本文主要研究内容
作者祁昊（2019）在《Met调节奶牛乳腺上皮细胞脂肪合成、细胞增殖及自噬的分子机理》一文中研究指出：氨基酸作为重要的有机小分子参与各项生命活动,既可合成蛋白质、脂质,又是调节细胞生长与分化、增殖与自噬等细胞代谢的关键信号分子。氨基酸作为外源信号可以激活磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)与哺乳动物雷帕霉素靶蛋白(the mammalian target of rapamycin,mTOR)等来调控细胞代谢。Na~+偶联中性氨基酸转运蛋白2(sodium-coupled neutral amino acid transporter 2,SNAT2)是溶质载体转运蛋白,可转运蛋氨酸(methionine,Met)、亮氨酸(leucine,Leu)等中性氨基酸进入细胞,并在奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)中高表达,促进BMECs乳合成,但其具体机理尚不清楚。实验室前期研究发现,SNAT2可以介导Met参与调节BMECs乳合成,而在泌乳过程中,BMECs的乳合成与细胞增殖、自噬及凋亡均相关,但其具体机理有待进一步研究。本研究将拟揭示Met调节BMECs乳脂合成、细胞增殖及自噬的分子机理,为进一步研究Met在乳生产中的重要作用提供实验依据。本研究以组织块法培养的BMECs为研究对象,免疫荧光结合Western blotting来鉴定BMECs中角蛋白18(cytokeratin 18,CK18)与β-酪蛋白(β-casein)的表达,证明获得纯化、可泌乳的体外培养BMECs。向体外培养的BMECs分别添加0、0.3、0.6、0.9和1.2 mM的Met,BODIPY染色及甘油三酯(triglyceride,TG)检测发现,随着Met浓度的增加,BMECs中脂滴及TG含量逐渐增加,并且在Met浓度为0.6 mM时达到峰值,随后下降。结果表明Met以剂量依赖性方式促进乳脂肪合成;CASY细胞计数及流式细胞仪检测细胞数和细胞周期变化,发现随着Met浓度增加,细胞数及S期和G2/M期的细胞比例逐渐增加,并且在Met浓度为0.6 mM时达到峰值,随后降低。结果表明Met以剂量依赖性影响BMECs增殖及细胞周期;Western blotting检测相关蛋白表达变化,结果发现p-mTOR、SREBP-1c、Cyclin D1的蛋白水平显著增加,在Met浓度为0.6 mM时最高,随后逐渐下降。结果说明Met对mTOR,SREBP-1c和Cyclin D1信号传导途径的激活也具有剂量依赖性,添加0.6 mM的Met为最适浓度,过量的Met可能具有细胞毒性。为了研究Met对BMECs自噬的影响,Western blotting检测SNAT2抑制后自噬标志蛋白LC3-I/II表达量的影响,并利用免疫荧光检测细胞自噬斑点(LC3-II)变化。结果显示,SNAT2抑制时,p-mTOR表达量下降,LC3-II表达量增加,免疫荧光检测自噬斑点增多。添加自噬增强剂海藻糖(trehalose,Tre)和Met后,Met组同Met+Tre组比较,p-mTOR表达量增加,LC3-II表达量降低,自噬斑点减少;添加Tre和Met并抑制SNAT2时,p-mTOR表达量下降,LC3-II表达量增多,自噬斑点增多。以上结果表明Met通过SNAT2抑制自噬。SNAT2基因进行过表达和干扰表明SNAT2可以正向调节BMECs中甘油三脂的分泌和脂滴的形成,Western blotting检测结果表明SNAT2过表达可以上调p-mTOR、SREBP-1c、Cyclin D1的蛋白水平,而干扰SNAT2后,结果与过表达相反。表明SNAT2是mTOR、SREBP-1c和Cyclin D1的上游信号分子,并可正向调节乳脂肪合成和细胞增殖。为了研究Met调节SNAT2表达的分子机制,添加PI3K抑制剂-渥曼青霉素(Wortmannin,Wm)抑制PI3K信号通路的同时过表达SNAT2,Western blotting检测发现,PI3K抑制消除了SNAT2过表达对p-mTOR、SREBP-1c以及Cyclin D1的蛋白质水平的刺激。PI3K抑制实验表明,SNAT2激活mTOR,SREBP-1c和Cyclin D1信号通路需要PI3K激活。添加0.6 mM的Met同时抑制SNAT2,结果表明SNAT2敲低显著抑制Met对p-PI3K、p-mTOR、SREBP-1c和Cyclin D1的蛋白质水平的上调作用。Western blotting结合qRT-PCR检测发现,0.6 mM Met处理显著增加BMECs中SNAT2的蛋白质水平和mRNA水平,免疫荧光检测发现0.6mM Met处理显著增加SNAT2的细胞质定位。这些数据表明,Met通过SNAT2-PI3K调节mTOR,SREBP-1c和Cyclin D1信号通路。综上所述,Met剂量依赖性调节mTOR、SREBP-1c、Cyclin D1的表达,过量的Met对细胞具有毒性,Met通过SNAT2-PI3K途径调节mTOR、SREBP-1c、Cyclin D1信号通路,从而影响BMECs乳脂肪合成、细胞增殖与自噬。
Abstract
an ji suan zuo wei chong yao de you ji xiao fen zi can yu ge xiang sheng ming huo dong ,ji ke ge cheng dan bai zhi 、zhi zhi ,you shi diao jie xi bao sheng chang yu fen hua 、zeng shi yu zi shi deng xi bao dai xie de guan jian xin hao fen zi 。an ji suan zuo wei wai yuan xin hao ke yi ji huo lin zhi xian ji chun 3-ji mei (phosphatidylinositol 3-kinase,PI3K)yu bu ru dong wu lei pa mei su ba dan bai (the mammalian target of rapamycin,mTOR)deng lai diao kong xi bao dai xie 。Na~+ou lian zhong xing an ji suan zhuai yun dan bai 2(sodium-coupled neutral amino acid transporter 2,SNAT2)shi rong zhi zai ti zhuai yun dan bai ,ke zhuai yun dan an suan (methionine,Met)、liang an suan (leucine,Leu)deng zhong xing an ji suan jin ru xi bao ,bing zai nai niu ru xian shang pi xi bao (bovine mammary epithelial cells,BMECs)zhong gao biao da ,cu jin BMECsru ge cheng ,dan ji ju ti ji li shang bu qing chu 。shi yan shi qian ji yan jiu fa xian ,SNAT2ke yi jie dao Metcan yu diao jie BMECsru ge cheng ,er zai bi ru guo cheng zhong ,BMECsde ru ge cheng yu xi bao zeng shi 、zi shi ji diao wang jun xiang guan ,dan ji ju ti ji li you dai jin yi bu yan jiu 。ben yan jiu jiang ni jie shi Metdiao jie BMECsru zhi ge cheng 、xi bao zeng shi ji zi shi de fen zi ji li ,wei jin yi bu yan jiu Metzai ru sheng chan zhong de chong yao zuo yong di gong shi yan yi ju 。ben yan jiu yi zu zhi kuai fa pei yang de BMECswei yan jiu dui xiang ,mian yi ying guang jie ge Western blottinglai jian ding BMECszhong jiao dan bai 18(cytokeratin 18,CK18)yu β-lao dan bai (β-casein)de biao da ,zheng ming huo de chun hua 、ke bi ru de ti wai pei yang BMECs。xiang ti wai pei yang de BMECsfen bie tian jia 0、0.3、0.6、0.9he 1.2 mMde Met,BODIPYran se ji gan you san zhi (triglyceride,TG)jian ce fa xian ,sui zhao Metnong du de zeng jia ,BMECszhong zhi di ji TGhan liang zhu jian zeng jia ,bing ju zai Metnong du wei 0.6 mMshi da dao feng zhi ,sui hou xia jiang 。jie guo biao ming Metyi ji liang yi lai xing fang shi cu jin ru zhi fang ge cheng ;CASYxi bao ji shu ji liu shi xi bao yi jian ce xi bao shu he xi bao zhou ji bian hua ,fa xian sui zhao Metnong du zeng jia ,xi bao shu ji Sji he G2/Mji de xi bao bi li zhu jian zeng jia ,bing ju zai Metnong du wei 0.6 mMshi da dao feng zhi ,sui hou jiang di 。jie guo biao ming Metyi ji liang yi lai xing ying xiang BMECszeng shi ji xi bao zhou ji ;Western blottingjian ce xiang guan dan bai biao da bian hua ,jie guo fa xian p-mTOR、SREBP-1c、Cyclin D1de dan bai shui ping xian zhe zeng jia ,zai Metnong du wei 0.6 mMshi zui gao ,sui hou zhu jian xia jiang 。jie guo shui ming Metdui mTOR,SREBP-1che Cyclin D1xin hao chuan dao tu jing de ji huo ye ju you ji liang yi lai xing ,tian jia 0.6 mMde Metwei zui kuo nong du ,guo liang de Metke neng ju you xi bao du xing 。wei le yan jiu Metdui BMECszi shi de ying xiang ,Western blottingjian ce SNAT2yi zhi hou zi shi biao zhi dan bai LC3-I/IIbiao da liang de ying xiang ,bing li yong mian yi ying guang jian ce xi bao zi shi ban dian (LC3-II)bian hua 。jie guo xian shi ,SNAT2yi zhi shi ,p-mTORbiao da liang xia jiang ,LC3-IIbiao da liang zeng jia ,mian yi ying guang jian ce zi shi ban dian zeng duo 。tian jia zi shi zeng jiang ji hai zao tang (trehalose,Tre)he Methou ,Metzu tong Met+Trezu bi jiao ,p-mTORbiao da liang zeng jia ,LC3-IIbiao da liang jiang di ,zi shi ban dian jian shao ;tian jia Trehe Metbing yi zhi SNAT2shi ,p-mTORbiao da liang xia jiang ,LC3-IIbiao da liang zeng duo ,zi shi ban dian zeng duo 。yi shang jie guo biao ming Mettong guo SNAT2yi zhi zi shi 。SNAT2ji yin jin hang guo biao da he gan rao biao ming SNAT2ke yi zheng xiang diao jie BMECszhong gan you san zhi de fen bi he zhi di de xing cheng ,Western blottingjian ce jie guo biao ming SNAT2guo biao da ke yi shang diao p-mTOR、SREBP-1c、Cyclin D1de dan bai shui ping ,er gan rao SNAT2hou ,jie guo yu guo biao da xiang fan 。biao ming SNAT2shi mTOR、SREBP-1che Cyclin D1de shang you xin hao fen zi ,bing ke zheng xiang diao jie ru zhi fang ge cheng he xi bao zeng shi 。wei le yan jiu Metdiao jie SNAT2biao da de fen zi ji zhi ,tian jia PI3Kyi zhi ji -wo man qing mei su (Wortmannin,Wm)yi zhi PI3Kxin hao tong lu de tong shi guo biao da SNAT2,Western blottingjian ce fa xian ,PI3Kyi zhi xiao chu le SNAT2guo biao da dui p-mTOR、SREBP-1cyi ji Cyclin D1de dan bai zhi shui ping de ci ji 。PI3Kyi zhi shi yan biao ming ,SNAT2ji huo mTOR,SREBP-1che Cyclin D1xin hao tong lu xu yao PI3Kji huo 。tian jia 0.6 mMde Mettong shi yi zhi SNAT2,jie guo biao ming SNAT2qiao di xian zhe yi zhi Metdui p-PI3K、p-mTOR、SREBP-1che Cyclin D1de dan bai zhi shui ping de shang diao zuo yong 。Western blottingjie ge qRT-PCRjian ce fa xian ,0.6 mM Metchu li xian zhe zeng jia BMECszhong SNAT2de dan bai zhi shui ping he mRNAshui ping ,mian yi ying guang jian ce fa xian 0.6mM Metchu li xian zhe zeng jia SNAT2de xi bao zhi ding wei 。zhe xie shu ju biao ming ,Mettong guo SNAT2-PI3Kdiao jie mTOR,SREBP-1che Cyclin D1xin hao tong lu 。zeng shang suo shu ,Metji liang yi lai xing diao jie mTOR、SREBP-1c、Cyclin D1de biao da ,guo liang de Metdui xi bao ju you du xing ,Mettong guo SNAT2-PI3Ktu jing diao jie mTOR、SREBP-1c、Cyclin D1xin hao tong lu ,cong er ying xiang BMECsru zhi fang ge cheng 、xi bao zeng shi yu zi shi 。
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论文作者分别是来自东北农业大学的祁昊，发表于刊物东北农业大学2019-08-27论文，是一篇关于蛋氨酸论文,乳脂肪合成论文,细胞增殖论文,自噬论文,东北农业大学2019-08-27论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自东北农业大学2019-08-27论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：蛋氨酸论文; 乳脂肪合成论文; 细胞增殖论文; 自噬论文; 东北农业大学2019-08-27论文;