奶牛POTs的表达及小肽对乳腺上皮细胞乳蛋白合成的影响

发布时间：2018-03-25 05:23

本文选题：奶牛　切入点：小肽转运蛋白　出处：《扬州大学》2015年硕士论文

【摘要】：本试验研究了奶牛各组织小肽转运蛋白(proton-dependent oligopeptide transporters, POTs)的表达,并通过二肽培养奶牛乳腺上皮细胞系(bovine mammary epithelial cell, BMEC),研究小肽对BMEC乳蛋白合成的影响。为POTs在奶牛组织中的功能性研究提供依据,并为小肽在BMEC中的转运机制提供参考。研究包含3个试验,具体如下：[试验一]研究POTs在奶牛乳腺组织(mammary gland, MG)及BMEC中的定位和表达分析。在无菌条件下分离脂肪较少且富含乳腺小叶的MG进行纯化培养,应用免疫荧光法研究PepT1和PepT2蛋白在MG及BMEC中的分布,应用实时荧光定量PCR和Western blot法研究PepT1和PepT2在MG及BMEC中的表达。结果表明,PepT1分布于MG的小叶间结缔组织,PepT2分布于MG的乳导管及腺泡腔上皮细胞；PepT1和PepT2均分布于BMEC的细胞质和细胞膜；实时荧光定量PCR和Western blot分析发现,PepT1和PepT2在MG和BMEC中均有表达。[试验二]研究POTs在奶牛组织中的定量分析。屠宰4头健康的3月龄荷斯坦犊牛,采集奶牛心脏、肝脏、肺脏、肾脏、脾脏、胸腺、乳腺(采自2头泌乳后期淘汰奶牛)、肌肉组织及瘤胃、网胃、瓣胃、皱胃、十二指肠、空肠、回肠、盲肠、结肠的黏膜层。应用实时荧光定量PCR法研究PepT1、PepT2、PHT1和PHT2 mRNA在各组织中的表达差异。结果表明,PepT1 mRNA在奶牛空肠和十二指肠中表达量极显著高于回肠、瘤胃、.肺脏、结肠、瓣胃、肝脏、盲肠、网胃、胸腺、脾脏、皱胃、肾脏、乳腺、心脏和肌肉(P0.01)；PepT2 mRNA在奶牛乳腺中表达量极显著高于肾脏、肝脏、肺脏、脾脏、回肠、皱胃、胸腺、十二指肠、网胃、盲肠、心脏、空肠、结肠、瓣胃、肌肉和瘤胃(P0.01)；PHT1 mRNA在奶牛脾脏、盲肠和肺脏中表达量极显著高于心脏和肌肉(P0.01)；PHT2 mRNA在奶牛肺脏、盲肠和胸腺中表达量极显著高于瘤胃、皱胃、网胃、心脏和肌肉(P0.01)。[试验三]研究二肽对BMEC乳蛋白合成的影响。不同浓度Met-Met、Val-Met和Leu-Met分别培养BMEC,在最佳二肽浓度基础上,试验分为二肽组(Met-Met, Val-Met, Leu-Met),1/2二肽+1/2氨基酸组(Met-Met/Met、Met、Val-Met/Val、Met、Leu-Met/Leu、 Met),氨基酸组(Met、Met、Val、Met、Leu、Met),分别刺激培养BMEC,比较研究蛋白合成相关基因mRNA表达差异。结果表明：Met-Met/Met、Met组mTOR、EIF-4E、 S6K1和SOCS-3基因表达量极显著高于Met-Met组和Met、Met组(P0.01)。Val-Met/Val、 Met组K-酪蛋白、EIF-4E mRNA表达量极显著高于Val-Met组和Val、Met组(P0.01)：Val-Met/Val、Met组PepT2 mRNA表达量显著高于Val-Met组和Val、Met组(P0.05);Leu-Met组PHT1 mRNA表达量显著高于Leu-Met/Leu、Met组(P0.05),Leu-Met/Leu、Met组K-酪蛋白mRNA表达量极显著高于Leu-Met组和Leu、Met组(P0.01),Leu-Met组EIF-4E mRNA表达量显著高于Leu、Met组(P0.05)。HPLC检测BMEC培养前后培养液中二肽浓度变化,Met-Met/Met、Met组和Leu-Met/Leu、Met组二肽消失量高于Met-Met组和Leu-Met。结论：(1)PepT1分布于奶牛MG小叶间结缔组织；PepT2分布于MG叶间导管及腺泡腔内侧上皮细胞。(2)PepT1和PepT2蛋白分布于BMEC的细胞质。(3)PepT1在奶牛空肠和十二指肠,PepT2在乳腺,PHT1在脾脏、盲肠和胸腺,PHT2在肺脏、盲肠和胸腺中mRNA表达量最高。(4)二肽与氨基酸混合培养可促进乳K-酪蛋白和蛋白合成相关基因转录：(5)Met、Val和Leu可促进BMEC对Met-Met、Val-Met和Leu-Met二肽的转运。
[Abstract]:The study of small peptide transporter organizations (proton-dependent oligopeptide transporters, POTs cow) expression, and by two peptide cultured bovine mammary epithelial cell line (bovine mammary epithelial cell, BMEC), the research of small peptide ring effect on milk protein synthesis of BMEC. To provide the basis for the functional studies of POTs in cow tissues, and provide a reference for the transport mechanism of small peptide in BMEC. The study included 3 experiments as follows: [a] Experimental Study of POTs in mammary tissue (mammary gland, MG) and the analysis of the location and expression of BMEC. The separation of less fat and rich in mammary glands under sterile conditions were purified and cultured MG immunofluorescence was used to study the distribution, PepT1 and PepT2 protein in MG and BMEC's expression by fluorescence quantitative real-time PCR and Western blot and PepT2 PepT1 method in MG and BMEC. The results show that the distribution of PepT1 In MG the interlobular connective tissue, the distribution of PepT2 in MG breast ductal and acinar epithelial cells; PepT1 and PepT2 BMEC were distributed in the cytoplasm and cell membrane; real time fluorescence quantitative PCR and Western blot analysis, quantitative analysis in cow tissue PepT1 and PepT2 in both MG and BMEC expression in experiment two. Study POTs. Slaughter 4 healthy 3 month old Holstein calves, collecting cow heart, liver, lung, kidney, spleen, thymus, breast (collected from 2 lactating cows, late elimination) of muscle tissue and rumen, reticulum, omasum, abomasum, twelve duodenal, jejunum, ileum, cecum, colon mucosa the application of real time fluorescence quantitative PCR analysis of PepT1, PepT2, PHT1 and PHT2 mRNA expression in different tissues. The results showed that the expression of PepT1 mRNA in the jejunum and duodenum of dairy cattle was much higher than that of ileum, rumen. Lung, colon, liver, omasum, cecum, net The stomach, thymus, spleen, kidney, breast, abomasum, heart and muscle (P0.01); the expression of PepT2 mRNA in cow mammary gland volume was significantly higher than that of kidney, liver, lung, spleen, thymus, abomasum, ileum, duodenum, stomach, jejunum, cecum, colon, heart, muscle of rumen and omasum, (P0.01 PHT1; mRNA) in bovine spleen and lung, expression of cecum was much higher than that in heart and muscle (P0.01); PHT2 mRNA in cow lung, expression of cecum and thymus weight were significantly higher than that of rumen, abomasum, stomach, heart and muscle (P0.01) three] effect of two peptide synthesis on BMEC milk protein "test. Different concentrations of Met-Met, Val-Met and Leu-Met were cultured in BMEC, the optimal concentration of two peptide on the basis of the experiment was divided into two groups (Met-Met, Val-Met peptide, Leu-Met), 1/2 two (Met-Met/Met, +1/2 peptide amino acids Met, Val-Met/Val, Met, Leu-Met/Leu, Met), amino acids (Met, Met, Val group Met, Leu, Met. ), were cultured BMEC, comparative study on protein synthesis related gene mRNA expression. The results showed that: Met-Met/Met, EIF-4E, Met group mTOR, S6K1 and SOCS-3 gene expression was significantly higher than that in Met-Met group and Met group (P0.01), Met.Val-Met/Val, Met K- EIF-4E group of casein, the expression of mRNA was significantly higher than that in Val-Met group and Val, group Met (P0.01):Val-Met/Val, PepT2 mRNA expression in Met group was significantly higher than that in Val-Met group and Val Met group (P0.05); PHT1 mRNA expression of Leu-Met group was significantly higher than that in Leu-Met/Leu group (P0.05), Met, Leu-Met/Leu, K-, mRNA in Met group the expression of casein was significantly higher than that in Leu-Met group and Leu group (Met. P0.01, EIF-4E, mRNA) expression of Leu-Met group was significantly higher than that in Leu group, Met (P0.05).HPLC detection of BMEC cells before and after culture change, two peptide concentration of Met-Met/Met, Met and Leu-Met/Leu group, Met group of two peptides was higher than that in Met-Met group and Leu-Met.. Conclusion: (1) PepT1 Cloth in the cow MG interlobular connective tissue; PepT2 distribution in MG interlobar duct and acinar cavity inside epithelial cells. (2) PepT1 and PepT2 protein distributed in the cytoplasm. BMEC (3) PepT1 in the jejunum and duodenum of dairy cows, PepT2 in breast, PHT1 in spleen, thymus and cecum, PHT2 in lung volume. The highest expression of mRNA in cecum and thymus. (4) two peptides and amino acids in mixed culture can promote milk casein and K- protein synthesis related gene transcription: (5) Met, Val and Leu can promote the BMEC of Met-Met, Val-Met and Leu-Met two peptide transporters.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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