本文關(guān)鍵詞： 奶牛乳腺上皮細(xì)胞 乳蛋白 蛋氨酸 二肽　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：本試驗(yàn)運(yùn)用體外培養(yǎng)的奶牛乳腺上皮細(xì)胞為模型,研究蛋氨酸及含蛋氨酸二肽等量替代其所含的游離氨基酸后對(duì)乳腺上皮細(xì)胞中乳蛋白合成及相關(guān)基因表達(dá)以及細(xì)胞內(nèi)外氨肽酶蛋白含量的影響,為深入研究小肽對(duì)奶牛乳蛋白合成機(jī)理及改善乳品質(zhì)提供了理論基礎(chǔ)。試驗(yàn)由兩個(gè)試驗(yàn)構(gòu)成：試驗(yàn)一主要研究了蛋氨酸濃度及培養(yǎng)時(shí)間對(duì)奶牛乳腺上皮細(xì)胞乳蛋白基因表達(dá)的影響,篩選出最適培養(yǎng)濃度及培養(yǎng)時(shí)間。取健康的荷斯坦奶牛乳腺組織,分離純化得到乳腺上皮原代細(xì)胞,待細(xì)胞融合度達(dá)到80%時(shí)進(jìn)行傳代。試驗(yàn)采用單因子完全隨機(jī)試驗(yàn)設(shè)計(jì),將第3代乳腺上皮細(xì)胞隨機(jī)分為6個(gè)處理組,每組6個(gè)重復(fù),每組分別加入不同劑量的游離蛋氨酸,使其培養(yǎng)液中的終濃度分別為0、20、40、60、80 和 100μg/mL,其中Oμg/mL為對(duì)照組,其余為試驗(yàn)組。將細(xì)胞培養(yǎng)板置于37℃、5%的CO2培養(yǎng)箱中分別培養(yǎng)24h、48h和72h。試驗(yàn)結(jié)果表明：用不同濃度的游離蛋氨酸培養(yǎng)奶牛乳腺上皮細(xì)胞,當(dāng)培養(yǎng)48h時(shí),乳腺上皮細(xì)胞活力和αs1-酪蛋白(αs1-casein, CSN1S1)、k-酪蛋白(κ-casein, CSN3)和β-乳球蛋白(β-lactoglobulin, LGB)基因表達(dá)量隨著蛋氨酸濃度的增加呈顯著的一元二次曲線增加,當(dāng)濃度達(dá)到60μg/mL時(shí),表達(dá)量最高；但蛋氨酸的添加抑制了β-酪蛋白(p-casein, CSN2)基因的表達(dá)；當(dāng)培養(yǎng)72h時(shí),所有濃度的蛋氨酸均抑制了細(xì)胞增殖。蛋氨酸的添加濃度為60μg/mL、培養(yǎng)時(shí)間為48h時(shí),乳腺上皮細(xì)胞的活力以及細(xì)胞中乳蛋白基因的表達(dá)量均較高。試驗(yàn)二研究了八種含蛋氨酸二肽等量替代其所含的游離氨基酸后對(duì)奶牛乳腺上皮細(xì)胞乳蛋白基因(CSN1S1、CSN2、CSN3、LGB)、Ⅱ型小肽轉(zhuǎn)運(yùn)載體基因(PEPT2)、氨肽酶氮基因(APN)表達(dá)以及細(xì)胞內(nèi)外氨肽酶(APA)含量的影響。在試驗(yàn)一的基礎(chǔ)上,將蛋氨酸與其它必需氨基酸分別組成八種二肽(蛋.氨酸-蛋氨酸(P-Met-Met)、蛋氨酸-賴氨酸(P-Met-Lys)、蛋氨酸-色氨酸(P-Met-Trp)、蛋氨酸-苯丙氨酸(P-Met-Phe)、蛋氨酸-蘇氨酸(P-Met-Thr)、蛋氨酸-異亮氨酸(P-Met-Ile)、蛋氨酸-亮氨酸(P-Met-Leu)、蛋氨酸-纈氨酸(P-Met-Val)),等量替代相應(yīng)的游離氨基酸(F-Met-Met、F-Met-Lys、F-Met-Trp、F-Met-Phe、F-Met-Thr、 F-Met-Ile、F-Met-Leu、F-Met-Val)。試驗(yàn)二分為三個(gè)部分：第一部分研究了八種二肽對(duì)奶牛乳腺上皮細(xì)胞CSN1S1、CSN2、CSN3、LGB、PEPT2、APN基因表達(dá)以及細(xì)胞內(nèi)外APA活性的影響。采用單因子隨機(jī)試驗(yàn)設(shè)計(jì)分為9個(gè)組,8個(gè)二肽組與空白對(duì)照組。結(jié)果表明：P-Met-Met和P-Met-Lys組較對(duì)照組和其它二肽處理組顯著上調(diào)了CSN1S1、CSN2、和 CSN3基因的表達(dá),P-Met-Met組優(yōu)于P-Met-Lys組。P-Met-Thr、P-Met-Leu、P-Met-Ile 和 P-Met-Val組抑制了CSN2基因和CSN3基因的表達(dá)。第二部分研究了與八種二肽對(duì)應(yīng)的游離氨基酸對(duì)奶牛乳腺上皮細(xì)胞乳蛋白基因、PEPT2、APN表達(dá)以及細(xì)胞內(nèi)外APA活性的影響。采用單因子隨機(jī)試驗(yàn)設(shè)計(jì)分為9個(gè)組,8個(gè)氨基酸組與空白對(duì)照組。結(jié)果表明,F-Met-Met 和 F-Met-Lys組較對(duì)照組和其它氨基酸處理組顯著地促進(jìn)了CSN1S1基因的表達(dá)。F-Met-Thr組較其它氨基酸處理組顯著抑制了CSN3基因的表達(dá)。PEPT2基因表達(dá)量的結(jié)果表明,F-Met-Ile組顯著低于空白對(duì)照組,但F-Met-Met組顯著高于F-Met-Trp、 F-Met-Leu、 F-Met-Ile和F-Met-Val氨基酸處理組,但與對(duì)照組無顯著差異。不同氨基酸處理組對(duì)APN基因表達(dá)無顯著的影響。第三部分研究了二肽等量替代相應(yīng)游離氨基酸對(duì)奶牛乳腺上皮細(xì)胞乳蛋白基因、PEPT2、APN表達(dá)以及細(xì)胞內(nèi)外APA活性的影響。結(jié)果表明,除P-Met-Val 和 P-Met-Leu,不同二肽組合替代氨基酸后均不同程度地促進(jìn)了乳蛋白基因和PEPT2基因的表達(dá)量,以P-Met-Met表現(xiàn)出較好的促進(jìn)效果,顯著上調(diào)了乳蛋白CSN1S1、CSN2、CSN3基因的表達(dá)量,PEPT2的基因表達(dá)量有趨于顯著的提高；以P-Met-Trp、 P-Met-Phe、P-Met-Lys的促進(jìn)效果次之。二肽等量替換游離氨基酸能夠顯著的促進(jìn)乳蛋白基因的表達(dá),P-Met-Met、 P-Met-Trp、P-Met-Phe 和 P-Met-Lys的促進(jìn)效果較好,其中尤以P-Met-Met的效果最好。
[Abstract]:This experiment using cultured bovine mammary epithelial cells as a model of methionine and methionine containing two peptide instead of free amino acids contained in the milk protein expression of mammary epithelial cells and synthesis related genes and effects of intracellular aminopeptidase protein content, for the further study of small peptides provides a theoretical basis for the synthesis of mechanism the cow milk protein and improve milk quality. The test consists of two components: a test of test of effect of methionine concentration and culture time on the expression of dcmecs protein gene, screened the optimum culture concentration and culture time. From healthy Holstein cow mammary tissue, purified mammary epithelial cells when the cells were passaged, up to 80% degrees. This test uses a single factor completely randomized design, the third generation of mammary epithelial cells were randomly divided into 6 treatments Group, with 6 replicates in each group respectively with different doses of free methionine, the final concentration of the culture medium were 0,20,40,60,80 and 100 g/mL, the O g/mL as the control group, the other as the experimental group. The cell culture plate is arranged at 37 DEG C, 5% CO2 culture 24h were cultured in the 48H box. And the 72h. test results showed that the culture of bovine mammary epithelial cells with different concentrations of free methionine, when cultured in 48h, epithelial cell viability and alpha s1- casein breast (alpha s1-casein, CSN1S1), k- (kappa casein -casein, CSN3) and beta lactoglobulin (beta -lactoglobulin, LGB) with gene expression significantly one of the two curves increase methionine concentration, when the concentration reached 60 g/mL, the highest expression level; but the addition of methionine inhibits beta casein (p-casein, CSN2) gene expression; when cultured 72h, all concentrations of methionine was inhibited by fine Cell proliferation. Methionine concentration is 60 g/mL, when the culture time was 48h, the expression activity of mammary epithelial cells and cells in milk protein genes were higher. Experiment two studied eight kinds of free amino acids containing two methionine peptide replacing the content of milk protein gene of bovine mammary epithelial cells (on CSN1S1, CSN2, CSN3, LGB), type II peptide transporter gene (PEPT2), aminopeptidase N (APN) gene expression and intracellular aminopeptidase (APA) were studied. On the basis of Experiment 1, the methionine and other essential amino acids were composed of eight species and two peptide (egg. The amino acid methionine (P-Met-Met) - methionine, lysine, methionine (P-Met-Lys) - tryptophan (P-Met-Trp) - phenylalanine, methionine, threonine methionine (P-Met-Phe) - (P-Met-Thr) - methionine, isoleucine, leucine methionine (P-Met-Ile) - (P-Met-Leu) - P-Met-Va (valine, methionine L)), instead of the corresponding free amino acids (F-Met-Met, F-Met-Lys, F-Met-Trp, F-Met-Phe, F-Met-Thr, F-Met-Ile, F-Met-Leu, F-Met-Val) two. The test is divided into three parts: the first part studies eight two peptides in bovine mammary epithelial cells CSN1S1, CSN2, CSN3, LGB, PEPT2, APN gene expression and the effect of the intracellular APA activity. Using single factor randomized design was divided into 9 groups, 8 two peptide group and blank control group. The results showed that the P-Met-Met and P-Met-Lys group than in the control group and the other two peptide treatment group was significant upregulation of CSN1S1, CSN2, and CSN3 gene expression, P-Met-Met of group P-Met-Lys was superior to.P-Met-Thr. P-Met-Leu, P-Met-Ile and P-Met-Val inhibited the expression of CSN2 gene and CSN3 gene. The second part studies the PEPT2 free amino acids and eight kinds of two peptides corresponding to dcmecs protein gene, APN, expression and intracellular Effect of the activity of APA. By using the single factor randomized design was divided into 9 groups, 8 amino acid group and blank control group. The results showed that the F-Met-Met and F-Met-Lys group than in the control group and the treatment group of other amino acids significantly enhanced the expression of.F-Met-Thr CSN1S1 gene significantly than other amino acid treatment inhibited the expression of.PEPT2 gene CSN3 gene expression results showed that the F-Met-Ile group was significantly lower than the control group, but F-Met-Met group was significantly higher than that of F-Met-Trp, F-Met-Leu, F-Met-Ile and F-Met-Val amino acid treatment group, but no significant difference with the control group. The treatment group of amino acid on the expression of APN gene had no significant effect. The third part studies the two peptides corresponding instead of PEPT2 the free amino acids of dcmecs protein gene, APN, expression and effect of intracellular and extracellular APA activity. The results showed that, in addition to P-Met-Val and P-Met-Leu, Two different combinations of amino acid substitution peptides are different levels to promote the expression of milk protein gene and PEPT2 gene, the P-Met-Met showed better effect, increased milk protein CSN1S1, CSN2, CSN3 gene expression, the gene expression of PEPT2 was significantly improved tends to P-Met-Trp, P-Met-Phe, P-Met-Lys; the effect of the second. Two peptide equivalent substitution free amino acid can significantly promote the expression of milk protein gene, P-Met-Met, P-Met-Trp, P-Met-Phe and P-Met-Lys to promote good effect, especially the effect of P-Met-Met is best.

【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 曹志軍,李勝利,丁志民;日糧中添加小肽對(duì)奶牛產(chǎn)奶性能影響的研究[J];飼料工業(yè);2004年04期

2 張春;趙學(xué)梅;周麗;牛英才;劉吉成;;MTT法測定低分子姬松茸多糖體外抗腫瘤活性[J];齊齊哈爾醫(yī)學(xué)院學(xué)報(bào);2009年01期

3 黃建國;高學(xué)軍;佟慧麗;張宇翔;謝小來;伍浩楠;扈光輝;;蛋白飼料源小肽對(duì)奶牛產(chǎn)奶量和乳品質(zhì)的影響[J];中國乳品工業(yè);2009年09期

4 李喜艷;王加啟;魏宏陽;卜登攀;胡菡;周凌云;;MTT比色法檢測賴氨酸、蛋氨酸對(duì)體外培養(yǎng)的奶牛乳腺上皮細(xì)胞增殖的影響[J];生物技術(shù)通報(bào);2010年03期

5 畢微微;高學(xué)軍;林葉;李慶章;;營養(yǎng)素調(diào)控奶牛乳蛋白合成的研究進(jìn)展[J];乳業(yè)科學(xué)與技術(shù);2012年04期

6 王恬,貝水榮,傅永明,呂俊龍,楊州,陳漢根;小肽營養(yǎng)素對(duì)奶牛泌乳性能的影響[J];中國奶牛;2004年02期

7 張春剛;王加啟;劉光磊;程金波;趙國琦;卜登攀;魏宏陽;周凌云;;乳蛋白基因及乳蛋白綜合調(diào)控技術(shù)[J];中國奶牛;2007年10期

8 王加啟;趙圣國;;我國牛奶質(zhì)量安全的現(xiàn)狀、問題和對(duì)策[J];中國奶牛;2009年11期

9 劉桂瑞;李正洪;李兆林;;影響生鮮乳蛋白質(zhì)含量的因素及調(diào)控措施[J];中國奶牛;2011年17期

10 姜寧,張愛忠,苗樹君,曹志軍,宋屹,郝剛;補(bǔ)充蛋氨酸和小肽及過瘤胃保護(hù)處理對(duì)奶牛血液生化指標(biāo)和氨基酸濃度的影響[J];中國畜牧雜志;2005年05期

相關(guān)碩士學(xué)位論文 前1條

1 孫康玉;小肽對(duì)奶牛乳腺細(xì)胞乳蛋白合成的影響[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2012年

，

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