸”代謝通路是調(diào)控乳腺上皮細(xì)胞中酪蛋白合成的關(guān)鍵通路。試驗(yàn)3：在泌乳中期的荷斯坦奶牛中,通過頸靜脈灌注nor-NOHA, nor-NOHA+Arg后,檢測奶牛血液中精氨酸代謝相關(guān)酶活力,泌乳奶牛產(chǎn)奶量,乳品質(zhì),乳中酪蛋白濃度,乳腺中酪蛋白基因以及精氨酸轉(zhuǎn)運(yùn)載體蛋白基因表達(dá)量。結(jié)果發(fā)現(xiàn),頸靜脈灌注nor-NOHA后,奶牛血液中總精氨酸酶和鳥氨酸脫羧酶活力顯著降低(P0.05),而總的NO合酶的活力卻沒有顯著變化(P0.05),乳中乳蛋白率及乳脂率也有所降低(P0.05),但是產(chǎn)奶量卻沒有變化(P0.05)；此外乳中酪蛋白濃度及乳腺中酪蛋白基因表達(dá)量也有所下降(P0.05),但是乳腺中精氨酸轉(zhuǎn)運(yùn)載體蛋白的基因表達(dá)量卻沒有顯著變化(P0.05)。而當(dāng)補(bǔ)充灌注精氨酸處理后,奶牛血液中總精氨酸酶和鳥氨酸脫羧酶活力沒有顯著變化(P0.05),但是總的NO合酶活力顯著上升(P0.05),而乳中乳蛋白率,乳脂率和酪蛋白濃度卻沒有顯著變化(P0.05),此外當(dāng)補(bǔ)充灌注精氨酸后,奶牛乳腺中部分精氨酸轉(zhuǎn)運(yùn)載體的基因表達(dá)量有了顯著上升(P0.05),奶牛的產(chǎn)奶量也有所提高(P0.05)。動(dòng)物在體試驗(yàn)得出與體外細(xì)胞試驗(yàn)一致的結(jié)論,即精氨酸通過“精氨酸—鳥氨酸”代謝通路調(diào)控酪蛋白生成。試驗(yàn)4：本試驗(yàn)主要是研究灌注nor-NOHA和nor-NOHA+Arg處理后,奶牛飼料消化率,奶牛體內(nèi)氮素循環(huán)以及奶牛血液指標(biāo)的變化。具體試驗(yàn)方法同試驗(yàn)3。本試驗(yàn)發(fā)現(xiàn),灌注nor-NOHA對飼料中養(yǎng)分的表觀消化率沒有顯著影響(P0.05),但是尿氮的含量卻有所上升(P0.05),而乳氮卻有所下降(P0.05),并且血中尿素氮含量也有所上升(P0.05)。而當(dāng)補(bǔ)充灌注精氨酸后,飼料中粗脂肪的消化率有所提高(P0.05),而尿氮、乳氮和血中尿素氮濃度均恢復(fù)至正常水平(P0.05)。此外,灌注nor-NOHA和nor-NOHA+Arg對奶牛血常規(guī)指標(biāo)和免疫球蛋白水平均沒有顯著影響(P0.05)。本試驗(yàn)結(jié)果提示,不同灌注處理并不影響奶牛機(jī)體健康狀況,但是當(dāng)“精氨酸—鳥氨酸”通路抑制后奶牛體內(nèi)氮素循環(huán)受到顯著影響。綜合以上體內(nèi)外試驗(yàn)結(jié)果提示,精氨酸對奶牛乳腺中酪蛋白合成主要是通過“精氨酸—鳥氨酸”代謝通路實(shí)現(xiàn)。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S823

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