本文關(guān)鍵詞： 奶牛乳腺上皮細(xì)胞 酪蛋白 蛋氨酸寡肽 基因表達(dá)　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：本論文以體外培養(yǎng)的奶牛乳腺上皮細(xì)胞(BMECs)為模型,研究不同蛋氨酸寡肽(Op-Met)濃度及Op-Met代替不同比例的游離蛋氨酸(F-Met)后對(duì)BMECs中酪蛋白基因及其相關(guān)基因表達(dá)以及細(xì)胞內(nèi)外氨肽酶(AP)含量的影響,為揭示寡肽對(duì)BMECs中酪蛋白合的影響及其機(jī)理奠定理論基礎(chǔ)。論文由三大部分構(gòu)成:第一部分包含兩個(gè)試驗(yàn),試驗(yàn)一研究了不同培養(yǎng)時(shí)間的條件下,培養(yǎng)基中添加不同濃度的蛋氨酸二肽(Met-Met)和蛋氨酸三肽(Met-Met-Met)對(duì)BMECs中酪蛋白基因表達(dá)的影響,以期篩選出最適培養(yǎng)時(shí)間及培養(yǎng)濃度。利用消化法對(duì)BMECs進(jìn)行原代培養(yǎng),利用第3代細(xì)胞進(jìn)行指標(biāo)測(cè)定。將細(xì)胞隨機(jī)分為11個(gè)處理組,每組5個(gè)重復(fù),一組為對(duì)照組,試驗(yàn)組分別向培養(yǎng)基中加入終濃度為40、50、60、70和80μg/mL的Op-Met。將細(xì)胞培養(yǎng)24h、48h和72h。結(jié)果表明:用添加不同濃度的Op-Met的培養(yǎng)基培養(yǎng)細(xì)胞24h后,BMECs整體活力最高;當(dāng)Op-Met的添加濃度為60μg/mL,培養(yǎng)時(shí)間為24h時(shí),αs1-酪蛋白基因(CSN1S1)和β-酪蛋白基因(CSN2)表達(dá)量顯著提高(P0.05)。試驗(yàn)二在確定的適宜培養(yǎng)時(shí)間及培養(yǎng)濃度后,測(cè)定細(xì)胞中兩種小肽轉(zhuǎn)運(yùn)載體(PepTs)基因和氨肽酶氮基因(APN)的表達(dá)和細(xì)胞內(nèi)外AP含量的變化。將細(xì)胞隨機(jī)分為3個(gè)處理組,每組3個(gè)重復(fù),其中一組為對(duì)照組,試驗(yàn)組分別加入60μg/mL的Met-Met或Met-Met-Met后將細(xì)胞培養(yǎng)24h。結(jié)果表明:Met-Met組的小肽轉(zhuǎn)運(yùn)載體2基因(PepT2)和APN表達(dá)顯著提高(P0.05),細(xì)胞外的AP含量極顯著升高(P0.01);Met-Met-Met組的小肽轉(zhuǎn)運(yùn)載體1基因(PepT1)和PepT2表達(dá)量顯著提高(P0.05)。第二部分根據(jù)以前學(xué)者確定的BMECs培養(yǎng)基中適宜F-Met添加濃度為60μg/mL為基礎(chǔ),測(cè)定在培養(yǎng)BMECs的過(guò)程中添加適宜濃度的F-Met、Met-Met和Met-Met-Met的情況下各酪蛋白基因的表達(dá)量。將細(xì)胞隨機(jī)分為4個(gè)處理組,每組3個(gè)重復(fù),一組為對(duì)照組,試驗(yàn)組分別加入適宜濃度的F-Met、Met-Met或Met-Met-Met后培養(yǎng)細(xì)胞24h。結(jié)果表明:添加F-Met組對(duì)CSN1S1、CSN2和κ-酪蛋白基因(CSN3)均有極顯著促進(jìn)作用(P0.01);添加Met-Met組對(duì)CSN1S1表達(dá)有極顯著的促進(jìn)作用(P0.01);添加Met-Met-Met組對(duì)CSN3有極顯著的抑制作用(P0.01)。第三部分測(cè)定Op-Met在替代不同比例的F-Met時(shí),BMECs中酪蛋白基因及其相關(guān)基因表達(dá)和AP含量的變化。將細(xì)胞隨機(jī)分為10個(gè)處理組,每組3個(gè)重復(fù),一組為對(duì)照組,試驗(yàn)組培養(yǎng)基中分別添加0%、5%、10%、15%和20%的Op-Met代替其中的F-Met,培養(yǎng)24h。結(jié)果表明:Op-Met代替F-Met的最適代替比例為15%;當(dāng)Op-Met代替量為0%時(shí),細(xì)胞外AP含量顯著升高(P0.05)。這些試驗(yàn)結(jié)果證明小肽的添加能使BMECs酪蛋白基因表達(dá)量提高,適宜的Op-Met添加濃度為60μg/mL時(shí),CSN1S1和CSN2顯著提高,PepT2基因表達(dá)顯著提高;用Op-Met代替15%F-Met時(shí),細(xì)胞中各酪蛋白基因表達(dá)量顯著提高。
[Abstract]:In this study, the bovine mammary epithelial cells (BMECs) were cultured in vitro. The effects of different concentrations of methionine oligoseptide Op-Meta and Op-Met on the expression of casein gene and its related genes in BMECs and the content of aminopeptidase (APP) in and out of BMECs were studied. In order to reveal the effect of oligoseptide on casein synthesis in BMECs and its mechanism, the thesis consists of three parts: the first part consists of two experiments. Effects of different concentrations of methionine dipeptide Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met-Met@@. The cells were randomly divided into 11 treatment groups with 5 repeats in each group and a control group. In the experimental group, the final concentration of Op-Meta was 40,50,6070 and 80 渭 g / mL, respectively. The cells were cultured for 24 h for 48 h and 72 h. The results showed that the whole activity of BMECs was the highest in the culture medium supplemented with different concentrations of Op-Met for 24 h. When the concentration of Op-Met was 60 渭 g / mL and the culture time was 24 hours, the expression of 偽 s1-casein gene (CSN1S1) and 尾 -casein gene (CSN2) increased significantly. The expression of PepTsgene and aminopeptidase nitrogen gene (APN) and the changes of AP content in and out of cells were measured. The cells were randomly divided into three treatment groups with 3 repeats in each group, one of which was a control group. The cells in the experimental group were cultured for 24 h with 60 渭 g / mL Met-Met or Met-Met-Met, respectively. The results showed that the expression of PepT2 and APN in the small peptide transport vector 2 (PepT2) and the expression of APN were significantly increased, and the AP content in the extracellular AP was significantly higher than that in the P0.01Met-Met Met-Met group (PepT1). And PepT2 expression increased significantly. The second part was based on the suitable F-Met concentration of 60 渭 g / mL in BMECs medium, which was determined by previous scholars. The expression of casein genes was determined by adding appropriate concentrations of F-MetMet-Met and Met-Met-Met in the culture of BMECs. The cells were randomly divided into four treatment groups with 3 repeats in each group and one as control group. In the experimental group, the cells were cultured at the appropriate concentration of F-Met-Met or Met-Met-Met for 24 h. The results showed that the addition of F-Met could significantly promote the expression of CSN1S1CSN2 and 魏 -casein gene (CSN3), the addition of Met-Met could significantly promote the expression of CSN1S1 (P0.01), and the addition of F-Met could significantly promote the expression of CSN1S1. In the third part, the expression of casein gene and its related genes and the content of AP in the BMECs of Op-Met were measured when replacing F-Met with different proportions. The cells were randomly divided into 10 treatment groups. Each group had 3 repetitions and one group was the control group. In the experimental group, 15% and 20%% Op-Met were added to the culture medium to replace F-Met.The results showed that the optimum substitution ratio of the F-Met was 15g when the Op-Met substitution was 0. The results showed that the addition of small peptide could increase the expression of BMECs casein gene. When the appropriate concentration of Op-Met was 60 渭 g / mL, CSN1S1 and CSN2 significantly increased the expression of PepT2 gene, and when Op-Met was used instead of 15F-Met, the expression of Op-Met casein gene was increased significantly. The expression of casein genes increased significantly.
【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】：2017
【分類號(hào)】：S823

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鄭永唐,賁昆龍;測(cè)定細(xì)胞存活和增殖的MTT方法的建立[J];免疫學(xué)雜志;1992年04期

2 史清河;奶牛氨基酸營(yíng)養(yǎng)與乳蛋白含量和產(chǎn)量的關(guān)系[J];中國(guó)飼料;2000年08期

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

4 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

5 John J.Kennely,趙改名;開發(fā)改變?nèi)槌煞值臐摿J];國(guó)外畜牧科技;2001年04期

6 王夢(mèng)芝;王洪榮;李國(guó)祥;張潔;;反芻動(dòng)物肽代謝的研究進(jìn)展[J];草食家畜;2006年04期

7 王俊鋒;王中華;梁國(guó)義;謝寶柱;;影響奶牛乳蛋白質(zhì)含量的因素及營(yíng)養(yǎng)調(diào)控技術(shù)研究[J];飼料工業(yè);2006年15期

8 李廣運(yùn);預(yù)防奶牛冷應(yīng)激的措施[J];養(yǎng)殖技術(shù)顧問(wèn);2005年01期

9 楊金勇;吳躍明;劉建新;;蛋氨酸和蛋氨酸二肽對(duì)奶牛乳腺上皮細(xì)胞酪蛋白α_(s1)基因表達(dá)的影響[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2007年01期

10 張春;趙學(xué)梅;周麗;牛英才;劉吉成;;MTT法測(cè)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

相關(guān)會(huì)議論文 前1條

1 張居農(nóng);張春禮;;奶牛熱應(yīng)激的發(fā)生機(jī)制與預(yù)防策略[A];第三屆中國(guó)牛業(yè)發(fā)展大會(huì)論文集[C];2008年

相關(guān)博士學(xué)位論文 前2條

1 崔艷;泌乳奶牛乳腺中小肽轉(zhuǎn)運(yùn)載體的鑒定及其生理特性的研究[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2015年

2 周苗苗;奶牛乳腺中小肽的攝取及其在乳蛋白合成中的作用[D];浙江大學(xué);2011年

相關(guān)碩士學(xué)位論文 前3條

1 常晨城;蛋氨酸及含蛋氨酸二肽對(duì)奶牛乳腺上皮細(xì)胞內(nèi)乳蛋白合成相關(guān)基因表達(dá)的影響[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2015年

2 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

3 楊金勇;蛋氨酸、賴氨酸及其二肽對(duì)奶牛乳腺上皮細(xì)胞酪蛋白α_（s1）基因表達(dá)的影響[D];浙江大學(xué);2006年

，

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