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作者徐哲（2019）在《在Ⅰ型干扰素反应细胞中特异敲除RACK1对造血干细胞的影响》一文中研究指出：目的:利用干扰素诱导型MX-Cre在Ⅰ型干扰素反应细胞中特异敲除含有7个WD40 repeat的接头蛋白RACK1（基因名Gnb2l1）,分析这种小鼠模型对病毒的抗感染能力如何。通过流式细胞术检测并分析在Ⅰ型干扰素反应细胞中特异敲除RACK1对造血干细胞、各谱系造血祖细胞的影响,分析RACK1缺失对造血干细胞的凋亡及增殖的影响。通过蛋白-蛋白相互作用分析探讨RACK1调控造血干细胞的分子机制。方法:1.在Ⅰ型干扰素反应细胞中敲除RACK1的小鼠模型,用Gnb2l1F/F,MX-Cre表示,对照小鼠用Gnb2l1F/F表示,课题组前期工作已经建立相应模型。在正式实验开始前,剪鼠尾鉴定基因型,以区分Gnb2l1F/F,MX-Cre小鼠和Gnb2l1F/F小鼠。Gnb2l1F/F,MX-Cre小鼠和Gnb2l1F/F小鼠腹腔注射Poly（I:C）,以实现Gnb2l1F/F,MX-Cre小鼠的体内诱导敲除。并通过WB鉴定在蛋白水平上是否敲除。2.腹腔注射Poly（I:C）后,Gnb2l1F/F,MX-Cre小鼠和Gnb2l1F/F小鼠尾静脉注射VSV,后取小鼠外周血以得到血清,检测血清中的IFNβ。取肺和肝并称重,分别计算肺/体重和肝/体重。通过流式细胞术检测Gnb2l1F/F,MX-Cre小鼠和Gnb2l1F/F小鼠脾和肝中I型干扰素主要产生细胞pDC和髓系细胞的表达情况。3.取小鼠股骨骨髓,通过流式细胞术分选造血干细胞、巨核细胞/红细胞系祖细胞、粒细胞/巨噬细胞祖细胞、髓系细胞祖细胞、淋巴细胞系祖细胞和B淋巴细胞、髓系细胞、红系细胞,通过Real-Time PCR检测各个阶段RACK1、c-Myc及N-Myc的基因表达水平,并通过多色荧光组化分析RACK1与造血干细胞的表面标记CD117是否为共定位关系。Gnb2l1F/F,MX-Cre小鼠及Gnb2l1F/F小鼠股骨石蜡切片进行HE染色分析骨髓的病理损伤情况。通过流式细胞术分析Gnb2l1F/F,MX-Cre小鼠和Gnb2l1F/F小鼠的造血干细胞、各谱系造血祖细胞比例和绝对数,RACK1缺失对造血干细胞的凋亡及增殖的影响。制备嵌合体小鼠验证上述变化是否由于细胞的内在缺陷所引起。流式细胞术检测Gnb2l1F/F,MX-Cre小鼠和Gnb2l1F/F小鼠造血干细胞中c-Myc和N-Myc的表达量,并给小鼠腹腔注射Myc inhibitor进行逆转实验。为接下来的机制探讨提供基础。4.基于上述实验基础,进行体外实验,免疫共沉淀（CO-IP）分析c-Myc、N-Myc分别与RACK1是否有相互作用。使用纯化的GST及GST-RACK1直接pull down,分析是否为两者的直接相互作用。纯化Gnb2l1F/F,MX-Cre小鼠及Gnb2l1F/F小鼠造血干细胞,沉淀c-Myc或N-Myc,来分析E3泛素连接酶Fbxw7在其中产生的作用。结果:1.小鼠基因型鉴定成功。Gnb2l1F/F,MX-Cre小鼠在Poly（I:C）诱导后蛋白水平上也实现敲除。2.VSV感染后,与Gnb2l1F/F小鼠相比,Gnb2l1F/F,MX-Cre小鼠血清中的IFNβ水平降低,肝肺湿重/体重比值有增高趋势。流式细胞术检测结果显示Gnb2l1F/F,MX-Cre小鼠肝脾中Ⅰ型干扰素主要产生细胞pDC明显少于Gnb2l1F/F小鼠,并且髓系细胞也明显低于Gnb2l1F/F小鼠。3.Real-Time PCR结果显示,在mRNA转录水平上,造血干细胞、各谱系造血祖细胞及成熟阶段细胞都含有RACK1、c-Myc和N-Myc。从数据上来看,幼稚阶段表达水平明显高于成熟阶段。通过Gnb2l1F/F小鼠股骨石蜡切片进行多色荧光组化的结果显示RACK1与CD117具有荧光共定位。将Gnb2l1F/F小鼠及Gnb2l1F/F,MX-Cre小鼠股骨石蜡切片进行HE染色分析,在Ⅰ型干扰素反应细胞中特异敲除RACK1使骨髓腔内造血细胞稀少。Gnb2l1F/F,MX-Cre小鼠中造血干细胞、CMP和CLP在细胞比例上和细胞总数上都显著减少,GMP及MEP在绝对数统计结果中有明显降低。HSC的凋亡及增殖在比例上都显著增多。嵌合体小鼠实验证明以上变化是由于小鼠细胞内在缺陷所引起。Ⅰ型干扰素反应细胞中特异敲除RACK1会使HSC中表达c-Myc和N-Myc的细胞比例增多。注射Myc inhibitor可以部分逆转HSC。4.CO-IP结果显示,RACK1与c-Myc、N-Myc之间都有相互作用,并且证明为直接相互作用。纯化的小鼠造血干细胞沉淀c-Myc或N-Myc,结果显示在Ⅰ型干扰素反应细胞中特异敲除RACK1后,c-Myc或N-Myc与E3泛素连接酶Fbxw7的相互作用减弱。结论:1.小鼠模型鉴定成功,区分出Gnb2l1F/F,MX-Cre小鼠和Gnb2l1F/F小鼠;2.在Ⅰ型干扰素反应细胞中特异敲除RACK1使小鼠的抗感染能力减弱;3.在Ⅰ型干扰素反应细胞中特异敲除RACK1使骨髓中的HSC和各谱系造血祖细胞大幅度减少。Gnb2l1F/F,MX-Cre小鼠的HSC凋亡比例显著增加,增殖也大幅增加。制备嵌合体小鼠证实了这种现象为细胞内在缺陷所引起。逆转实验提示我们在Ⅰ型干扰素反应细胞中特异敲除RACK1使骨髓中HSC产生一系列影响是由于c-Myc和N-Myc大量积聚所致;4.RACK1与c-Myc和N-Myc都具有直接相互作用,沉淀c-Myc或N-Myc,在Ⅰ型干扰素反应细胞中特异敲除RACK1后,与c-Myc或N-Myc相互作用的Fbxw7蛋白量减少。c-Myc或N-Myc与Fbxw7相互结合作用减弱,导致c-Myc或N-Myc大量积聚。为以后深入研究RACK1影响HSC的机制提供有利参考。
Abstract
mu de :li yong gan rao su you dao xing MX-Crezai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu han you 7ge WD40 repeatde jie tou dan bai RACK1（ji yin ming Gnb2l1）,fen xi zhe chong xiao shu mo xing dui bing du de kang gan ran neng li ru he 。tong guo liu shi xi bao shu jian ce bing fen xi zai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1dui zao xie gan xi bao 、ge pu ji zao xie zu xi bao de ying xiang ,fen xi RACK1que shi dui zao xie gan xi bao de diao wang ji zeng shi de ying xiang 。tong guo dan bai -dan bai xiang hu zuo yong fen xi tan tao RACK1diao kong zao xie gan xi bao de fen zi ji zhi 。fang fa :1.zai Ⅰxing gan rao su fan ying xi bao zhong qiao chu RACK1de xiao shu mo xing ,yong Gnb2l1F/F,MX-Crebiao shi ,dui zhao xiao shu yong Gnb2l1F/Fbiao shi ,ke ti zu qian ji gong zuo yi jing jian li xiang ying mo xing 。zai zheng shi shi yan kai shi qian ,jian shu wei jian ding ji yin xing ,yi ou fen Gnb2l1F/F,MX-Crexiao shu he Gnb2l1F/Fxiao shu 。Gnb2l1F/F,MX-Crexiao shu he Gnb2l1F/Fxiao shu fu qiang zhu she Poly（I:C）,yi shi xian Gnb2l1F/F,MX-Crexiao shu de ti nei you dao qiao chu 。bing tong guo WBjian ding zai dan bai shui ping shang shi fou qiao chu 。2.fu qiang zhu she Poly（I:C）hou ,Gnb2l1F/F,MX-Crexiao shu he Gnb2l1F/Fxiao shu wei jing mai zhu she VSV,hou qu xiao shu wai zhou xie yi de dao xie qing ,jian ce xie qing zhong de IFNβ。qu fei he gan bing chen chong ,fen bie ji suan fei /ti chong he gan /ti chong 。tong guo liu shi xi bao shu jian ce Gnb2l1F/F,MX-Crexiao shu he Gnb2l1F/Fxiao shu pi he gan zhong Ixing gan rao su zhu yao chan sheng xi bao pDChe sui ji xi bao de biao da qing kuang 。3.qu xiao shu gu gu gu sui ,tong guo liu shi xi bao shu fen shua zao xie gan xi bao 、ju he xi bao /gong xi bao ji zu xi bao 、li xi bao /ju shi xi bao zu xi bao 、sui ji xi bao zu xi bao 、lin ba xi bao ji zu xi bao he Blin ba xi bao 、sui ji xi bao 、gong ji xi bao ,tong guo Real-Time PCRjian ce ge ge jie duan RACK1、c-Mycji N-Mycde ji yin biao da shui ping ,bing tong guo duo se ying guang zu hua fen xi RACK1yu zao xie gan xi bao de biao mian biao ji CD117shi fou wei gong ding wei guan ji 。Gnb2l1F/F,MX-Crexiao shu ji Gnb2l1F/Fxiao shu gu gu dan la qie pian jin hang HEran se fen xi gu sui de bing li sun shang qing kuang 。tong guo liu shi xi bao shu fen xi Gnb2l1F/F,MX-Crexiao shu he Gnb2l1F/Fxiao shu de zao xie gan xi bao 、ge pu ji zao xie zu xi bao bi li he jue dui shu ,RACK1que shi dui zao xie gan xi bao de diao wang ji zeng shi de ying xiang 。zhi bei qian ge ti xiao shu yan zheng shang shu bian hua shi fou you yu xi bao de nei zai que xian suo yin qi 。liu shi xi bao shu jian ce Gnb2l1F/F,MX-Crexiao shu he Gnb2l1F/Fxiao shu zao xie gan xi bao zhong c-Myche N-Mycde biao da liang ,bing gei xiao shu fu qiang zhu she Myc inhibitorjin hang ni zhuai shi yan 。wei jie xia lai de ji zhi tan tao di gong ji chu 。4.ji yu shang shu shi yan ji chu ,jin hang ti wai shi yan ,mian yi gong chen dian （CO-IP）fen xi c-Myc、N-Mycfen bie yu RACK1shi fou you xiang hu zuo yong 。shi yong chun hua de GSTji GST-RACK1zhi jie pull down,fen xi shi fou wei liang zhe de zhi jie xiang hu zuo yong 。chun hua Gnb2l1F/F,MX-Crexiao shu ji Gnb2l1F/Fxiao shu zao xie gan xi bao ,chen dian c-Mychuo N-Myc,lai fen xi E3fan su lian jie mei Fbxw7zai ji zhong chan sheng de zuo yong 。jie guo :1.xiao shu ji yin xing jian ding cheng gong 。Gnb2l1F/F,MX-Crexiao shu zai Poly（I:C）you dao hou dan bai shui ping shang ye shi xian qiao chu 。2.VSVgan ran hou ,yu Gnb2l1F/Fxiao shu xiang bi ,Gnb2l1F/F,MX-Crexiao shu xie qing zhong de IFNβshui ping jiang di ,gan fei shi chong /ti chong bi zhi you zeng gao qu shi 。liu shi xi bao shu jian ce jie guo xian shi Gnb2l1F/F,MX-Crexiao shu gan pi zhong Ⅰxing gan rao su zhu yao chan sheng xi bao pDCming xian shao yu Gnb2l1F/Fxiao shu ,bing ju sui ji xi bao ye ming xian di yu Gnb2l1F/Fxiao shu 。3.Real-Time PCRjie guo xian shi ,zai mRNAzhuai lu shui ping shang ,zao xie gan xi bao 、ge pu ji zao xie zu xi bao ji cheng shou jie duan xi bao dou han you RACK1、c-Myche N-Myc。cong shu ju shang lai kan ,you zhi jie duan biao da shui ping ming xian gao yu cheng shou jie duan 。tong guo Gnb2l1F/Fxiao shu gu gu dan la qie pian jin hang duo se ying guang zu hua de jie guo xian shi RACK1yu CD117ju you ying guang gong ding wei 。jiang Gnb2l1F/Fxiao shu ji Gnb2l1F/F,MX-Crexiao shu gu gu dan la qie pian jin hang HEran se fen xi ,zai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1shi gu sui qiang nei zao xie xi bao xi shao 。Gnb2l1F/F,MX-Crexiao shu zhong zao xie gan xi bao 、CMPhe CLPzai xi bao bi li shang he xi bao zong shu shang dou xian zhe jian shao ,GMPji MEPzai jue dui shu tong ji jie guo zhong you ming xian jiang di 。HSCde diao wang ji zeng shi zai bi li shang dou xian zhe zeng duo 。qian ge ti xiao shu shi yan zheng ming yi shang bian hua shi you yu xiao shu xi bao nei zai que xian suo yin qi 。Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1hui shi HSCzhong biao da c-Myche N-Mycde xi bao bi li zeng duo 。zhu she Myc inhibitorke yi bu fen ni zhuai HSC。4.CO-IPjie guo xian shi ,RACK1yu c-Myc、N-Myczhi jian dou you xiang hu zuo yong ,bing ju zheng ming wei zhi jie xiang hu zuo yong 。chun hua de xiao shu zao xie gan xi bao chen dian c-Mychuo N-Myc,jie guo xian shi zai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1hou ,c-Mychuo N-Mycyu E3fan su lian jie mei Fbxw7de xiang hu zuo yong jian ruo 。jie lun :1.xiao shu mo xing jian ding cheng gong ,ou fen chu Gnb2l1F/F,MX-Crexiao shu he Gnb2l1F/Fxiao shu ;2.zai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1shi xiao shu de kang gan ran neng li jian ruo ;3.zai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1shi gu sui zhong de HSChe ge pu ji zao xie zu xi bao da fu du jian shao 。Gnb2l1F/F,MX-Crexiao shu de HSCdiao wang bi li xian zhe zeng jia ,zeng shi ye da fu zeng jia 。zhi bei qian ge ti xiao shu zheng shi le zhe chong xian xiang wei xi bao nei zai que xian suo yin qi 。ni zhuai shi yan di shi wo men zai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1shi gu sui zhong HSCchan sheng yi ji lie ying xiang shi you yu c-Myche N-Mycda liang ji ju suo zhi ;4.RACK1yu c-Myche N-Mycdou ju you zhi jie xiang hu zuo yong ,chen dian c-Mychuo N-Myc,zai Ⅰxing gan rao su fan ying xi bao zhong te yi qiao chu RACK1hou ,yu c-Mychuo N-Mycxiang hu zuo yong de Fbxw7dan bai liang jian shao 。c-Mychuo N-Mycyu Fbxw7xiang hu jie ge zuo yong jian ruo ,dao zhi c-Mychuo N-Mycda liang ji ju 。wei yi hou shen ru yan jiu RACK1ying xiang HSCde ji zhi di gong you li can kao 。
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论文作者分别是来自安徽医科大学的徐哲，发表于刊物安徽医科大学2019-09-02论文，是一篇关于造血干细胞论文,安徽医科大学2019-09-02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，资料来自安徽医科大学2019-09-02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。