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:第一部分包含兩個試驗,試驗一研究了不同培養(yǎng)時間的條件下,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BMECs中酪蛋白基因表達(dá)的影響,以期篩選出最適培養(yǎng)時間及培養(yǎng)濃度。利用消化法對BMECs進(jìn)行原代培養(yǎng),利用第3代細(xì)胞進(jìn)行指標(biāo)測定。將細(xì)胞隨機(jī)分為11個處理組,每組5個重復(fù),一組為對照組,試驗組分別向培養(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)時間為24h時,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)。試驗二在確定的適宜培養(yǎng)時間及培養(yǎng)濃度后,測定細(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個處理組,每組3個重復(fù),其中一組為對照組,試驗組分別加入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),測定在培養(yǎng)BMECs的過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個處理組,每組3個重復(fù),一組為對照組,試驗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CSN1S1表達(dá)有極顯著的促進(jìn)作用(P0.01);添加Met-Met-Met組對CSN3有極顯著的抑制作用(P0.01)。第三部分測定Op-Met在替代不同比例的F-Met時,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個處理組,每組3個重復(fù),一組為對照組,試驗組培養(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,細(xì)胞外AP含量顯著升高(P0.05)。這些試驗結(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,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,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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823

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