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作者刘亚杰（2019）在《自噬参与心外膜祖细胞向冠状动脉平滑肌细胞分化的实验研究》一文中研究指出：研究冠脉血管的发育,明确冠脉血管平滑肌细胞(coronary smooth muscle cells,CoSMCs)的起源及分化机制,将为先天性心脏病的防治及心肌梗死后冠脉侧枝循环血管的形成等研究领域提供重要信息。目前研究提示,心外膜祖细胞(Epicardial progenitor cells,EPCs)是CoSMCs的主要起源,但EPCs向CoSMCs分化的具体机制尚不完全明确。自噬(autophagy)是由溶酶体介导的蛋白质及其他细胞器的分解过程,与衰老的细胞器及破坏的蛋白质的清除和维持细胞内环境稳定有密切关系。既往研究证实自噬在哺乳动物的心脏发育与重塑组织和细胞分化过程起着重要作用,但尚无证据表明自噬是否能够参与调控EPCs向CoSMCs分化。在本研究中,我们成功体外培育了小鼠原代EPCs,并通过自噬的诱导和抑制实验观察自噬对EPCs向平滑肌细胞分化的影响,同时进一步在体观察自噬对小鼠冠脉平滑肌发育的影响,以期证实自噬参与调控心外膜祖细胞向冠状动脉平滑肌细胞分化。第一部分:原代EPCs的培养与鉴定目的:探讨构建高纯度心外膜祖细胞的方法,为体外培养并揭示心外膜祖细胞的分化机制奠定基础。方法:首先分离E11.5小鼠胚胎心脏,利用组织贴壁法建立心外膜祖细胞体外培养模型。镜下观察培养细胞的自身特性,进一步通过qRT-PCR技术及免疫荧光技术检测心外膜祖细胞标志基因Tbx18及WT1基因的表达,借此来检测培养的细胞纯度。结果:成功培养出原代心外膜祖细胞群。并首先以形态学观察证实该原代细胞群具有典型上皮形态,并连续观察72小时证实该细胞群分化的形态学变化。进一步以心肌细胞为对照组,通过qPCR及免疫荧光证实该细胞能同时表达心外膜祖细胞标志性基因Tbx18及WT1。结论:通过组织贴壁法进行原代培养所得心外膜祖细胞方法相对简单且同时表达多个心外膜上皮标志基因,细胞纯度高。体外培养的原代心外膜祖细胞爬出后形成的细胞群形态和排列均呈典型上皮形态,且在形态学上观察到能够进一步分化,为后续心外膜祖细胞的分化研究打下基础。第二部分:体外实验揭示细胞自噬对心外膜祖细胞向平滑肌细胞分化的影响目的:探讨细胞自噬对心外膜心外膜祖细胞向平滑肌细胞分化的影响,及对EPCs分化而来的SMCs功能的影响。方法:在原代心外膜祖细胞模型店基础上,用自噬诱导剂RAPA及抑制剂3-MA分别建立了自噬诱导和抑制模型。观察自噬诱导/抑制对心外膜祖细胞分化的形态学影响,并通过qPCR测定自噬相关Akt及mTOR的mRNA水平变化。进一步通过MDC染色及LC3B蛋白荧光染色及Westernblot实验揭示心外膜祖细胞分化过程是否细胞自噬。后以qPCR及免疫荧光检测培养72h后细胞平滑肌标志蛋白a-SMA及MYH11水平,MTT实验检测各组细胞增殖能力。再收集培养96h细胞,以胶原凝胶收缩实验评价其收缩功能,划痕实验评价细胞迁移能力。结果:我们的实验观察到,RAPA在72h内显著诱导心外膜祖细胞自噬水平上调,进而导致心外膜祖细胞增殖明显减慢,但长梭形大体积异形细胞增多。免疫荧光染色见RAPA组a-SMA及MYH11峰值提前,提示自噬可能诱导心外膜祖细胞提前分化,在细胞培养48h即出现大量梭形,异形的分化细胞,结合免疫染色结果可见RAPA诱导的自噬使平滑肌细胞的分化提前,但增殖较NORMAL组及3-MA组明显减弱。而在3-MA组其自噬水平明显下调后,出现细胞增殖及迁移上调,而分化延迟的表现。提示抑制自噬可能抑制心外膜祖细胞向平滑肌细胞分化,而促进其增殖及迁移。结论:细胞自噬参与调控心外膜祖细胞向平滑肌细胞分化,自噬上调可能诱导心外膜祖细胞提前分化,而使平滑肌细胞收缩能力增强,增殖迁移能力下降,而抑制则可能抑制心外膜祖细胞向平滑肌细胞分化,而促进其增殖及迁移。第三部分:体内实验揭示自噬对小鼠冠状动脉发育的影响目的:在组织水平明确心外膜祖细胞对冠脉血管平滑肌发育的影响及自噬标识信号分子与平滑肌标志基因的空间定位关系,从而在体探讨自噬对心外膜祖细胞对冠状动脉发育的影响。方法:应用羊膜腔注射给药的方式建立小鼠自噬诱导/抑制模型,通过HE染色观察自噬诱导/抑制对小鼠E12.5-E14.5冠脉发育的结构影响,通过qPCR及免疫荧光观察自噬对小鼠冠状动脉平滑肌分化过程中a-SMA的RNA及蛋白表达的影响,进而将自噬标志蛋白LC3B与平滑肌细胞标志蛋白MYH11共染,观察两者的空间定位关系。结果:自噬诱导后,心脏几乎未能形成管腔发育成熟的冠状动脉血管,但可见心内膜下逐渐出现的血窦样变化,提示小鼠心脏发育未能发育出成熟冠脉及建立有效的冠脉循环。同时亦观察到自噬诱导后出现的心室流出道发育障碍,心脏循环缺陷及心室壁变薄。qPCR及组织免疫荧光结果提示,小鼠E12.5-14.5的自噬现象可同时出现在心外膜及心肌组织中。而自噬诱导组我们可见在E12.5天时,心外膜内的心肌组织中a-SMA的表达量略高于NORMAL组,但在E14.5天时,RAPA组a-SMA表达量明显低于NORMAL组,并且未能发育为成熟冠状动脉管腔。而在E12.5-14.5天,自噬抑制组a-SMA表达量各时间段均低于NORMAL组,最终冠状动脉周围a-SMA表达量也比NORMAL组更低。荧光共染实验证实,多处心外膜及心外膜下MYH11阳性细胞能够与LC3B蛋白共表达。结论:自噬在冠脉发育中需要适宜的强度。过度激活自噬可能导致导平滑肌细胞的提前分化和迁移能力增强,有可能使内皮细胞周围平滑肌细胞的募集失调,不能形成有效的冠脉管腔。总体来说,冠状动脉发育受到抑制。而自噬抑制可能使心外膜祖细胞分化延迟,从而使冠脉形成的数量减少。心外膜祖细胞向冠脉平滑肌分化的过程需要适宜的自噬强度。
Abstract
yan jiu guan mai xie guan de fa yo ,ming que guan mai xie guan ping hua ji xi bao (coronary smooth muscle cells,CoSMCs)de qi yuan ji fen hua ji zhi ,jiang wei xian tian xing xin zang bing de fang zhi ji xin ji geng si hou guan mai ce zhi xun huan xie guan de xing cheng deng yan jiu ling yu di gong chong yao xin xi 。mu qian yan jiu di shi ,xin wai mo zu xi bao (Epicardial progenitor cells,EPCs)shi CoSMCsde zhu yao qi yuan ,dan EPCsxiang CoSMCsfen hua de ju ti ji zhi shang bu wan quan ming que 。zi shi (autophagy)shi you rong mei ti jie dao de dan bai zhi ji ji ta xi bao qi de fen jie guo cheng ,yu cui lao de xi bao qi ji po huai de dan bai zhi de qing chu he wei chi xi bao nei huan jing wen ding you mi qie guan ji 。ji wang yan jiu zheng shi zi shi zai bu ru dong wu de xin zang fa yo yu chong su zu zhi he xi bao fen hua guo cheng qi zhao chong yao zuo yong ,dan shang mo zheng ju biao ming zi shi shi fou neng gou can yu diao kong EPCsxiang CoSMCsfen hua 。zai ben yan jiu zhong ,wo men cheng gong ti wai pei yo le xiao shu yuan dai EPCs,bing tong guo zi shi de you dao he yi zhi shi yan guan cha zi shi dui EPCsxiang ping hua ji xi bao fen hua de ying xiang ,tong shi jin yi bu zai ti guan cha zi shi dui xiao shu guan mai ping hua ji fa yo de ying xiang ,yi ji zheng shi zi shi can yu diao kong xin wai mo zu xi bao xiang guan zhuang dong mai ping hua ji xi bao fen hua 。di yi bu fen :yuan dai EPCsde pei yang yu jian ding mu de :tan tao gou jian gao chun du xin wai mo zu xi bao de fang fa ,wei ti wai pei yang bing jie shi xin wai mo zu xi bao de fen hua ji zhi dian ding ji chu 。fang fa :shou xian fen li E11.5xiao shu pei tai xin zang ,li yong zu zhi tie bi fa jian li xin wai mo zu xi bao ti wai pei yang mo xing 。jing xia guan cha pei yang xi bao de zi shen te xing ,jin yi bu tong guo qRT-PCRji shu ji mian yi ying guang ji shu jian ce xin wai mo zu xi bao biao zhi ji yin Tbx18ji WT1ji yin de biao da ,jie ci lai jian ce pei yang de xi bao chun du 。jie guo :cheng gong pei yang chu yuan dai xin wai mo zu xi bao qun 。bing shou xian yi xing tai xue guan cha zheng shi gai yuan dai xi bao qun ju you dian xing shang pi xing tai ,bing lian xu guan cha 72xiao shi zheng shi gai xi bao qun fen hua de xing tai xue bian hua 。jin yi bu yi xin ji xi bao wei dui zhao zu ,tong guo qPCRji mian yi ying guang zheng shi gai xi bao neng tong shi biao da xin wai mo zu xi bao biao zhi xing ji yin Tbx18ji WT1。jie lun :tong guo zu zhi tie bi fa jin hang yuan dai pei yang suo de xin wai mo zu xi bao fang fa xiang dui jian chan ju tong shi biao da duo ge xin wai mo shang pi biao zhi ji yin ,xi bao chun du gao 。ti wai pei yang de yuan dai xin wai mo zu xi bao pa chu hou xing cheng de xi bao qun xing tai he pai lie jun cheng dian xing shang pi xing tai ,ju zai xing tai xue shang guan cha dao neng gou jin yi bu fen hua ,wei hou xu xin wai mo zu xi bao de fen hua yan jiu da xia ji chu 。di er bu fen :ti wai shi yan jie shi xi bao zi shi dui xin wai mo zu xi bao xiang ping hua ji xi bao fen hua de ying xiang mu de :tan tao xi bao zi shi dui xin wai mo xin wai mo zu xi bao xiang ping hua ji xi bao fen hua de ying xiang ,ji dui EPCsfen hua er lai de SMCsgong neng de ying xiang 。fang fa :zai yuan dai xin wai mo zu xi bao mo xing dian ji chu shang ,yong zi shi you dao ji RAPAji yi zhi ji 3-MAfen bie jian li le zi shi you dao he yi zhi mo xing 。guan cha zi shi you dao /yi zhi dui xin wai mo zu xi bao fen hua de xing tai xue ying xiang ,bing tong guo qPCRce ding zi shi xiang guan Aktji mTORde mRNAshui ping bian hua 。jin yi bu tong guo MDCran se ji LC3Bdan bai ying guang ran se ji Westernblotshi yan jie shi xin wai mo zu xi bao fen hua guo cheng shi fou xi bao zi shi 。hou yi qPCRji mian yi ying guang jian ce pei yang 72hhou xi bao ping hua ji biao zhi dan bai a-SMAji MYH11shui ping ,MTTshi yan jian ce ge zu xi bao zeng shi neng li 。zai shou ji pei yang 96hxi bao ,yi jiao yuan ning jiao shou su shi yan ping jia ji shou su gong neng ,hua hen shi yan ping jia xi bao qian yi neng li 。jie guo :wo men de shi yan guan cha dao ,RAPAzai 72hnei xian zhe you dao xin wai mo zu xi bao zi shi shui ping shang diao ,jin er dao zhi xin wai mo zu xi bao zeng shi ming xian jian man ,dan chang suo xing da ti ji yi xing xi bao zeng duo 。mian yi ying guang ran se jian RAPAzu a-SMAji MYH11feng zhi di qian ,di shi zi shi ke neng you dao xin wai mo zu xi bao di qian fen hua ,zai xi bao pei yang 48hji chu xian da liang suo xing ,yi xing de fen hua xi bao ,jie ge mian yi ran se jie guo ke jian RAPAyou dao de zi shi shi ping hua ji xi bao de fen hua di qian ,dan zeng shi jiao NORMALzu ji 3-MAzu ming xian jian ruo 。er zai 3-MAzu ji zi shi shui ping ming xian xia diao hou ,chu xian xi bao zeng shi ji qian yi shang diao ,er fen hua yan chi de biao xian 。di shi yi zhi zi shi ke neng yi zhi xin wai mo zu xi bao xiang ping hua ji xi bao fen hua ,er cu jin ji zeng shi ji qian yi 。jie lun :xi bao zi shi can yu diao kong xin wai mo zu xi bao xiang ping hua ji xi bao fen hua ,zi shi shang diao ke neng you dao xin wai mo zu xi bao di qian fen hua ,er shi ping hua ji xi bao shou su neng li zeng jiang ,zeng shi qian yi neng li xia jiang ,er yi zhi ze ke neng yi zhi xin wai mo zu xi bao xiang ping hua ji xi bao fen hua ,er cu jin ji zeng shi ji qian yi 。di san bu fen :ti nei shi yan jie shi zi shi dui xiao shu guan zhuang dong mai fa yo de ying xiang mu de :zai zu zhi shui ping ming que xin wai mo zu xi bao dui guan mai xie guan ping hua ji fa yo de ying xiang ji zi shi biao shi xin hao fen zi yu ping hua ji biao zhi ji yin de kong jian ding wei guan ji ,cong er zai ti tan tao zi shi dui xin wai mo zu xi bao dui guan zhuang dong mai fa yo de ying xiang 。fang fa :ying yong yang mo qiang zhu she gei yao de fang shi jian li xiao shu zi shi you dao /yi zhi mo xing ,tong guo HEran se guan cha zi shi you dao /yi zhi dui xiao shu E12.5-E14.5guan mai fa yo de jie gou ying xiang ,tong guo qPCRji mian yi ying guang guan cha zi shi dui xiao shu guan zhuang dong mai ping hua ji fen hua guo cheng zhong a-SMAde RNAji dan bai biao da de ying xiang ,jin er jiang zi shi biao zhi dan bai LC3Byu ping hua ji xi bao biao zhi dan bai MYH11gong ran ,guan cha liang zhe de kong jian ding wei guan ji 。jie guo :zi shi you dao hou ,xin zang ji hu wei neng xing cheng guan qiang fa yo cheng shou de guan zhuang dong mai xie guan ,dan ke jian xin nei mo xia zhu jian chu xian de xie dou yang bian hua ,di shi xiao shu xin zang fa yo wei neng fa yo chu cheng shou guan mai ji jian li you xiao de guan mai xun huan 。tong shi yi guan cha dao zi shi you dao hou chu xian de xin shi liu chu dao fa yo zhang ai ,xin zang xun huan que xian ji xin shi bi bian bao 。qPCRji zu zhi mian yi ying guang jie guo di shi ,xiao shu E12.5-14.5de zi shi xian xiang ke tong shi chu xian zai xin wai mo ji xin ji zu zhi zhong 。er zi shi you dao zu wo men ke jian zai E12.5tian shi ,xin wai mo nei de xin ji zu zhi zhong a-SMAde biao da liang lve gao yu NORMALzu ,dan zai E14.5tian shi ,RAPAzu a-SMAbiao da liang ming xian di yu NORMALzu ,bing ju wei neng fa yo wei cheng shou guan zhuang dong mai guan qiang 。er zai E12.5-14.5tian ,zi shi yi zhi zu a-SMAbiao da liang ge shi jian duan jun di yu NORMALzu ,zui zhong guan zhuang dong mai zhou wei a-SMAbiao da liang ye bi NORMALzu geng di 。ying guang gong ran shi yan zheng shi ,duo chu xin wai mo ji xin wai mo xia MYH11yang xing xi bao neng gou yu LC3Bdan bai gong biao da 。jie lun :zi shi zai guan mai fa yo zhong xu yao kuo yi de jiang du 。guo du ji huo zi shi ke neng dao zhi dao ping hua ji xi bao de di qian fen hua he qian yi neng li zeng jiang ,you ke neng shi nei pi xi bao zhou wei ping hua ji xi bao de mu ji shi diao ,bu neng xing cheng you xiao de guan mai guan qiang 。zong ti lai shui ,guan zhuang dong mai fa yo shou dao yi zhi 。er zi shi yi zhi ke neng shi xin wai mo zu xi bao fen hua yan chi ,cong er shi guan mai xing cheng de shu liang jian shao 。xin wai mo zu xi bao xiang guan mai ping hua ji fen hua de guo cheng xu yao kuo yi de zi shi jiang du 。
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论文作者分别是来自重庆医科大学的刘亚杰，发表于刊物重庆医科大学2019-09-24论文，是一篇关于自噬论文,心外膜祖细胞论文,冠状动脉论文,平滑肌论文,分化论文,重庆医科大学2019-09-24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，资料来自重庆医科大学2019-09-24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。