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

【摘要】：本試驗運用體外培養(yǎng)的奶牛乳腺上皮細胞為模型,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乳腺上皮細胞中乳蛋白合成及相關(guān)基因表達以及細胞內(nèi)外氨肽酶蛋白含量的影響,為深入研究小肽對奶牛乳蛋白合成機理及改善乳品質(zhì)提供了理論基礎(chǔ)。試驗由兩個試驗構(gòu)成：試驗一主要研究了蛋氨酸濃度及培養(yǎng)時間對奶牛乳腺上皮細胞乳蛋白基因表達的影響,篩選出最適培養(yǎng)濃度及培養(yǎng)時間。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胞,待細胞融合度達到80%時進行傳代。試驗采用單因子完全隨機試驗設(shè)計,將第3代乳腺上皮細胞隨機分為6個處理組,每組6個重復,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為對照組,其余為試驗組。將細胞培養(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)奶牛乳腺上皮細胞,當培養(yǎng)48h時,乳腺上皮細胞活力和αs1-酪蛋白(αs1-casein, CSN1S1)、k-酪蛋白(κ-casein, CSN3)和β-乳球蛋白(β-lactoglobulin, LGB)基因表達量隨著蛋氨酸濃度的增加呈顯著的一元二次曲線增加,當濃度達到60μg/mL時,表達量最高；但蛋氨酸的添加抑制了β-酪蛋白(p-casein, CSN2)基因的表達；當培養(yǎng)72h時,所有濃度的蛋氨酸均抑制了細胞增殖。蛋氨酸的添加濃度為60μg/mL、培養(yǎng)時間為48h時,乳腺上皮細胞的活力以及細胞中乳蛋白基因的表達量均較高。試驗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奶牛乳腺上皮細胞乳蛋白基因(CSN1S1、CSN2、CSN3、LGB)、Ⅱ型小肽轉(zhuǎn)運載體基因(PEPT2)、氨肽酶氮基因(APN)表達以及細胞內(nèi)外氨肽酶(APA)含量的影響。在試驗一的基礎(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)。試驗二分為三個部分：第一部分研究了八種二肽對奶牛乳腺上皮細胞CSN1S1、CSN2、CSN3、LGB、PEPT2、APN基因表達以及細胞內(nèi)外APA活性的影響。采用單因子隨機試驗設(shè)計分為9個組,8個二肽組與空白對照組。結(jié)果表明：P-Met-Met和P-Met-Lys組較對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,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。第二部分研究了與八種二肽對應(yīng)的游離氨基酸對奶牛乳腺上皮細胞乳蛋白基因、PEPT2、APN表達以及細胞內(nèi)外APA活性的影響。采用單因子隨機試驗設(shè)計分為9個組,8個氨基酸組與空白對照組。結(jié)果表明,F-Met-Met 和 F-Met-Lys組較對照組和其它氨基酸處理組顯著地促進了CSN1S1基因的表達。F-Met-Thr組較其它氨基酸處理組顯著抑制了CSN3基因的表達。PEPT2基因表達量的結(jié)果表明,F-Met-Ile組顯著低于空白對照組,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,但與對照組無顯著差異。不同氨基酸處理組對APN基因表達無顯著的影響。第三部分研究了二肽等量替代相應(yīng)游離氨基酸對奶牛乳腺上皮細胞乳蛋白基因、PEPT2、APN表達以及細胞內(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了乳蛋白基因和PEPT2基因的表達量,以P-Met-Met表現(xiàn)出較好的促進效果,顯著上調(diào)了乳蛋白CSN1S1、CSN2、CSN3基因的表達量,PEPT2的基因表達量有趨于顯著的提高；以P-Met-Trp、 P-Met-Phe、P-Met-Lys的促進效果次之。二肽等量替換游離氨基酸能夠顯著的促進乳蛋白基因的表達,P-Met-Met、 P-Met-Trp、P-Met-Phe 和 P-Met-Lys的促進效果較好,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.

【學位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S823.5

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