蛋氨酸及含蛋氨酸二肽对奶牛乳腺上皮细胞内乳蛋白合成相关基因表达的影响

发布时间：2018-01-23 22:28

本文关键词： 奶牛乳腺上皮细胞乳蛋白蛋氨酸二肽　出处：《内蒙古农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：本试验运用体外培养的奶牛乳腺上皮细胞为模型,研究蛋氨酸及含蛋氨酸二肽等量替代其所含的游离氨基酸后对乳腺上皮细胞中乳蛋白合成及相关基因表达以及细胞内外氨肽酶蛋白含量的影响,为深入研究小肽对奶牛乳蛋白合成机理及改善乳品质提供了理论基础。试验由两个试验构成：试验一主要研究了蛋氨酸浓度及培养时间对奶牛乳腺上皮细胞乳蛋白基因表达的影响,筛选出最适培养浓度及培养时间。取健康的荷斯坦奶牛乳腺组织,分离纯化得到乳腺上皮原代细胞,待细胞融合度达到80%时进行传代。试验采用单因子完全随机试验设计,将第3代乳腺上皮细胞随机分为6个处理组,每组6个重复,每组分别加入不同剂量的游离蛋氨酸,使其培养液中的终浓度分别为0、20、40、60、80 和 100μg/mL,其中Oμg/mL为对照组,其余为试验组。将细胞培养板置于37℃、5%的CO2培养箱中分别培养24h、48h和72h。试验结果表明：用不同浓度的游离蛋氨酸培养奶牛乳腺上皮细胞,当培养48h时,乳腺上皮细胞活力和αs1-酪蛋白(αs1-casein, CSN1S1)、k-酪蛋白(κ-casein, CSN3)和β-乳球蛋白(β-lactoglobulin, LGB)基因表达量随着蛋氨酸浓度的增加呈显著的一元二次曲线增加,当浓度达到60μg/mL时,表达量最高；但蛋氨酸的添加抑制了β-酪蛋白(p-casein, CSN2)基因的表达；当培养72h时,所有浓度的蛋氨酸均抑制了细胞增殖。蛋氨酸的添加浓度为60μg/mL、培养时间为48h时,乳腺上皮细胞的活力以及细胞中乳蛋白基因的表达量均较高。试验二研究了八种含蛋氨酸二肽等量替代其所含的游离氨基酸后对奶牛乳腺上皮细胞乳蛋白基因(CSN1S1、CSN2、CSN3、LGB)、Ⅱ型小肽转运载体基因(PEPT2)、氨肽酶氮基因(APN)表达以及细胞内外氨肽酶(APA)含量的影响。在试验一的基础上,将蛋氨酸与其它必需氨基酸分别组成八种二肽(蛋.氨酸-蛋氨酸(P-Met-Met)、蛋氨酸-赖氨酸(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-苏氨酸(P-Met-Thr)、蛋氨酸-异亮氨酸(P-Met-Ile)、蛋氨酸-亮氨酸(P-Met-Leu)、蛋氨酸-缬氨酸(P-Met-Val)),等量替代相应的游离氨基酸(F-Met-Met、F-Met-Lys、F-Met-Trp、F-Met-Phe、F-Met-Thr、 F-Met-Ile、F-Met-Leu、F-Met-Val)。试验二分为三个部分：第一部分研究了八种二肽对奶牛乳腺上皮细胞CSN1S1、CSN2、CSN3、LGB、PEPT2、APN基因表达以及细胞内外APA活性的影响。采用单因子随机试验设计分为9个组,8个二肽组与空白对照组。结果表明：P-Met-Met和P-Met-Lys组较对照组和其它二肽处理组显著上调了CSN1S1、CSN2、和 CSN3基因的表达,P-Met-Met组优于P-Met-Lys组。P-Met-Thr、P-Met-Leu、P-Met-Ile 和 P-Met-Val组抑制了CSN2基因和CSN3基因的表达。第二部分研究了与八种二肽对应的游离氨基酸对奶牛乳腺上皮细胞乳蛋白基因、PEPT2、APN表达以及细胞内外APA活性的影响。采用单因子随机试验设计分为9个组,8个氨基酸组与空白对照组。结果表明,F-Met-Met 和 F-Met-Lys组较对照组和其它氨基酸处理组显著地促进了CSN1S1基因的表达。F-Met-Thr组较其它氨基酸处理组显著抑制了CSN3基因的表达。PEPT2基因表达量的结果表明,F-Met-Ile组显著低于空白对照组,但F-Met-Met组显著高于F-Met-Trp、 F-Met-Leu、 F-Met-Ile和F-Met-Val氨基酸处理组,但与对照组无显著差异。不同氨基酸处理组对APN基因表达无显著的影响。第三部分研究了二肽等量替代相应游离氨基酸对奶牛乳腺上皮细胞乳蛋白基因、PEPT2、APN表达以及细胞内外APA活性的影响。结果表明,除P-Met-Val 和 P-Met-Leu,不同二肽组合替代氨基酸后均不同程度地促进了乳蛋白基因和PEPT2基因的表达量,以P-Met-Met表现出较好的促进效果,显著上调了乳蛋白CSN1S1、CSN2、CSN3基因的表达量,PEPT2的基因表达量有趋于显著的提高；以P-Met-Trp、 P-Met-Phe、P-Met-Lys的促进效果次之。二肽等量替换游离氨基酸能够显著的促进乳蛋白基因的表达,P-Met-Met、 P-Met-Trp、P-Met-Phe 和 P-Met-Lys的促进效果较好,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.

【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.5

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