CN113092774B - mtEF4蛋白作为毛发生长和脱发的生物标志物的用途 - Google Patents
mtEF4蛋白作为毛发生长和脱发的生物标志物的用途 Download PDFInfo
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Abstract
本公开涉及角质形成细胞中mtEF4蛋白作为毛发生长和脱发的生物标志物的用途,以及使细胞中的mtEF4蛋白编码基因过表达的试剂在制备用于促进毛发生长的药物中的用途。
Description
技术领域
本公开涉及分子生物学领域。具体涉及角质形成细胞中mtEF4蛋白作为毛发生长和脱发的生物标志物的用途,以及使细胞中的mtEF4蛋白编码基因过表达的试剂在制备用于促进毛发生长的药物中的用途。
背景技术
线粒体是一种复杂的细胞器,由双层膜包裹。内膜向内凸起形成嵴,嵴内部蛋白含量十分丰富。线粒体嵴是氧化磷酸化的主要发生部位。电子沿传递链传递的时候伴随有质子梯度的形成,最终形成H2O及ATP。线粒体基质中含有其自身的遗传系统,人线粒体DNA是长16569bp的环形分子,编码2种rRNA,22种tRNA及13种蛋白,参与呼吸链复合物亚基组装(整个呼吸链亚基共67种蛋白)(Craven等人,2017)。
线粒体核糖体起源于变形杆菌,在进化过程中,某些核糖体RNA(Ribosome RNA,rRNA)丢失,而蛋白种类增加(Englmeier等人,2017)。线粒体核糖体蛋白可能代替了部分丢失了的rRNA的功能。约50%的线粒体核糖体蛋白,无法从序列上找到同源细菌核糖体蛋白。所有的线粒体核糖体蛋白都是细胞核DNA编码的,且其线粒体定位信号多位于氨基酸序列N端。16S rRNA与MRPL蛋白组装形成线粒体核糖体大亚基;12S rRNA与MRPS蛋白组装形成线粒体核糖体小亚基;成熟的SSU再与LSU组装形成功能性线粒体核糖体。结构数据显示,线粒体核糖体rRNA几乎完全被蛋白所包裹。与大肠杆菌核糖体(分子量2.3MDa)相比,线粒体核糖体分子量更大,但其沉降系数却较小,这是因为线粒体核糖体蛋白之间的空隙比较大,结构更加松散导致其沉降系数变小(Greber and Ban,2016)。
mRNA的编码信息可以被翻译成蛋白质,这项工作由大分子机器——核糖体进行的。核糖体一次读取一个密码子(三个碱基)的信息,核糖体本质上是一种酶,通过识别每个密码子的tRNA的作用将其翻译成蛋白质以插入适当的氨基酸,tRNA实际上是翻译过程中的“核心翻译官”。整个翻译过程可以被分为四部分:起始、延伸、终止及再循环。虽然翻译核心步骤在真核、细菌及原生动物之间高度保守,但在具体细节上有所不同。最早期阶段,许多真核翻译起始因子指导80S核糖体在AUG起始位点组装,之后起始氨酰tRNA结合在p位点。翻译起始之后迅速进入延伸过程,标志是80S核糖体定位在AUG起始密码子处,P位点有一个甲酰甲硫氨酸氨酰tRNA(N-formylmethionyl transfer RNA,fMet-tRNA,也叫Met-tRNAi)。在延伸阶段,80S核糖体逐渐沿着mRNA移动,每一步前进三个碱基,利用氨酰tRNA与真核延伸因子(eukaryotic elongation factors,eEFs)的相互作用一次合成一个氨基酸。在这一过程中,tRNA一次沿着核糖体内部的三个位点移动,这三个位点由大亚基(60S)及小亚基(40S)共同包裹形成,即:A、P、E位点。在开放阅读框的末端(open reading frame,ORF),核糖体移至终止密码子,这些终止密码子被真核多肽链释放因子(eukaryotic releasefactors,eRFs)识别,引起新生肽链从肽基-tRNA上释放,离开核糖体。最后是再循环步骤,终止翻译之后的80S核糖体复合物被ABCE1(ATP-binding cassette sub-family E member1)识别,最终解离成40S和60S亚基,进入新一轮翻译(Tahmasebi等人,2018)。
脱发是一种常见皮肤疾病,其特征是毛囊干细胞分化异常。线粒体参与毛囊干细胞分化调控,脱发的发生与线粒体密切相关(Garcin等人,2016;Lemasters等人,2017;等人,2016;Tang等人,2016;Vidali等人,2016)。此外,随着年龄的增加,眉毛及胡须等其它特征性体表毛发也会脱落,对人的心理造成一定压力,容易引发精神紧张及其它健康问题。
mtEF4是线粒体翻译延伸因子,mtEF4蛋白是所有已知的基因组中最保守的蛋白,在原核生物中,它可以与正在进行蛋白延伸过程中的核糖体结合引发tRNA的反向转位。同时,研究发现mtEF4还具有GTP水解酶活性和分子伴侣活性,降低了蛋白翻译的出错率。在酵母中的研究发现mtEF4在应激条件下能够影响线粒体的蛋白合成,但是在人源化细胞中对mtEF4功能的研究还未见报道。
由上可知,mtEF4蛋白在人类细胞中发挥重要功能,其亚细胞定位为线粒体内膜。
发明内容
本公开的目的在于提供哺乳动物毛发生长和脱发的生物标志物mtEF4。mtEF4其是线粒体翻译延伸因子,它可以使核糖体在翻译蛋白时,遇到错配的碱基及时倒退重新合成,避免产生错误的多肽链。发明人构建了在表皮细胞中mtEF4基因敲除鼠,观察对该基因敲除对小鼠毛囊生长的影响,根据皮肤外观观察结果显示,mtEF4基因敲除鼠皮肤毛干脱落,皮肤裸露,毛囊处于静止期。另外构建了小鼠mtEF4蛋白的过表达AAV病毒,结果表明mtEF4过表达可以显著增加毛囊生长期的比例。因此,mtEF4可以作为哺乳动物毛发生长和脱发的生物标志物。
本公开的上述目的是通过下面的技术方案得以实现的:
在一个方面,本公开提供mtEF4蛋白的表达量检测试剂在制备用于诊断所述受试者的毛发生长和脱发的产品中的用途,其中上述受试者是哺乳动物,例如人或小鼠,优选地,检测的对象为所述受试者的角质形成细胞。人mtEF4蛋白的氨基酸序列如SEQ ID No.1所示。
在一个实施方案中,所述人mtEF4蛋白的核苷酸编码序列如SEQ ID No.2所示。
在一个实施方案中,所述表达量检测试剂是mtEF4蛋白的特异性抗体。
在一个实施方案中,所述表达量检测试剂包含mtEF4蛋白的核苷酸编码序列的特异性引物。在一个实施方案中,当所述mtEF4蛋白是人或鼠源的,所述特异性引物的序列为:SEQ ID No.3和SEQ ID No.4。
本领域技术人员已知,人或鼠mtEF4蛋白的核苷酸编码序列的特异性引物记载在NCBI数据库中,https://www.ncbi.nlm.nih.gov/nuccore/NM_001310631.1中,能够通用。
在一个实施方案中,所述mtEF4蛋白的表达量的升高指示受试者的毛发生长。
在一个实施方案中,所述mtEF4蛋白的表达量的降低指示受试者的脱发的风险。
在另一个方面,本公开提供使mtEF4蛋白编码基因过表达的试剂在制备用于促进受试者的毛发生长的药物中的用途,所述受试者是哺乳动物,例如人或小鼠。
使mtEF4蛋白编码基因过表达的试剂,为本领域中常用的使目的基因过表达的试剂,其包含针对所述mtEF4蛋白编码基因的过表达载体,其中用于包装所述过表达载体的载体是慢病毒或逆转录病毒,例如腺相关病毒,优选血清型AAV2的腺相关病毒。
附图说明
图1:人及小鼠皮肤角质形成细胞mtEF4表达和定位情况,其中箭头所指的荧光颜色为红色,该红色代表mtEF4蛋白。
图1a显示在人皮肤毛囊角质形成细胞中mtEF4的免疫荧光染色结果图。
图1b显示针对mtEF4的小鼠皮肤整个毛囊结构的组织的免疫荧光染色结果图。
图2:表皮细胞mtEF4敲除对小鼠毛囊生长周期的影响图。
图2a显示敲除表皮细胞中的mtEF4基因的敲除鼠的毛囊生长周期变化图。
图2b显示7周龄表皮细胞mtEF4敲除鼠和野生型小鼠的皮肤外观观察结果图。
图2c显示7周龄表皮细胞mtEF4敲除鼠和野生型小鼠在拔毛后0天和8天的皮肤观察结果图以及拔毛后8天针对毛囊形态的HE染色图。
图3:mtEF4的过表达对毛囊生长的影响。
图4:小鼠皮肤中mtEF4基因的过表达效率检测。
图4a显示瞬转腺相关病毒包装的过表达mtEF4基因载体后,在小鼠的表皮细胞中mtEF4基因表达的总mRNA表达量。
图4b显示瞬转腺相关病毒包装的过表达mtEF4基因载体后,在小鼠的表皮细胞中mtEF4基因表达的总蛋白表达量。
图5:mtEF4蛋白在毛囊组织中的亚细胞定位图。
图5a:mtEF4蛋白在毛囊组织中的亚细胞定位的免疫荧光合并(merge)图。蓝色信号指示为细胞核,实线箭头所指的颜色为红色,代表mtEF4,点划线箭头所指的颜色为绿色,代表Tomm20,为线粒体外膜蛋白,可确定线粒体大致位置,图中点虚线箭头所指的颜色为黄色,其为红色的mtEF4和绿色的线粒体外膜蛋白合并得到的。
图5b表示mtEF4在毛囊形态结构中定位的卡通图。毛囊的结构大致可分为外根鞘(ORS)和内根鞘(IRS)两部分,其中用灰色表示外根鞘部分。
图6:mtEF4过表达对线粒体呼吸链复合物功能的影响。
图6a为在mtEF4过表达的情况下线粒体蛋白的SDS PAGE胶图。
图6b表示mtEF4过表达对线粒体呼吸链复合物I、II、IV和V的活性的影响。
具体实施方式
下面结合实施例进一步阐述本发明,但并不以此限定本发明保护范围。尽管参照实施例对本发明做了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。实施例中未注明具体条件的实验方法,通常按照常规条件,例如按照常规条件,或按照制造厂商所建议的条件。
实施例1:人及小鼠皮肤角质形成细胞mtEF4表达和定位
1对象和方法
1.1样本来源
在取得健康的提供毛发者的知情同意后,收集其人头部毛囊,从中分离并培养人原代皮肤毛囊角质形成细胞
C57BL/6小鼠购自维通利华
1.2主要的仪器和设备
共聚焦显微镜(Nikon)
结构照明显微镜(DeltaVision OMX 3D)
1.3主要实验试剂
一抗:抗人或鼠的兔抗Guf1(EF4)抗体(Abcam,ab171161)
二抗:Alexa Fluor 488偶联驴抗兔抗体(Invitrogen,W10811)
1.4分离人原代皮肤毛囊角质形成细胞
(1)通过拔出法获得本实施例1.1中所述的人头部毛囊,剪掉毛干部分,放入培养皿,用眼科镊轻轻剥除毛乳头及毛干前体细胞,留下毛囊外根鞘部分。
(2)将毛囊外根鞘部分置于DMEM培养基中培养,培养基成分:DMEM高糖+10%胎牛血清+1%双抗,培养条件:37℃,5%CO2。
(3)培养5-10天后,人原代皮肤毛囊角质形成细胞从组织块中爬向细胞培养皿表面,成为贴壁生长细胞,正常传代4-6次期间不进行细胞冻存。
1.5细胞免疫荧光实验
(1)将10mm直径的细胞爬片置于六孔板中,加入2ml培养基后,将1x 103个本实施例1.4得到人原代皮肤毛囊角质形成细胞接种于六孔板中,37℃,5%CO2条件中培养16h后。
(2)取出六孔板,吸走培养基,加入新鲜配制的4%多聚甲醛,室温固定5min,PBS(pH7.2)洗涤3次,每次5min。
(3)加入封闭液,封闭液配方:5ml驴血清+95ml PBS(pH7.2),室温封闭30min后,PBS(pH7.2)洗涤3次,每次5min。
(4)加入一抗(兔抗Guf1(EF4)抗体),1∶100稀释,稀释液成分:PBS(pH7.2),4℃孵育过夜。第二天取出样品,PBS(pH7.2)洗涤3次,每次5min。
(5)加入二抗(Alexa Fluor 488偶联驴抗兔抗体),1∶100稀释,稀释液成分:PBS(pH7.2),4℃孵育4h,PBS(pH7.2)洗涤3次,每次5min,抗荧光淬灭封片剂封片,荧光显微镜下观察并拍照。
1.6小鼠背部皮肤取材及切片实验:
(1)随机选取出生后32天C57BL/6小鼠(从维通利华购买)40只,1%戊巴比妥钠麻醉后,断颈法处死小鼠,用眼科镊剪掉小鼠背部毛干后,剪下小鼠背部皮肤,于4%多聚甲醛中固定过夜。
(2)取出固定后的小鼠背部皮肤,经酒精梯度脱水,酒精梯度设置:50%-75%-85%-95%-100%,每个梯度2h。
(3)之后进行二甲苯透明,共透明2次,每次15min。石蜡渗透4h后,石蜡包埋及切片,得到石蜡切片。
1.7组织免疫荧光
(1)将本实施例1.6中得到的石蜡切片置于二甲苯中脱蜡30min。
(2)酒精梯度复水(100%-95%-85%-75%-50%)至蒸馏水,加入抗原修复液(北京百灵克生物科技有限责任公司),经微波炉加热沸腾7min后冷却至室温。
(3)PBS(pH 7.2)洗3次后,用5%BSA封闭液于室温封闭30min。
(4)一抗(兔抗Guf1(EF4)抗体)4℃孵育过夜。
(5)PBS(pH 7.2)洗涤3次,于4℃孵育二抗(Alexa Fluor 488偶联驴抗兔抗体)4h。
(6)DAPI染色10min,PBS(pH 7.2)洗涤3次后,抗荧光淬灭封片剂(北京兰博利德商贸有限公司)封片。
(7)利用Nikon激光共聚焦显微镜观察并拍照。
2.结果
2.1免疫荧光实验结果
利用人皮肤毛囊角质形成细胞的免疫荧光实验(如图1a)可见红色代表的mtEF4蛋白在人皮肤毛囊角质形成细胞中有较高水平的蛋白表达。其次,我们利用动物实验观察了mtEF4在小鼠皮肤毛囊中的表达及定位情况(如图1b),对小鼠整个毛囊结构的组织免疫荧光染色,红色代表mtEF4蛋白的表达,结果显示,mtEF4在小鼠毛囊中,特异性高表达于毛囊外根鞘细胞。
实施例2:mtEF4敲除对小鼠毛囊生长的影响
1对象和方法
1.1样本来源:
mtEF4f1/f1小鼠购自南京大学模式动物研究所
Krt14-CreERT-2小鼠购自维通利华
1.3主要仪器设备
普通体视镜:Olympus公司
倒置荧光显微镜:Zeiss公司
1.4主要的实验试剂
1%戊巴比妥钠(西陇化工)
苏木素(碧云天生物技术研究所)
二甲苯(西陇化工)
酒精(西陇化工)
1.5实验方法
1.5.1敲除表皮细胞mtEF4基因的敲除鼠构建
(1)委托南京大学模式动物研究所针对mtEF4基因生成嵌合体小鼠mtEF4f1/fl小鼠。
(2)将所述mtEF4fl/fl小鼠与Krt14-CreER-2小鼠交配,得到能够敲除表皮细胞mtEF4基因的敲除鼠。因Cre是一种位点特异性的DNA重组酶,能特异识别loxp位点,介导loxp位点间的序列删除或重组,使mtEF4fl/f1小鼠与Krt14-CreERT-2小鼠杂交后得到的后代可得到敲除表皮细胞mtEF4基因的敲除鼠,以C57BL/6小鼠为对照小鼠。
(3)通过对上述能够敲除表皮细胞mtEF4基因的敲除鼠进行腹腔注射Tamoxifen,诱导Cre酶进入细胞核,介导loxp位点间的mtEF4基因敲除。得到已敲除表皮细胞mtEF4基因的Krt14-CreERT-2/mtEF4f1/fl小鼠(在本文中简称为敲除鼠)。
1.5.2通过石蜡切片和HE染色的方法,每天观察出生后C57BL/6小鼠及本实施例1.5.1(3)得到的敲除表皮细胞mtEF4基因的敲除鼠的背部毛囊生长周期变化,结果如图5a。
1.5.3将C57BL/6小鼠及本实施例1.5.1(3)培养至7周龄,观察已敲除表皮细胞mtEF4基因的敲除鼠的皮肤外观,结果如图5b。
1.5.4小鼠拔毛
(1)将松香:蜂蜡按体积1∶1混合后,置于金属杯中,于电磁炉中加热至混合物融化。
(2)用1%戊巴比妥钠将7周龄的C57BL/6小鼠及本实施例1.5.3得到的敲除鼠麻醉,用1ml移液枪枪尖涂抹松香和蜂蜡混合物至小鼠背部皮肤上,待凝固后,拔除毛干,分别于拔毛后的0天及8天后观察小鼠背部皮肤特征变化,结果如图2c所示。
1.5.5HE染色
(1)依次对本实施例中1.5.4得到的拔毛8天后的小鼠背部皮肤组织进行取材、固定、脱水、浸蜡、包埋、切片操作。
(2)取出背皮切片,二甲苯脱蜡。
(3)酒精梯度复水。
(4)蒸馏水洗涤两次后,经苏木素染色1min,自来水洗净后,用伊红染色液染色5s,自来水洗净后晾干并封片,病理显微镜下观察拍照。
2.结果
我们构建了敲除表皮细胞mtEF4基因的敲除鼠,并观察小鼠毛囊周期变化(图2a),图2a显示了敲除表皮细胞mtEF4基因的敲除鼠的毛囊生长周期包括毛囊生长期(从出生那天算起敲除鼠是第0-15天,WT是第0-17天),毛囊凋落期(从出生那天算起敲除鼠是第15-18天,WT为第17-19天)和毛囊静止期(从出生那天算起敲除鼠是第18-52天,WT为第19-29天),图中P代表天数。发现表皮细胞mtEF4敲除导致毛囊出生后生长周期延长,推迟进入下一轮毛囊生长。
另外,敲除表皮细胞mtEF4基因的敲除鼠的皮肤外观观察结果显示(图2b),mtEF4基因敲除鼠皮肤毛干脱落,皮肤裸露,而野生型对照小鼠皮肤覆盖毛被。
通过对7周龄小鼠进行背部皮肤毛发拔除,诱导毛囊进入下一轮生长期。结果显示(图2c),拔毛后8天的野生型小鼠背部皮肤呈明显的黑色,而mtEF4敲除鼠的拔毛模型并未诱导毛发生长。HE染色结果显示,拔毛后8天,野生型小鼠背皮毛囊已经进入生长期IV期(毛乳头完全被毛母质细胞包裹),而mtEF4基因敲除鼠的背皮毛囊始终处于静止期。
实施例3:mtEF4的过表达对毛囊生长的影响
1对象和方法
1.1样本来源
C57BL/6小鼠(来自南京生物医药研究院)
1.3主要仪器设备
普通体视镜:Olympus公司
荧光体视镜和倒置荧光显微镜:Zeiss公司
显微注射系统:差分干涉相差(DIC)显微镜(Leica公司,Eppendof公司),微量加压注射器(Eppendof公司),电极拉制仪(日本Narishige公司)等仪器组成。
1.4主要的实验试剂
腺相关病毒质粒构建和腺相关病毒包装均由吉玛公司完成
129BAC(bMQ-420K22)克隆(购自Welcome Trust Sanger Institute)
1%戊巴比妥钠(西陇化工)
T4 DNA连接酶(NEB)
Taq PCR Mastermix(KT201)(TIANGEN)
Trizoal reagent(Invitrogen)
蛋白酶抑制剂(Roche)
甘氨酸(生工)
感受态细胞(TaKaRa)
核酸marker(TianGen)
1.5实验方法
1.5.1构建mtEF4过表达腺相关病毒
(1)采用血清型AAV2的腺相关病毒,首先将小鼠mtEF4全长cDNA序列(是根据所述的小鼠mtEF4蛋白编码基因的序列(如SEQ ID No.6所示)(其编码的蛋白序列如SEQ IDNo.5所示)通过转录和逆转录合成的cDNA序列)构建至血清型AAV2的腺相关病毒的骨架质粒。
(2)之后分别将带有小鼠mtEF4全长cDNA序列的骨架质粒、AAV helper质粒、Adhepler质粒共转染293T细胞,得到mtEF4过表达AAV病毒,以不带有小鼠mtEF4全长cDNA序列的血清型AAV2的腺相关病毒骨架质粒、AAV helper质粒、Ad hepler质粒共转染293T细胞,得到对照AAV病毒。
1.5.2AAV病毒皮内注射
(1)随机取40只7周龄野生型C57BL/6小鼠,1%戊巴比妥钠麻醉后,用眼科剪移除背皮毛干,用1ml胰岛素注射器向小鼠皮内注射1×106U本实施例中1.5.1中得到的mtEF4过表达AAV病毒及对照AAV病毒,得到mtEF4过表达小鼠和对照组小鼠,饲养0、5、10、20天。
1.5.3取材及数据统计
(1)将本实施例中1.5.2中得到的mtEF4过表达小鼠和对照组小鼠麻醉并处死,背皮取材后进行石蜡切片实验。
(2)对切片进行脱蜡复水及HE染色处理。
(3)拍照并统计生长期毛囊比例。
1.6统计学分析
结果采用的是prism 8.0软件进行分析。将第10天和第20天的过表达组与各自的对照组的数据比较,比较采用t检验,***P<0.001,*P<0.05为差异有统计学意义。
2.结果
通过向小鼠皮内注射对照AAV病毒及mtEF4蛋白的过表达AAV病毒,我们发现mtEF4过表达可以显著增加生长期毛囊的比例(图3)。
实施例4:小鼠皮肤中mtEF4基因的过表达效率检测
1对象和方法
样本来源
C57BL/6小鼠(来自南京生物医药研究院)
1.3主要仪器设备
普通体视镜:Olympus公司
ABI StepOne Plus实时PCR系统(Applied Biosystems)
1.4主要的实验试剂
TRIzol试剂(Invitrogen)
oligo(dT)(Promega)
SYBR Green Master Mix(Takara)
内标抗体:ACBT抗体(Santa Cruz,sc-17764)
一抗:抗人或鼠的兔抗Guf1(EF4)抗体(Abcam,ab171161)
二抗:Alexa Fluor 488偶联驴抗兔抗体(Invitrogen,W10811)
蛋白酶抑制剂(Roche)
甘氨酸(生工)
抗体稀释液(东洋纺)
1.5引物设计
1.6实验方法
1.6.1总RNA的提取
取背皮毛囊处于静止期的7周龄野生型C57BL/6小鼠,以1×106U的剂量皮下注射实施例3中1.5.1步骤得到的mtEF4过表达腺相关病毒,分别于注射后0周、1周、2周及3周取小鼠的背部皮肤,液氮研磨后用Trizol提取法提取背部皮肤组织中的总RNA,测总RNA的浓度,OD值是1.8-2.0表示提取的RNA纯度较高可作为逆转录实验的模板。提取的RNA可放于-80℃保存。
1.6.2cDNA的合成
使用TAKARA试剂盒进行逆转录合成cDNA。全程使用无RNA酶的枪头,反应在冰上进行。首先去除基因组DNA,再进行逆转录反应形成mtEF4的cDNA。
逆转录反应体系和步骤如下:
37℃ 15min(RT反应)
85℃ 5sec(反转录酶的失活反应)
4℃ 保持
1.6.3实时荧光定量PCR
配置好反应体系后,按下列的方法进行qPCR反应:
荧光定量PCR反应参数设置:
1.6.4PCR数据处理
PCR的扩增结果用CT值来表示,CT值的含义是PCR反应液中荧光信号达到所设定的阈值时的循环数。样品目的基因的相对表达率(RQ)采用△△CT方法计算,
RQ=2ΔΔCT(CT表示反应的实时荧光强度显著大于背景值时的循环数)。
1.7统计学分析
采用Prism 8.0软件进行数据处理,计量资料以(x±s)表示,第3周实验组和0周的实验组比较采用t检验。***P<0.001为差异有统计学意义。
1.8western blot检测小鼠皮肤中mtEF4基因的过表达效率
1.8.1总蛋白的提取
取背皮毛囊处于静止期的7周龄野生型C57BL/6小鼠,皮下注射实施例3中1.5.1步骤得到的mtEF4过表达腺相关病毒,分别于注射后0周、1周、2周及3周取小鼠的背部皮肤,液氮研磨后提取背部皮肤组织中的总蛋白,最后放置于1.5ml的EP管中。蛋白裂解液为:RIPA∶蛋白酶抑制剂=100∶1。
1.8.2BCA法测蛋白浓度
使用BCA蛋白浓度测定试剂盒(购自购自碧云天),先配好A和B混合液体积比是50∶1,按照每孔100μl的体积加入到96孔板中,每个样品设置3个复孔,每孔分别加蛋白质标准品(0μg/μl、0.5μg/μl、0.75μg/μl、1μg/μl、1.5μg/μl、2μg/μl、3μg/μl、4μg/μl)和样品每孔2μl,用排枪加样,盖上盖子,放在摇床上摇晃30min,设置37℃ 300rpm。然后酶标仪选定波长630nm测定每孔的光度值。
最后,根据光度值和蛋白质标准品的浓度做出标准曲线。根据标准曲线计算出样品蛋白的浓度。加入5×SDS,使其在蛋白样品中是1×,煮沸5min。放-20℃保存。
1.8.3配制浓缩胶和分离胶
分别配8%和15%的分离胶,5%的浓缩胶。
1.8.4电泳
根据标准曲线计算出样品蛋白的浓度,每个样品上样50μg电泳。
1.8.5转膜
实验室采用湿转的方式进行恒流转膜。首先,将PVDF膜和滤纸剪成与分离胶一样大小,PVDF膜在使用前用无水甲醛浸泡10min使其激活,将滤纸和海绵垫放转膜液中浸泡,然后按照(阴极-黑色)海绵垫-三层滤纸-凝胶-PVDF膜-三层滤纸-海绵垫(阳极-白色)这样的顺序安装三明治夹。用试管轻轻滚动,保证其中没有气泡,最后盖上三明治夹,装配完备后放入电泳槽,黑色对黑色,红色对红色,加入转膜液,盖上电泳槽。特别注意的是:在转膜的过程中,一定要保持低温环境,所以要将电泳槽放至转满冰的的冰盒中。设置参数是在恒流的情况下,250mA转膜90min。
1.8.6孵育一抗
转膜结束后,将PVDF膜用ddH20洗两遍,将其放到丽春红溶液中,这时会看到条带,然后用1×TBST洗2遍放到5%的牛奶中封闭。放摇床中摇晃2h后,用1×TBST洗三遍,每次3min。然后对照Maker的相对分子质量所在的位置,切下目的条带,分别放在标记好的槽中,加入目的蛋白一抗,一抗配制:将内标ACTB抗体(Abcam)以1∶10000的比例稀释在TBST中;将能标记mtEF4的抗体抗人或鼠兔抗Guf1(EF4)抗体以1∶1000的比例稀释在TBST中。4℃过夜孵育带有目的条带的PVDF膜。
1.8.7孵育二抗
敷完一抗,用1×TBST洗三遍,每次5min,取出一抗孵育过的PVDF膜放到干净的烘干的孵育槽中,加入二抗,二抗配制:将二抗Alexa Fluor 488偶联驴抗兔抗体和抗体稀释液按照1∶1000的比例稀释,室温1h放摇床孵育2h。
1.8.8显影
二抗孵育完后,用1×TBST洗三次,每次5min。将膜取出放到黑板上,加入发光液(1mL发光液+3μlH2O2),反应2min后放入Bio-Rad ChemiDoc XRS+化学发光成像系统中,保存拍下照片。
2结果
我们首先向7周龄野生型小鼠背部皮肤内注射mtEF4过表达腺相关病毒,分别于注射后0周、1周、2周及3周取材,提取背部皮肤组织中的总RNA和总蛋白进行半定量分析,采用ACTB作为内标,经qRT-PCR实验结果表明(图4a)在注射后3周,mtEF4过表达小鼠背部皮肤内的mtEF4的mRNA拷贝数水平较高。Western-blot实验结果表明(图4b)在注射后2-3周,mtEF4过表达小鼠背部皮肤内的mtEF4蛋白表达较高,并且变化趋势与(图4a)中的mRNA相一致。
实施例5:mtEF4蛋白在毛囊细胞中的亚细胞定位
1对象和方法
1.1样本来源
C57BL/6小鼠(来自南京生物医药研究院)
1.2主要的仪器和设备
普通体视镜:Olympus公司
荧光体视镜和倒置荧光显微镜:Zeiss公司
激光共聚焦显微镜:Olympus公司,型号FV500或FV1000
主要的实验试剂
小鼠抗Tom20抗体(Santa Cruz,sc-17764)
抗人或鼠的兔抗Guf1(EF4)抗体(Abcam,ab171161)
Alexa Fluor 488偶联驴抗兔抗体(Invitrogen,W10811)
Alexa Fluor 555偶联驴抗小鼠抗体(Invitrogen,W10810)
蛋白酶抑制剂(Roche)
甘氨酸(生工)
抗体稀释液(东洋纺)
1.4小鼠背部皮肤取材及切片实验:
(1)取3只10周龄野生型C57BL/6小鼠,1%戊巴比妥钠麻醉后,断颈法处死小鼠,用眼科镊剪掉小鼠背部毛干后,剪下小鼠背部皮肤,于4%多聚甲醛中固定过夜。
(2)取出固定后的小鼠背部皮肤,经酒精梯度脱水,酒精梯度设置:50%-75%-85%-95%-100%,每个梯度2h。
(3)之后进行二甲苯透明,共透明2次,每次15min。石蜡渗透4h后,石蜡包埋及切片,得到石蜡切片。
1.6组织免疫荧光
(1)将石蜡切片置于二甲苯中脱蜡30min。
(2)酒精梯度复水(100%-95%-85%-75%-50%)至蒸馏水,加入抗原修复液,经微波炉加热沸腾7min后冷却至室温。
(3)PBS(pH 7.2)洗3次后,用5%BSA封闭液于室温封闭30min。
(4)一抗选择兔抗Guf1(EF4)抗体和小鼠抗Tom20抗体两种,其中Tom20为内参,一抗4℃孵育过夜。
(5)PBS(pH 7.2)洗涤3次,二抗选择Alexa Fluor 488偶联驴抗兔抗体(H+L)和Alexa Fluor 555偶联驴抗小鼠抗体(H+L)两种,于4℃孵育二抗4h。
(6)细胞核用hochest 33342进行染色,染色10min,PBS(pH 7.2)洗涤3次后,抗荧光淬灭封片剂封片。
(7)利用Nikon激光共聚焦显微镜观察并拍照。
2.结果
毛囊由外到内可大致分为外根鞘(Outer root sheath,ORS)和内根鞘(Innerroot sheath,IRS)两部分包裹着毛干(hair shaft,HS),毛囊外部是连接组织鞘(connective tissue sheath,CTS)我们通过免疫荧光技术观察了小鼠背部皮肤的mtEF4定位情况(图5a),蓝色信号指示为细胞核,实线箭头所指的颜色为红色,代表红色信号为mtEF4,点划线箭头所指的颜色为绿色,代表Tomm20,为线粒体外膜蛋白,可确定线粒体大致位置,图中点虚线箭头所指的颜色为黄色,其为红色的mtEF4和绿色的线粒体外膜蛋白合并得到的,结果表明,野生型小鼠mtEF4定位于毛囊外根鞘的线粒体中。图5b是mtEF4在毛囊形态结构中定位的卡通图,灰色表示毛囊的外根鞘位置,即mtEF4蛋白主要位于毛囊的外根鞘。
实施例6:mtEF4蛋白对线粒体呼吸链复合物功能的影响
1对象和方法
1.1材料
7周龄的C57BL/6小鼠购于北京维通利华实验动物技术有限公司
1.2主要仪器设备
普通体视镜:Olympus公司
主要的实验试剂
线粒体提取试剂盒(Abcam);
氯化硝基四氮唑(西陇化工)
烟酰胺腺嘌呤二核苷酸(西陇化工)
小鼠抗Tom20抗体(Santa Cruz,sc-17764)
兔抗Guf1(EF4)抗体(Abcam,ab171161)
1.4实验方法
1.4.1向7周龄的C57BL/6小鼠背部皮肤分别注射1X106U实施例3构建的mtEF4过表达腺相关病毒或对照AAV病毒,在1周时间后取小鼠背部皮肤组织样本用于分离线粒体呼吸链复合物。分离线粒体呼吸链复合物。
(1)使用线粒体提取试剂盒提取组织线粒体:
a、取50-100mg新鲜的本实施例1.4.1得到的组织样本,用PBS洗涤干净。
b、用剪刀尽可能剪碎,用4℃PBS洗涤两次。
c、加入400μl 4℃试剂A,置冰上10分钟。
d、用Dounce匀浆器匀浆30-40下,然后在4℃,500g的条件下离心5分钟。
e、快速将上清吸入另一个4℃预冷的干净的离心管。
f、在4℃,11000g的条件下离心20分钟。
g、在沉淀中加入400μl 4℃试剂B,混匀。
h、在4℃,11000g的条件以上的条件下离心20分钟。
i、弃上清,沉淀用线粒体保存液重悬,既得到线粒体样品。
j、置冰箱备用或直接用于下游实验。
(2)收集(1)中用线粒体提取试剂盒提取的400μg线粒体,加入40μl缓冲液A(50mM氯化钠,50mM咪唑/盐酸,1mM EDTA,pH 7.0)重悬。分别分为Null组(空白对照组)和OE组(实验组,也即mtEF4过表达组)。
(3)每管加入12μl digitonin,混匀冰上作用10分钟。
(4)100000g,4度离心30分钟,取上清,加入5μl甘油和6μl考马斯亮蓝。
(5)取40μl样品进行SDS PAGE(确定分离线粒体呼吸链复合物的存在及表达量)以及Blue-native PAGE(测定各线粒体呼吸链复合物的活性)。
1.4.3线粒体呼吸链复合物活性检测
线粒体呼吸链酶也称为线粒体呼吸链复合物或线粒体呼吸链复合酶。线粒体呼吸链位于线粒体内膜上,由5个复合物组成,分别为:NADH酶(也称为复合物I)、琥珀酸氧化还原酶(也称为复合物II)、细胞色素C氧化还原酶(复合物III)、细胞色素C氧化酶(也称为复合物IV)和ATP合成酶(也称为复合物V)。
(1)复合物I活性:将本实施例1.4.2(5)中得到的Blue-native PAGE分离胶孵育在50mM Tris-Hcl,pH 7.4缓冲液中(含有0.5mM NBT(Nitroblue tetrazolium chloride,氯化硝基四氮唑),5mM NADH(Nicotinamide adenine dinucleotide,烟酰胺腺嘌呤二核苷酸)),室温作用1小时。
(2)复合物II活性:将本实施例1.4.2(5)中得到的Blue-native PAGE分离胶孵育在50mM Tris-Hcl,pH 7.4缓冲液中(含有4.5mM EDTA,10mM KCN(氰化钾),0.2mMPMS(Methylphenaziniummethylsulfate,吩嗪甲基硫酸盐),84mM琥珀酸,50mM NBT),室温作用1小时。
(3)复合物IV活性:将本实施例1.4.2(5)中得到的Blue-native PAGE分离胶孵育在50mM Tris-Hcl,pH 7.4缓冲液中(含有0.1%二氨基联苯胺,24个单位/ml过氧化氢酶,0.1%细胞色素C),37度作用3-6小时。
(4)复合物V活性:将本实施例1.4.2(5)中得到的Blue-native PAGE分离胶用清水漂洗10分钟,放入0.1M的甘氨酸缓冲液中(pH8.6)作用1小时。再将分离胶放入含有下列成分的缓冲液中:35mM Tris碱,270mM甘氨酸,14mM硫酸镁,5mMATP,0.2%硝酸银,37度作用3-6小时。
2结果
SDS PAGE胶图显示了mtEF4过表达对各个线粒体呼吸链复合物的表达量几乎没有影响(图6a)。
图6b表示mtEF4过表达对线粒体呼吸链复合物I、II、IV和V的活性的影响:在内参Tom20表达量一致的前提下,与对照组(Null)相比,实验组(OE)中NAGHDHase的条带变深变粗,表示过表达mtEF4升高了线粒体的NAGHDHase的活性,实验组(OE)中Cytc Oxidase蛋白分子量增加,说明了过表达mtEF4使得Cytc Oxidase的组装得更好,蛋白稳定性变好,进一步提高了其活性。这些都表明mtEF4基因过表达,提高了线粒体呼吸链复合物的活性,进一步增强线粒体代谢能力。
参考文献:
1.Craven,L.,C.L.Alston,R.W.Taylor,and D.M.Tumbull.2017.RecentAdvances in Mitochondrial Disease.Annual Review of Genomics and HumanGenetics.18:257-275.
2.Englmeier,R.,S.Pfeffer,and F.2017.Structure of the HumanMitochondrial Ribosome Studied<em>In Situ</em>by CryoelectronTomography.Structure(London,England:1993).25:1574-1581.e1572.
3.Garcin,C.L.,D.M.Ansell,D.J.Headon,R.Paus,and M.J.Hardman.2016.HairFollicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re-epithelialization.Stem cells(Dayton,Ohio).34:1377-1385.
4.Greber,B.J.,and N.Ban.2016.Structure and Function oftheMitochondrial Ribosome.Annual review of biochemistry.85:103-132.
5.Lemasters,J.J.,V.K.Ramshesh,G.L.Lovelace,J.Lim,G.D.Wright,D.Harland,and T.L.Dawson,Jr.2017.Compartmentation of Mitochondrial andOxidative Metabolism in Growing Hair Follicles:A Ring of Fire.The Journal ofinvestigative dermatology.
6.Minjuan,W.,X.Jun,S.Shiyun,X.Sha,N.Haitao,and W.Yue.2016.HairFollicle Morphogenesis in the Treatment of Mouse Full-Thickness Skin DefectsUsing Composite Human Acellular Amniotic Membrane and Adipose DerivedMesenchymal Stem Cells.Stem cells international.2016:8281235.
7.Tahmasebi,S.,A.Khoutorsky,M.B.Mathews,andN.Sonenberg.2018.Translation deregulation in human disease.Nature ReviewsMolecular Cell Biology.
8.Tang,Y.,B.Luo,Z.Deng,B.Wang,F.Liu,J.Li,W.Shi,H.Xie,X.Hu,andJ.Li.2016.Mitochondrial aerobic respiration is activated during hair folliclestem cell differentiation,and its dysfunction retards hairregeneration.PeerJ.4:e1821.
9.Vidali,S.,J.Chéret,M.Giesen,S.Haeger,M.Alam,R.E.B.Watson,A.K.Langton,M.Klinger,J.Knuever,W.Funk,B.Kofler,and R.Paus.2016.ThyroidHormones Enhance Mitochondrial Function in Human Epidermis.Journal ofInvestigative Dermatology.136:2003-2012.
Claims (12)
1.mtEF4蛋白的表达量检测试剂在制备用于诊断受试者的毛发生长和脱发的产品中的用途,其中所述受试者是人或小鼠。
2.如权利要求1所述的用途,其中检测的对象为所述受试者的角质形成细胞。
3.如权利要求1所述的用途,其中所述表达量检测试剂是mtEF4蛋白的特异性抗体。
4.如权利要求1所述的用途,其中所述表达量检测试剂包含mtEF4蛋白的核苷酸编码序列的特异性引物。
5.如权利要求4所述的用途,当所述mtEF4蛋白是人源的,其中所述特异性引物的序列为:SEQ ID No.3和SEQ ID No.4。
6.如权利要求1-5中任一项所述的用途,其中所述mtEF4蛋白的表达量的升高指示受试者的毛发生长。
7.如权利要求1-5中任一项所述的用途,其中所述mtEF4蛋白的表达量的降低指示受试者的脱发的风险。
8.使mtEF4蛋白编码基因过表达的试剂在制备用于促进受试者的毛发的生长的药物中的用途,所述受试者是小鼠。
9.如权利要求8所述的用途,其中使mtEF4蛋白编码基因过表达的试剂包含针对所述mtEF4蛋白编码基因的过表达载体。
10.如权利要求9所述的用途,其中用于包装所述过表达载体的病毒是慢病毒或逆转录病毒。
11.如权利要求9所述的用途,其中用于包装所述过表达载体的病毒是腺相关病毒。
12.如权利要求9所述的用途,其中用于包装所述过表达载体的病毒是血清型AAV2的腺相关病毒。
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Non-Patent Citations (2)
Title |
---|
Mitochondrial diseases: the contribution of organelle stress responses to pathology;Anu Suomalainen等;《nature reviews molecular cell biology》;20170809;第19卷;第77-92页 * |
Mitochondrial translation factor EF4 regulates oxidative phosphorylation complexes and the production of ROS;Guichen Li等;《Free Radical Research》;20190129;第52卷;第1250-1255页 * |
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