CN110894508B - 一种调控白色脂肪棕色化的基因Adra1a的应用 - Google Patents
一种调控白色脂肪棕色化的基因Adra1a的应用 Download PDFInfo
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Abstract
本发明提供了一种调控白色脂肪棕色化的基因Adra1a的应用。所述基因Adra1a的核苷酸序列如SEQ ID NO.2所示。本发明从细胞水平和小鼠个体水平验证了Adra1a通过抑制白色脂肪细胞的增殖和分化减少白色脂肪含量,使白色脂肪明显出现“多室”的棕色样脂肪形态特征,促进白色脂肪中棕色脂肪标记基因的表达,抑制白色脂肪标记基因的表达。揭示Adra1a基因调控白色脂肪棕色化的作用机理是通过PI3K‑AKT信号通路影响棕色脂肪标志基因Pgc1α基因的表达,进而影响白色脂肪的棕色化。本发明提供了Adra1a基因在调控白色脂肪棕色化的新用途,在制备有效缓解和治疗肥胖的药物方面具有实用价值。
Description
技术领域
本发明属于基因工程领域,特别涉及一种调控白色脂肪棕色化的基因Adra1a的应用。
背景技术
肥胖是由多因素引起的脂肪代谢性失衡症,以体内的白色脂肪细胞数量增多、体积增大,并在局部过度沉积为特点。棕色脂肪为一类与白色脂肪功能相反的脂肪,其增多有利于消耗能量,可有效缓解和治疗肥胖,大量研究证明增加棕色脂肪的有效途径是让白色脂肪棕色化,目前研究较多的影响白色脂肪棕色化的有转录因子、分泌蛋白和小RNA等,但是白色脂肪的棕色化是由多因素或多基因调控,因此挖掘调控白色脂肪棕色化的关键基因,将为研究肥胖奠定坚实的基础。本研究为了探索白色脂肪棕色化的机理,通过转录组测序筛选得到Adra1a(Adrenoceptor Alpha 1A)这一调控白色脂肪棕色化的关键基因,该基因编码肾上腺素α-1A受体,可介导心脏、神经及其他组织器官的信号传导,但目前对白色脂肪棕色化的作用机理还不清楚。
目前虽已经发现了部分调控小鼠白色脂肪棕色化的基因,但白色脂肪棕色化的研究才刚刚起步,且白色脂肪棕色化为多因素或多基因调控,因此挖掘调控白色脂肪棕色化的关键基因显的至关重要。
Adra1a基因为G蛋白偶联受体家族的成员,编码多通路跨膜蛋白。能够与肾上腺素、去肾上腺素等多种激素结合共同调节心血管功能和交感神经活动,从而调节血压。
Adra1a编码的蛋白质与肾上腺素和去甲肾上腺素结合,以介导心脏、神经和其他器官系统细胞中的信号传导。α1-肾上腺素受体广泛分布于中枢神经系统和外周神经系统,它们在平滑肌收缩中起主要作用,同时促进有丝分裂反应并调节多种细胞的生长和增殖。
但目前肾上腺素α-1A受体对白色脂肪棕色化的研究及作用机制还未见报道。
发明内容
本发明的目的是提供一种调控白色脂肪棕色化的基因Adra1a的应用。
本发明通过RNA-seq技术,分析筛选得到C57BL/6小鼠白色脂肪和棕色脂肪的差异表达基因99个。q-PCR验证得到Adra1a、Cxcr5和Galnt6三个差异表达明显的候选基因,在白色脂肪和棕色脂肪细胞中过表达和干扰候选基因,通过q-PCR和Western Blot技术在RNA水平和蛋白水平确定候选基因对白色脂肪棕色化的影响,最终筛选得到Adra1a为影响白色脂肪棕色化的重要基因。随后本发明在细胞水平证实了Adra1a基因可有效促进白色脂肪棕色化,Adra1a基因的过表达有利于白色脂肪细胞的棕色化,抑制白色脂肪细胞的增殖和分化。CRISPR/Cas9技术介导的Adra1a基因敲除抑制棕色脂肪细胞中棕色标记基因的表达,抑制棕色脂肪的增殖与分化;同时制备Adra1a基因过表达小鼠在个体水平验证Adra1a过表达能够促进白色脂肪棕色化,可减轻小鼠体重和白色脂肪重量,有利于白色脂肪棕色化,能够使白色脂肪形成“多室”棕色样脂肪,同时可以增加棕色脂肪的表达,增加棕色脂肪重量及数量。再通过RNA-seq技术分析Adra1a过表达小鼠与野生小鼠的白色脂肪和棕色脂肪中的差异表达基因,对获得的差异基因进行GO功能富集及KEGG信号通路分析,预测得到4条Adra1a相关的调控白色脂肪棕色化的信号通路。对预测得到的AKT信号通路进行体外实验验证,结果表明Adra1a激活增加AKT的磷酸化水平表达,同时Adra1a激活促进Pgc1α基因的表达,但是添加AKT抑制剂后,Adra1a激活对Pgc1α影响减弱,证实Adra1a基因依赖PI3K-AKT信号通路影响Pgc1α基因的表达,进而影响白色脂肪的棕色化。
本发明提供的Adra1a基因的编码蛋白具有如下任一种氨基酸序列:
(1)如SEQ ID NO.1所示的氨基酸序列;
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列;
(3)与如SEQ ID NO.1所示的氨基酸序列具有至少80%同源性的氨基酸序列;优选地,所述同源性为至少90%;更优选为95%;
本发明提供的Adra1a基因的CDS序列具有如下任一种核苷酸序列:
(1)如SEQ ID NO.2所示的核苷酸序列;
(2)如SEQ ID NO.2所示的核苷酸序列经一个或多个核苷酸的替换、插入或缺失得到的编码相同功能蛋白的核苷酸序列。
本发明构建Adra1a的过表达载体并转染白色脂肪细胞,Adra1a过表达促进白色脂肪细胞中Ucp1、Cidea、Fndc5等棕色脂肪标记基因的表达,抑制Serpina3k、resistin、Asc1等白色脂肪标记基因的表达,同时抑制白色脂肪细胞的增殖和分化。
本发明通过原核注射的方法得到Adra1a转基因小鼠,同时扩繁获得Adra1a转基因小鼠后代34只。标准饲喂条件下,Adra1a转基因小鼠与野生小鼠相比,体内白色脂肪含量减少,且白色脂肪明显出现“多室”的棕色样脂肪形态特征。分子生物学检测表明,Adra1a过表达小鼠白色脂肪中棕色脂肪特异基因的表达增加,白色脂肪特异基因的表达减少。Adra1a过表达小鼠与野生小鼠相比,体内棕色脂肪与白色脂肪比值显著增加且棕色脂肪含量增加,分子生物学检测表明,Adra1a过表达小鼠体内棕色脂肪中Ucp1,Prdm16和Pgc1α等基因的表达量显著提高。小鼠个体水平结果与细胞水平结果相互印证,表明Adra1a基因调控白色脂肪棕色化。
本发明构建Adra1a的CRISPR/Cas9敲除载体并转染棕色脂肪细胞,Adra1a的敲除抑制棕色脂肪细胞中Ucp1,Prdm16和Cidea等基因的表达,同时抑制棕色脂肪的增殖与分化。
第一方面,基于上述发现,本发明提供了Adra1a基因或其编码的蛋白、或Adra1a基因表达促进剂在调控白色脂肪棕色化中的应用。所述应用具体为Adra1a基因的过表达抑制了白色脂肪细胞增殖、分化。
本发明提供了Adra1a基因或其编码的蛋白、或Adra1a基因表达促进剂在降低或抑制白色脂肪标记基因表达量中的应用。具体地,所述白色脂肪标记基因包括Serpina3k、Asc1,Leptin,Fad3,resistin,SCD,FASN,SREBP1,ACC和Psat1。
进一步地,本发明提供了Adra1a基因的表达抑制剂在促进体内白色脂肪细胞数量增多、体积增大、细胞增殖、分化中的应用。
更进一步地,本发明提供了Adra1a基因的表达抑制剂在制备使体内白色脂肪细胞增多、体积增大、细胞增殖、分化的药物中的应用
本领域技术人员基于本发明能够理解,Adra1a基因或其编码的蛋白、或Adra1a基因表达促进剂在提高或促进棕色脂肪标记基因表达量中的应用也属于本发明的保护范围。具体地,所述棕色脂肪标记基因包括Ucp1、Fndc5、Prdm16、Pgc1α、Pparγ、Fabp4、Adipoq和Cidea。
本发明提供了Adra1a基因的表达抑制剂在抑制体内棕色脂肪细胞数量增多、体积增大、或在制备抑制棕色脂肪细胞增殖和分化的药物中的应用。
本发明提供了Adra1a基因或其编码的蛋白通过PI3K-AKT信号通路提高或促进Pgc1α基因表达量中的应用。
第二方面,本发明还提供了Adra1a基因或其编码的蛋白、或Adra1a基因表达促进剂在缓解或治疗肥胖中的应用。
本发明提供了Adra1a基因、或其编码的蛋白、或Adra1a基因的表达促进剂在制备缓解或治疗肥胖的药物中的应用。
本发明所述的Adra1a基因表达促进剂是指任何能使Adra1a基因表达量增多或过表达的生物或化学制品。本发明所述的Adra1a基因表达抑制剂是指任何能使Adra1a基因表达量降低或干扰其表达的生物或化学制品。
第三方面,本发明提供了一种能够缓解或治疗肥胖的药物,所述药物含有能够促进Adra1a基因表达量提高的生物制品和/或化学制品。
第四方面,本发明提供了一种靶向Adra1a基因的sgRNA,其核苷酸序列如SEQ IDNO.45或SEQ ID NO.46所示。本发明实验发现,核苷酸序列如SEQ ID NO.44所示的sgRNA1打靶Adra1a基因后使得Adra1a基因的表达量降低为对照的72%,核苷酸序列如SEQ ID NO.45所示的sgRNA2使得Adra1a基因的表达量降低为对照的59%,核苷酸序列如SEQ ID NO.46所示的sgRNA3使得Adra1a基因的表达量降低为对照的55%,可见sgRNA2和sgRNA3的打靶效率高于其他sgRNA。
含有上述sgRNA的CRISPR/Cas9打靶载体属于本发明的保护范围。
本发明的优异效果在于,本发明于本领域内首次发现一种新的调控白色脂肪棕色化的基因Adra1a,在细胞水平构建该基因的过表达载体和敲除载体,从正反两个方面验证Adra1a基因调控小鼠白色脂肪的棕色化;本发明制备了Adra1a过表达小鼠模型,在个体水平验证该Adra1a基因能够调控小鼠白色脂肪向棕色脂肪转变;本发明进一步揭示Adra1a基因调控白色脂肪棕色化的作用机理,该基因通过PI3K-AKT信号通路影响棕色脂肪标志基因Pgc1α基因的表达,进而影响白色脂肪的棕色化。本发明提供了Adra1a基因在调控白色脂肪棕色化的新用途,在制备有效缓解和治疗肥胖的药物方面具有实用价值。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,本发明实施例中所用的实验材料、试剂和仪器等均可市售获得,若未具体指明,实施例中所用的技术手段均为本领域技术人员所熟知的常规手段。
实施例1调控白色脂肪棕色化关键基因的筛选及确定
本实施例通过转录组测序预测结合体外实验验证确定Adra1a基因在白色脂肪棕色化过程中起作用。
一、RNA-seq筛选白色脂肪和棕色脂肪中差异表达基因
取5月龄的C57BL/6小鼠的白色脂肪和棕色脂肪,每种样本取两只小鼠作为重复,进行RNA提取,对检测合格的样本使用Illumina(HiSeq Xten)进行建库测序。获得的数据进行纯化分析,包括数据质控、比对组装、差异表达和功能注释。
本实验的差异基因分析分为两组,第一种对C57BL/6小鼠的白色脂肪和棕色脂肪组织进行差异基因分析(G3 VS G4);第二种是对小鼠白色脂肪和棕色脂肪细胞进行差异基因分析,包括成脂诱导后的0d,4d,6d(B0 VS W0,B4 VS W4,B6 VS W6)进行分析。通过以上两种方法最终确定出影响白色脂肪棕色化的关键基因,结果如下:
在脂肪组织层面,实验共分析出3757个差异表达基因,其中上调基因1046个,下调基因2711个;在脂肪细胞层面,实验共分为三个阶段,其中0d中分析出差异表达基因7979个,上调基因4213个,下调基因3766个,4d中差异表达基因1799个,上调基因1156个,下调基因643个;6d中差异表达基因1317个,上调基因356,下调基因961个。对获得的差异基因进行共有和特有差异基因分析,白色、棕色脂肪组织和细胞共有99个差异基因。聚类分析差异表达基因,WAT day0、WAT day4和WAT day6聚类明显,为同一类细胞;BAT day0、BAT day4和BAT day6聚类明显,为同一类细胞,同时白色脂肪组织和棕色脂肪组织差异明显,与预期结果相符。
对获得的差异基因进行GO功能注释,在白色和棕色脂肪细胞诱导0d时,差异基因富集的GO term主要包括cell,intracellular和organelle;在白色和棕色脂肪细胞诱导4d时,差异基因富集的GO term主要包括ion binding,cellular developmental process和anatomical structure morphogenesis;在白色和棕色脂肪细胞诱导6d时,差异基因富集的GO term主要包括anatomical structure morphogenesis,cellular developmentalprocess和single-organism membrane organization。其中在白色脂肪和棕色脂肪细胞发育的4d和6d均参与的过程包括cellular developmental process和anatomicalstructure morphogenesis。cellular developmental process与细胞的发育过程,anatomical structure morphogenesis与解剖结构形态发生有关,与预期相符。
KEGG信号通路富集分析显示,白色脂肪和棕色脂肪高度相关的信号通路包括“PI3K-AKT signaling pathway”和“Metabolic pathways”。其中,“PI3K-AKT signalingpathway”为细胞增殖的关键信号通路,说明白色脂肪的棕色化可能与细胞增殖相关,而“Metabolic pathways”为代谢通路,本身与脂肪代谢有着密切的关系。
二、调控白色脂肪棕色化关键基因的筛选确定
取3只2周龄C57BL/6小鼠的白色脂肪组织和棕色脂肪组织,对RNA-Seq分析获得的部分差异表达基因进行q-PCR验证。Adra1a,Galnt6和Cxcr5基因表达差异倍数最为明显,且与转录组测序结果趋势一致。预测Adra1a、Cxcr5和Galnt6基因可能对白色脂肪的棕色化起作用,将以上三个基因作为影响白色脂肪棕色化的候选基因进行后续实验。
为了进一步确定这三个基因对白色脂肪棕色化的作用,实验构建了这三个基因的过表达载体和干扰载体转染白色脂肪细胞和棕色脂肪细胞。通过q-PCR方法检测目标基因及相关标志基因在RNA水平的表达量,进而确定Adra1a、Cxcr5和Galnt6基因对白色脂肪棕色化的作用。
1、过表达Adra1a、Cxcr5和Galnt6基因对白色脂肪棕色化的影响
将成功构建的Adra1a、Cxcr5和Galnt6基因的过表达载体转染小鼠白色和棕色脂肪细胞,转染后48h收样提取RNA,反转形成cDNA,检测白色标记基因和棕色标记基因的表达量。对于白色脂肪细胞,Adra1a基因的过表达量是对照的107.70倍,棕色脂肪细胞相关的特异表达基因Ucp1,Prdm16,Pgc1α,Fndc5整体呈升高趋势,白色脂肪标记基因Asc1为对照的0.0046倍(p<0.0001)呈下降趋势。白色脂肪细胞中过表达Cxcr5基因使得Ucp1,Prdm16,Pgc1α,Fndc5和Asc1基因均有了不同程度的提高。Galnt6基因的过表达使得棕色脂肪特异表达基因Prdm16,Pgc1α和Fndc5发生下调,白色脂肪标记基因Asc1发生上调。
对于棕色脂肪细胞,Adra1a基因的过表达量是对照细胞的98.7倍,提高Adra1a表达量使得棕色脂肪标记基因Ucp1,Prdm16,Fndc5和Pgc1α发生上调,白色脂肪标记基因Asc1发生下调。提高Cxcr5基因在棕色脂肪细胞中的表达量使得Ucp1,Prdm16,Pgc1α,Fndc5和Asc1基因均有了不同程度的提高,与白色脂肪细胞中结果一致。Galnt6基因在棕色脂肪细胞中的过表达使得Ucp1,Prdm16和Fndc5基因发生下调,结果显示Adra1a基因和Cxcr5基因可能影响白色脂肪的棕色化。
2、干扰Adra1a、Cxcr5和Galnt6基因对白色脂肪棕色化的影响
Adra1a、Cxcr5和Galnt6基因的siRNA由TAKARA合成,序列分别为siRNA-Adra1a:CCAAGAAUAAGACUCACUU;siRNA-Cxcr5:GCUCUGCACAAGAUCAAUU;siRNA-Galnt6:CCAUCGACCUUAAUACCUU;其中Adra1a-siRNA转染棕色脂肪细胞,Cxcr5-siRNA和Galnt6-siRNA转染白色脂肪细胞,通过q-PCR检测白色脂肪棕色化相关基因的表达情况。棕色脂肪细胞干扰Adra1a基因使得棕色脂肪特异表达基因Ucp1,Prdm16,Pgc1α和Fndc5发生下调,而白色脂肪特异表达基因Asc1表达上调。在白色脂肪细胞中对Cxcr5基因进行干扰使得Prdm16,Fndc5,Ucp1,Pgc1α、和Asc1基因均发生下调,且差异极显著。Galnt6干扰使得Prdm16基因表达量发生上调,Fndc5和Asc1基因表达量发生下调。结果表明Adra1a和Cxcr5基因可能对白色脂肪棕色化起作用。
3、Adra1a基因在蛋白水平影响白色脂肪棕色化
为了从Adra1a、Cxcr5和Galnt6三个候选基因中进一步筛选确定调控白色脂肪棕色化的关键基因,实验对Adra1a基因及棕色脂肪标记基因Ucp1和白色脂肪标记基因Asc1进行蛋白免疫印迹实验。结果表明在蛋白水平,白色脂肪细胞中过表达Adra1a基因可以促进棕色脂肪特异表达基因Ucp1表达,抑制白色脂肪特异表达基因Asc1表达;干扰Adra1a基因使得棕色脂肪标记基因Ucp1表达量发生下调,白色脂肪标记基因Asc1发生上调,免疫印迹结果与q-PCR实验结果一致,确定Adra1a基因为调控白色脂肪棕色化的关键基因。实施例2Adra1a基因调控白色脂肪细胞棕色化的功能分析
本实施例成功分离的白色和棕色脂肪细胞,细胞水平验证Adra1a基因的过表达有利于白色脂肪细胞的棕色化,抑制白色脂肪细胞的增殖和分化;CRISPR/Cas9技术介导的Adra1a基因敲除抑制棕色脂肪细胞中棕色标记基因的表达,同时抑制棕色脂肪的增殖与分化。
一、白色脂肪细胞和棕色脂肪细胞的分离鉴定
小鼠白色脂肪细胞和棕色脂肪细胞的分离:颈椎脱臼处死2周龄的C57BL/6小鼠,于75%的医用酒精清洗好后放于细胞超净工作台内100mm的细胞培养皿中,用灭菌的手术剪和手术镊取其腹股沟附近的白色脂肪和背部肩胛骨处的棕色脂肪,分别放于干净的100mm细胞培养皿中用含有1%PS的PBS清洗三次并去除血液残留和多余杂质,剪碎研磨后置于离心管内,同时加入适量含0.2%Ⅰ型胶原酶和1%BSA的PBS,隔5min震荡一次使组织和消化液充分混匀并使温度保持在37℃。40min后,1500rpm离心5min收集管底细胞并接种于100mm细胞培养皿中进行培养及形态学观察。
在荧光相差显微镜下观察已经分离好的细胞,白色脂肪细胞和棕色脂肪细胞均呈梭型,形态无明显差异,但棕色脂肪细胞生长速度明显比白色脂肪细胞慢。使用q-PCR、Western Blot和免疫荧光等分子生物学手段对获得的细胞进行鉴定,结果表明其符合白色脂肪细胞和棕色脂肪细胞的特点,同时测定二者的增殖和分化能力,确保可以用于后续实验。
二、过表达Adra1a基因促进白色脂肪细胞棕色化
构建Adra1a的过表达载体并转染白色脂肪细胞,Adra1a过表达促进白色脂肪细胞中Ucp1、Cidea、Fndc5等棕色脂肪标记基因的表达,抑制Serpina3k、resistin、Asc1等白色脂肪标记基因的表达,同时抑制白色脂肪细胞的增殖和分化。
1、构建Adra1a基因过表达载体
以C57BL/6小鼠脑组织的cDNA为模板,通过PCR技术克隆大小为1401bp的Adra1a基因(SEQ ID NO.2),送华大基因测序,结果显示克隆片段与SEQ ID NO.2中公布的小鼠Adra1a基因的cDNA相似度为100%,可以用于后续实验。将扩增好的cDNA连接到PMD-19T,测序正确后开始后续实验。
过表达载体p3×FLAG-CMV-10-Adra1a主要包括CMV启动子,目的基因Adra1a及标签蛋白FLAG。使用含有EcoRI和BamHI酶切位点的引物扩增Adra1a基因,引物信息见表1,通过酶切连接等方法将扩增好的片段连接到含有标签蛋白FLAG的p3×FLAG-CMV-10骨架载体上,为防止移码突变,在上游引物的3’端添加碱基C,构建好的载体进行初步酶切鉴定,初步确定载体构建成功,后送华大测序进一步确定载体构建成功。
表1.PCR检测引物的序列信息
2、Adra1a过表达抑制白色脂肪细胞增殖
将构建好的p3×FLAG-CMV-10-Adra1a过表达载体通过Invitrogen公司的2000Reagent转染试剂盒转染白色脂肪细胞,转染后的细胞进行EDU增殖实验,激光共聚焦显微镜显示,与对照组相比,过表达Adra1a基因使得白色脂肪增殖速度变慢,同时,对细胞荧光数量进行统计,统计分析表明,Adra1a过表达的细胞增殖速率明显低于对照组,增殖实验结果与细胞实际生长情况相符。
3、Adra1a过表达抑制白色脂肪细胞成脂分化
分别将适量脂肪细胞接种于6孔细胞培养皿中,体外培养24h后进行成脂诱导。成脂诱导液(DMEM培养液,20%FBS,10μg/mL胰岛素,1μmol地塞米松,0.5mmol IBMX,0.1mmol吲哚美辛)处理2d,维持液(DMEM培养液,20%FBS,10μg/mL胰岛素)处理2d,后加入正常培养液(DMEM培养液,20%FBS)至细胞出现脂滴。对细胞形成的脂滴进行油红O染色,使用荧光倒置显微镜进行脂滴观察并拍照,最终于550nm处使用酶标仪确定细胞的吸光值,检测成脂诱导情况。
结果显示在成脂诱导前,白色脂肪细胞生长状态良好,细胞核明显,细胞贴壁状况良好,成典型的梭型。成脂诱导3d后,细胞质开始回缩,细胞核周围出现细小的颗粒物质。4d-6d时,白色脂肪细胞开始出现细小的微脂滴。8d时细胞开始出现大的单泡脂滴,具有明显的形态,接下来进行油红O染色,同时测定分化的脂肪细胞中脂滴的含量,检测结果表明Adra1a基因的过表达抑制白色脂肪细胞的成脂。
4、Adra1a过表达促进棕色脂肪特异基因表达
研究白色脂肪细胞是否发生棕色化的一个主要方面是看白色脂肪细胞中棕色脂肪特异表达的标记基因是否发生变化。为了进一步研究Adra1a基因的过表达是否引起白色脂肪的棕色化,实验将Adra1a基因在白色脂肪细胞中过表达,通过q-PCR、Western Blot和细胞免疫荧光实验检测白色脂肪细胞中棕色脂肪特异基因的表达量变化。
q-PCR方法检测Adra1a基因对白色脂肪细胞的影响。q-PCR引物见表2,检测结果显示,与对照相比,Adra1a基因在白色脂肪细胞中的表达量提高了118.74倍(p<0.0001),棕色脂肪标记基因整体呈现上升趋势,其中Prdm16,Pparγ,Fabp4,Pgc1α,Ucp1,Adipoq和Cidea基因在Adra1a过表达的白色脂肪细胞中的表达量分别是对照细胞的3.48倍(p=0.00014),3.40倍(p=0.00018),3.39倍(p<0.0001),1.63倍(p=0.0036),4.90倍(p=0.0068),2.10倍(p<0.0001)和2.37倍(p=0.00058)。该实验结果说明过表达Adra1a基因有利于白色脂肪细胞棕色化。
表2.q-PCR引物
通过Western Blot技术检测不同标记基因在Adra1a基因过表达和对照细胞中的蛋白表达量,检测结果与q-PCR结果趋势一致,棕色脂肪标记基因在Adra1a基因过表达细胞中蛋白表达量呈上升趋势。
5、Adra1a过表达抑制白色脂肪特异基因表达
为了进一步研究Adra1a基因对白色脂肪的作用,实验通过q-PCR、Western Blot和免疫荧光方法检测了白色脂肪特异基因表达量的变化。
q-PCR检测Adra1a基因对白色脂肪细胞中白色脂肪特异表达基因的的影响。结果表明白色脂肪细胞相关的标记基因整体呈下降趋势,其中白色脂肪标记基因Asc1,Leptin,Fad3,Serpina3k,resistin和Psat1在Adra1a过表达的白色脂肪细胞中的表达量分别是对照细胞的0.27倍(p<0.0001),0.85倍(p=0.168219),0.63倍(p=0.00050),0.24倍(p<0.0001),0.34倍(p<0.0001)和0.68倍(p<0.0001),整体呈下降趋势。
通过Western Blot技术检测不同标记基因在Adra1a基因过表达和对照细胞中的蛋白表达量,检测结果与q-PCR结果趋势一致。结果显示,白色脂肪标记基因在Adra1a过表达细胞中的表达量整体呈下降趋势。其中Scd、Srebp1和Asc1基因呈显著差异。
本实施例对Adra1a过表达的白色脂肪细胞和对照细胞进细胞免疫荧光鉴定,实验分别使用白色脂肪标记基因Leptin,棕色脂肪标记基因Ucp1和内参基因Gapdh,其中,Leptin基因在Adra1a过表达白色脂肪细胞中的表达量低于对照细胞,Ucp1基因在Adra1a过表达白色脂肪细胞中的表达量高于对照细胞。实验结果与q-PCR和Western Blot实验结果相互印证,表明在白色脂肪细胞中提高Adra1a基因的表达量有助于白色脂肪细胞向棕色脂肪细胞转化。
三、CRISPR/Cas9技术敲除Adra1a基因抑制棕色脂肪细胞形成
本实施例构建Adra1a的CRISPR/Cas9敲除载体并转染棕色脂肪细胞,Adra1a的敲除抑制棕色脂肪细胞中Ucp1,Prdm16和Cidea等基因的表达,同时抑制棕色脂肪的增殖与分化。细胞水平证实Adra1a基因可有效促进白色脂肪棕色化。
1、构建CRISPR/Cas9-Adra1a高效敲除载体
使用两种方法设计针对Adra1a基因CDS序列的sgRNA。两种方法各有优劣,其中方法一在设计sgRNA的同时,对潜在脱靶位点进行了评估,而方法二则对设计好的sgRNA进行分数评估,使得结果更加明了直观。因此本实验同时使用两种方法以确保设计的sgRNA更加准确,切割效率更高,脱靶效率更低。两种方法共获得了31对sgRNA,所有的sgRNA均位于Adra1a基因CDS序列的前200bp,该设计更有利于破坏Adra1a基因的功能区,达到Adra1a基因敲除的作用。通过两种方法的综合考量,最终获得四对sgRNA,sgRNA序列信息分别为sgRNA1:cgagtgcagatgccgatgacagg;sgRNA2:ggggggcctcatcattttcgggg;sgRNA3:tattttagtgatcctctcggtgg;sgRNA4:gccgatgacaggccaccgagagg。
将设计好的sgRNA连接到骨架载体pCas-Guide-EF1a-GFP,送华大测序确定载体的成功构建,同时,对构建成功的骨架载体进行效率检测,分别将四对sgRNA转染小鼠棕色脂肪细胞,转染24h后收样通过q-PCR检测敲除效率,结果表明sgRNA1打靶Adra1a基因后使得Adra1a基因的表达量降低为对照的72%,sgRNA2使得Adra1a基因的表达量降低为对照的59%,sgRNA3使得Adra1a基因的表达量降低为对照的55%,sgRNA4没有发生切割,sgRNA2和sgRNA3的切割效率更高,可用于后续实验。
2、CRISPR/Cas9-Adra1a抑制BAT细胞增殖
本实验利用CRISPR/Cas9技术敲除BAT细胞中的Adra1a基因,将敲除载体转染棕色脂肪细胞后通过EDU细胞增殖检测方法检测细胞的增殖能力。将EDU处理后的细胞放于激光共聚焦显微镜下观察荧光情况,结果显示BAT细胞中Adra1a基因敲除后细胞的增殖能力减弱。对获得的荧光结果进行统计,选取10000左右细胞进行计数,对照细胞和基因敲除细胞没有明显差异,但是增殖的细胞数差异明显,P=0.00019,对细胞的增殖率进行统计,Adra1a基因敲除细胞增殖能力明显减弱,说明BAT细胞中Adra1a基因的敲除抑制细胞的增殖。
3、CRISPR/Cas9-Adra1a抑制BAT细胞成脂分化
将pCas-Guide-EF1a-GFP载体和pCas-Guide-EF1a-sgRNA2载体分别转染BAT细胞,转染6h后加入成脂诱导液进行成脂诱导,使用荧光倒置显微镜随时观察细胞成脂情况。在成脂诱导前,BAT细胞生长状态良好,细胞核明显,细胞贴壁状况良好,成典型的梭型。成脂诱导3d后,细胞质开始回缩,细胞核周围出现细小的颗粒物质。4d-6d时,BAT细胞开始出现细小的微脂滴。8d时细胞开始出现大的单泡脂滴。对脂滴进行油红O染色同时测定分化的脂肪细胞中脂滴的含量情况,检测结果表明Adra1a基因敲除细胞的OD值显著低于对照组,表明Adra1a基因的敲除抑制棕色脂肪细胞的成脂。
4、CRISPR/Cas9-Adra1a抑制BAT细胞中棕色脂肪标记基因的表达
实验利用CRISPR/Cas9技术将Adra1a基因在BAT细胞中敲除,通过q-PCR、WesternBlot和细胞免疫荧光实验检测棕色脂肪细胞中棕色脂肪特异基因的表达量变化。
q-PCR实验检测棕色脂肪标记基因在RNA水平的表达整体呈现下降趋势,其中Prdm16,Pparγ,Fabp4,Pgc1α,Ucp1,Adipoq和Cidea基因在Adra1a敲除的棕色脂肪细胞中的表达量分别是对照细胞的0.13倍,0.14倍,0.59倍,0.62倍,0.66倍,0.58倍和0.14倍。该实验结果说明棕色脂肪细胞中敲除Adra1a基因使得棕色脂肪的标记基因下调。
Western Blot检测棕色脂肪不同标记基因在Adra1a-KO和对照细胞中的蛋白表达量的变化,检测结果与q-PCR结果趋势一致,结果显示,棕色脂肪标记基因在Adra1a-KO细胞中的表达量整体呈下降趋势。
细胞免疫荧光分别使用白色脂肪标记基因Leptin,棕色脂肪标记基因Ucp1和内参基因Gapdh,其中,Leptin基因在棕色脂肪细胞中几乎不表达,Ucp1基因在Adra1a-KO棕色脂肪细胞中的表达量低于对照细胞。实验结果与q-PCR和Western Blot实验结果相互印证,说明棕色脂肪细胞中敲除Adra1a基因抑制棕色脂肪的表达。
实施例3小鼠个体水平Adra1a对小鼠白色脂肪棕色化的作用
本实施例在小鼠个体水平验证Adra1a基因过表达可减轻小鼠体重和白色脂肪重量,有利于白色脂肪棕色化,能够使白色脂肪形成“多室”棕色样脂肪,同时可以增加棕色脂肪的表达,增加棕色脂肪重量及数量。
一、成功制备Adra1a基因过表达小鼠模型
1、Adra1a基因过表达小鼠模型的制备及鉴定
实验利用显微注射技术将小鼠Adra1a基因载体导入C57BL/6小鼠受精卵中,实验将20只小鼠进行超数排卵,获得240枚卵,受精后获得155枚受精卵,存活126枚,移植到4只假孕母鼠,最终获得7只后代。
通过PCR方法检测F0代小鼠的基因型。设计2对引物对F0代小鼠进行Adra1a基因检测(表3),7只存活小鼠中有2只为阳性小鼠,编号为23#,24#,后将PCR产物送华大测序,测序结果进一步确定该小鼠为转基因小鼠。
表3.PCR引物序列
2、Adra1a基因过表达小鼠的建系
对获得的Adra1a基因过表达小鼠进行建系,本实验采用经典的育种方式结合PCR鉴定方法筛选转基因纯合子小鼠。将原核注射获得的F0代阳性小鼠与野生小鼠扩繁,以此来获得F1代小鼠,经鉴定为阳性后同窝交配产生F2,以此类推最终将获得纯合小鼠,对交配获得的每一只小鼠进行PCR鉴定。共获得F1代小鼠11只(阳性小鼠6)。对F1中阳性小鼠同胞交配,获得F2代小鼠47只(阳性小鼠17),F3代小鼠共22只(阳性小鼠11)。目前实验共获得后代小鼠80只,其中阳性转基因小鼠34只,转基因小鼠阳性率为42.5%。
3、转基因小鼠的个体生理水平检测
转基因小鼠的个体生理水平鉴定主要从两个方面进行,第一是称量小鼠不同时间段的体重,第二是检测小鼠各脏器的体重,确定Adra1a基因过表达是否对小鼠的生理指标产生影响。主要结果如下:
对实验获得的转基因小鼠体重进行实时监测,检测时间为小鼠出生后的27-67d,检测结果显示,转基因母鼠的体重均小于非转基因母鼠,转基因公鼠体重也均小于非转基因的公鼠,其中在47d、51d、59d、63d和67d呈显著差异。说明Adra1a基因的过表达有利于小鼠体重的减少。
针对F1代转基因小鼠37#号、42#号和其对应的野生型小鼠进行解剖,同时对小鼠各脏器进行称量,检测Adra1a基因过表达是否对小鼠各脏器大小及重量产生影响,结果显示转基因小鼠(Adra1a)与野生型小鼠(WT)相比,组织器官重量无明显差异。
4、转基因小鼠的q-PCR鉴定
对获得的Adra1a过表达小鼠和野生型小鼠的各脏器的Adra1a基因表达量进行q-PCR鉴定,检测结果表明,在转基因小鼠中,Adra1a基因在肝脏、大脑、脾脏、大肠、肺、胃、卵巢及棕色脂肪中的表达量提高极显著,在小脑中表达量与野生型相比没有差异。Adra1a过表达小鼠相对于野生型小鼠,Adra1a基因在各组织中整体呈提高趋势,说明获得的转基因小鼠为Adra1a基因过表达小鼠。
二、Adra1a基因过表达促进小鼠白色脂肪棕色化
研究Adra1a基因对白色脂肪棕色化是否起作用,主要通过三个方面,第一是看小鼠白色脂肪组织的重量是否发生变化,第二是看Adra1a过表达小鼠白色脂肪组织的形态与野生型相比是否发生变化,第三是看白色脂肪组织中白色脂肪和棕色脂肪中特异表达的标记基因是否发生变化,下面将从以上几个方面进行验证。
1、Adra1a基因过表达诱导白色脂肪重量减少
前期实验表明,在细胞水平,Adra1a基因可以促进白色脂肪细胞棕色化,但是该基因在个体水平是否影响白色脂肪棕色化还未见报道。因此实验制备了Adra1a基因的过表达小鼠,对编号为24#号,37#号和42#号的Adra1a基因过表小鼠进行称重及体内腹股沟处白色脂肪进行称重,结果表明,在标准饲养条件下,与同窝同性别小鼠相比Adra1a基因过表达小鼠的体重均显降低,其中24#号鼠体重减少3.55g,37#号鼠体重减少6.35g,42#号鼠体重减少9.11g,同时白色脂肪组织的重量也减轻,结果表明Adra1a基因的过表达减少体内白色脂肪的含量,对降低肥胖及肥胖引起的代谢疾病有一定的促进作用。
2、Adra1a基因过表达诱导白色脂肪形成多室脂肪
研究表明,相比于内脏脂肪,腹股沟处的白色脂肪更容易棕色化,实验制备了Adra1a基因过表达小鼠模型,取转基因小鼠的腹股沟白色脂肪进行切片,HE染色,结果显示,与相同条件下的对照小鼠相比,Adra1a基因过表达小鼠皮下腹股沟白色脂肪组织中出现大量的多室脂肪细胞,出现明显的棕色化特征,尤其是24#号转基因小鼠更为明显,这类细胞又被称作为米色脂肪细胞。结果表明Adra1a基因过表达可以促使小鼠体内米色脂肪的生成,与前期WAT细胞的结果一致。
3、Adra1a基因过表达增加白色脂肪中棕色脂肪特异基因表达
研究白色脂肪是否发生棕色化现象的一个主要方面是看白色脂肪组织中棕色脂肪特异表达的标记基因是否发生变化。为了进一步研究Adra1a基因的过表达是否引起白色脂肪的棕色化,实验通过q-PCR技术、Western Blot技术以及免疫组化技术对白色脂肪中的棕色标记基因进行不同层面的检测。
q-PCR的方法检测棕色脂肪标记基因Prdm16、Pparγ、Fabp4、Pgc1α、Ucp1、Adipoq和Cidea在RNA水平的相对表达量。检测结果表明,Adra1a基因过表达小鼠的白色脂肪组织中,以上棕色标记基因的表达量均有了不同程度的提高。其中Ucp1基因和Cidea基因表达极显著,P<0.0001。Fabp4基因以及Pgc1α基因均有显著性差异,P<0.05。
Western Blot技术检测小鼠的白色脂肪中棕色脂肪标记基因Fndc5、Prdm16、Pparγ、Pgc1α、Ucp1和Cidea在蛋白水平的相对表达量,检测样本为24#号转基因小鼠、37#号转基因小鼠以及相同条件下饲养的两只对照小鼠。Western Blot检测结果表明,Adra1a基因在24#号和37#号小鼠中的表达量与野生型小鼠相比较均提高。而棕色脂肪标记基因Prdm16、Fndc5、Cidea在24#和37#转基因小鼠中的表达量明显高于对照小鼠,结果表明Adra1a基因的过表达有助于白色脂肪的棕色化。
4、Adra1a基因过表达降低白色脂肪中白色脂肪特异基因表达
实验在小鼠个体水平检测了白色脂肪组织中白色脂肪标记基因的变化情况,使得体内外实验结果相互印证,验证Adra1a基因的功能。实验主要通过q-PCR技术、WesternBlot技术以及免疫组化技术对白色脂肪中的白色标记基因进行检测。
实验通过q-PCR的方法检测白色脂肪标记基因Asc1、Leptin、Adipoq、Fasn、Scd和Acc在RNA水平的相对表达量。Adra1a基因在转基因小鼠中表达量提高,同时白色脂肪标记基因的表达量均有了不同程度的下降,其中Leptin、Adipoq、Fasn、Scd和Acc基因表达差异极显著,P<0.001,Asc1基因具有显著的差异表达P<0.05。
Western Blot技术检测小鼠的白色脂肪中白色脂肪标记基因Srebp1、Psat1、Dlgap1、Asc1、Fasn、Scd和Acc在蛋白水平的相对表达量,检测样本为24#号转基因小鼠、37#号转基因小鼠以及相同条件下饲养的两只对照小鼠。Western Blot检测结果显示Adra1a基因在24#号和37#号小鼠中的表达量相对于野生型小鼠均有所提高,内参基因α-Tublin的表达量差异不显著,而白色脂肪标记基因在整体水平上呈下降趋势,与q-PCR结果相互印证。对37#号转基因小鼠及相同条件下喂养的对照小鼠进行免疫组化实验,分别使用目标基因Adra1a,棕色脂肪标记基因Ucp1和白色脂肪标记基因Leptin的抗体进行孵育,使用ImagePro Plus分析光密度值,将免疫组化结果量化,结果显示与对照组相比,过表达小鼠的Adra1a基因的表达量有所提高,棕色脂肪标记基因Ucp1表达量提高,白色脂肪标记基因Leptin变化不明显。
三、Adra1a基因过表达诱导小鼠棕色脂肪增加
1、Adra1a基因过表达诱导棕色脂肪重量增加
前期研究表明,Adra1a过表达小鼠可以促进白色脂肪棕色化,但对棕色脂肪的影响还未知,本实验将通过基因型鉴定的野生型小鼠(WT)和Adra1a基因过表达小鼠(Adra1a)进行标准日粮喂养,解剖后对小鼠的不同脂肪组织进行称重比较。结果显示WT小鼠和Adra1a小鼠的棕色脂肪组织重量增加,白色脂肪组织重量减少,棕色脂肪与白色脂肪重量的比值,相对于WT均差异明显。
2、Adra1a基因过表达增加棕色脂肪细胞数量
对Adra1a及WT型小鼠棕色脂肪组织进行切片及HE染色,结果显示相对于WT,Adra1a过表达小鼠的棕色脂肪组织脂肪细胞数量增加,细胞体积缩小,同时BAT组织切片单位面积细胞核的数量也有了明显的增加,结果表明Adra1a诱导棕色脂肪重量增加可能是由于增加棕色脂肪细胞的数量引起,该结果与前期细胞增殖实验结果相吻合,Adra1a引起棕色脂肪细胞增殖加快。
3、Adra1a基因过表达促进棕色脂肪特异基因表达
实验通过q-PCR技术、Western Blot技术以及免疫组化技术对小鼠棕色脂肪特异表达的基因进行检测,检测表明Adra1a基因过表达促进小鼠棕色脂肪特异基因表达,与前期脂肪组织形态学观察结果一致。同时,细胞实验中在BAT细胞中敲除Adra1a基因,引起BAT细胞中棕色标记基因下调,在本节中棕色脂肪组织中过表达Adra1a基因,引起棕色脂肪特异表达基因上调,两种结果相互应证,相互补充。具体结果如下:
通过q-PCR的方法检测Adra1a基因过表达小鼠和对照小鼠棕色脂肪组织中棕色脂肪标记基因Prdm16、Pparγ、Fabp4、Pgc1α、Ucp1和Cidea在RNA水平的相对表达量。检测结果表明,Adra1a基因在转基因小鼠中表达量有所提高,同时以上棕色脂肪标记基因的表达量均有了不同程度的提高。其中Pparγ、Fabp4、Ucp1和Cidea基因表达差异极显著,Prdm16基因差异表达显著。以上结果说明,在RNA水平,Adra1a基因的过表达在促进白色脂肪棕色化的同时促进棕色脂肪的表达。
Western Blot技术检测小鼠的白色脂肪中棕色脂肪标记基因Fndc5、Prdm16、Pparγ、Pgc1α、Ucp1和Cidea在蛋白水平的相对表达量,检测样本为24#号转基因小鼠、37#号转基因小鼠以及相同条件下饲养的两只对照小鼠。Western Blot检测结果表明,Adra1a基因在24#号和37#号小鼠中的表达量相对于野生型小鼠均有所提高,内参基因α-Tublin的表达量差异不显著。而棕色脂肪标记基因Fndc5、Pgc1α、Ucp1在Adra1a小鼠中的表达量明显有所提高。
对37#号转基因小鼠及相同条件下喂养的对照小鼠进行免疫组化实验,分别使用目标基因Adra1a,棕色脂肪标记基因Ucp1和白色脂肪标记基因Leptin的抗体进行孵育,使用Image Pro Plus对获得的免疫组化结果进行分析,结果显示与对照组相比,过表达小鼠的Adra1a基因的表达量有所提高,棕色脂肪标记基因Ucp1表达量提高,白色脂肪标记基因Leptin变化不明显。
实施例4Adra1a基因通过PI3K-AKT信号通路调控白色脂肪棕色化
本实施例对Adra1a调控白色脂肪棕色化的机制进行研究。通过RNA-seq测序结合KEGG分析预测得到4条Adra1a基因相关的调控白色脂肪棕色化的信号通路,体外实验进一步验证确定Adra1a基因通过PI3K-AKT信号通路影响Pgc1α基因的表达,进而影响白色脂肪的棕色化。
一、Adra1a基因控白色脂肪棕色化的生物信息分析
1、数据质控
实验提取测序样品的RNA,检测RNA浓度及完整性,确定样品合格后利用Illumina(HiSeq Xten)进行测序、建库。获得的实验数据进行测序质量分析和碱基含量分布分析,去除少量接头污染的reads及低质量的reads,获得可以用于后续分析的Clean Date,最终过滤后的数据占原始数据的比例均大于90%,最高比例达93.12%,错误率为0.03%;Q20均值为98.57%,Q30均值为94.66%;GC含量均值为46.58%,结果表明测序数据可以用于后续实验分析。
2、比对组装
使用Hisat2软件,将过滤后数据与小鼠参考基因组序列进行比较。结果显示Totalmapped均大于70%,最高达92.77%,说明参考基因组序列选择合适且样品不存在污染;Multiple mapped为6.20%-19.21%,Uniquely mapped为73.80%-85.10%,Non-splicereads为50%左右,说明有一半以上的序列可以比对到外显子上,也有部分数据比对到内含子上。
3、差异表达
(1)表达定量分析
对各样品进行基因表达水平的统计分析,FPKM在0-1区间内的基因均高于70%,FPKM在1-3,3-5和大于60的基因均低于6%,总体而言,脂肪组织中低表达的基因含量较高。RNA-Seq相关性进行检测结果显示组内差异高于组间差异,更加接近于1,说明样本重复性较好,符合重复样本设置条件。同时,通过FPKM分布图来展示不同条件下基因表达水平,一般情况下,差异基因数量在整体基因数量中只占据小部分,不会影响到所有基因的表达量分布,根据每个样品的基因表达量及整体分布趋势,得到小鼠脂肪转录组的表达量分布情况。
(2)差异基因分析
实验共分为四组,G1:BAT_37,BAT_42,G2:WAT_37,WAT_42,G3:WT37_BAT,WT42_BAT和G4:WT37_WAT,WT42_WAT。同时进行两组比较分析差异基因,G1VSG2和G3VSG4。差异分析结果如下:
G1和G2组比较获得4401个差异基因,其中上调1300个基因,下调3101个基因;G3VSG4共获得3757个差异表达基因,其中上调基因1046个,下调基因2711个。对获得的差异基因进行共有和特有差异基因分析。野生型小鼠与转基因小鼠脂肪共有2922个差异基因,其中包括Adra1a基因及棕色脂肪相关标志基因Ucp1、Hoxc10等。聚类分析差异表达的基因,结果显示G2和G4为一类组织,均为白色脂肪,同源性较高,G1和G3为一类组织,均为棕色脂肪,同源性较高,差异基因只占其中一小部分。该结果与预期相符,为后续研究白色脂肪棕色化奠定了一定的基础。
4、功能注释
(1)GO富集分析
GO富集分析有利于确定差异基因的分子生物学功能,为筛选调控白色脂肪棕色化的关键基因奠定了基础。实验对获得的差异显著GO term绘制柱状图,结果显示G1VSG2主要富集的GO包括single-organism process、cytoplasm和immune system process,G3VSG4同样主要富集的GO包括single-organism process和immune system process。说明肥胖可能与免疫应答有关。
(2)RNA-seq结合KEGG预测白色脂肪棕色化相关信号通路
实验利用RNA-seq技术测定了野生C57BL/6小鼠的白色脂肪和棕色脂肪组织中的差异表达基因,同时相同条件下测定Adra1a过表达小鼠的相同部位,对差异表达的基因进行KEGG信号通路富集。分析统计出Adra1a参与的与白色脂肪和棕色脂肪高度相关的信号通路包括“PI3K-AKT signaling pathway”、“Metabolic pathways”、“Cardiacmusclecontraction”和“Adrenergic signaling in cardiomyocytes”信号通路。其中“PI3K-AKT signaling pathway”和“Metabolic pathways”经综合分析,相关性最高,而“Adrenergic signaling in cardiomyocytes”信号通路为Adra1a基因相关信号通路,该基因通过ERK影响肥大表型,同时表明Adra1a基因与AKT有直接或间接的作用关系。
二、Adra1a基因依赖PI3K-AKT调控白色脂肪棕色化机制研究
1、Adra1a激动剂HY最佳浓度的确定
在Adra1a过表达小鼠中及Adra1a过表达的白色脂肪细胞中,我们均已发现该基因的过表达会引起小鼠白色脂肪棕色化,为了验证Adra1a基因的作用机理,我们对相关蛋白的磷酸化水平进行蛋白免疫印迹检测,在进行磷酸化水平检测之前,摸索了α1-肾上腺素受体激动剂(HY)的最佳转染条件,分别使用不同浓度的HY处理白色脂肪细胞,处理6h后收样,进行Ucp1基因表达量的检测,检测结果表明,相对于对照组,处理组的Ucp1表达量均有所提高,其中浓度为100ng/mL时结果最为明显,因此确定HY处理白色脂肪细胞的最佳浓度为100ng/mL。
2、Adra1a基因影响AKT信号通路
使用最适浓度100ng/mL的HY处理白色脂肪细胞,处理后每隔5min收一次样并提取细胞总蛋白,后检测AKT、ERK1/2及P38的磷酸化水平和非磷酸化水平,检测结果表明,在处理后的15min内,AKT的非磷酸化没有发生明显变化,15min后,非磷酸化水平开始出现变化,磷酸化水平在HY处理10min后开始升高,后呈持续升高状态。ERK1/2的非磷酸水平变化不明显,磷酸化水平整体呈上升趋势。P38非磷酸化水平变化不明显,磷酸化水平在HY处理20min时达到最高点,25min时又出现下降趋势。AMPK非磷酸化水平在15min后出现变化,总之,Adra1a基因促进白色脂肪的棕色化可能通过增加AKT、ERK和P38的磷酸化水平,AMPK的非磷酸化水平实现。
3、Adra1a增多引起棕色脂肪标记基因Pgc1α上调
使用最佳浓度的Adra1a激动剂HY处理白色脂肪细胞,处理12h和24h后收样并提取细胞总蛋白,后检测白色脂肪棕色化关键基因Pgc1α和Fndc5的蛋白表达情况,检测结果表明,在24h内,Pgc1α和Fndc5随处理时间延长而升高,该结果与前期在白色脂肪细胞中转入Adra1a基因过表达载体结果一致。说明Adra1a基因通过Pgc1α和Fndc5基因影响白色脂肪的棕色化。
4、Adra1a基因依赖PI3K-AKT信号通路影响Pgc1α的表达
前期研究结果表明,在白色脂肪细胞中加入Adra1a激动剂后,会影响AKT的磷酸化水平,同时,转录组测序结果表明白色脂肪的棕色化可能受到“PI3K-AKT”信号通路的影响,研究结果表明白色脂肪细胞中激活Adra1a基因引起Pgc1α和Fndc5基因的上调,那么,Adra1a基因是否通过“PI3K-AKT signaling pathway”信号通路影响Pgc1α和Fndc5基因的表达,从而影响白色脂肪棕色化。为了验证这一实验结果,我们使用了AKT信号通路的抑制剂DIH,该药物抑制AKT1/AKT2/AKT3的IC50分别为5nM/12nM/65nM。实验先在白色脂肪细胞中加入100ng/mL AKT抑制剂DIH 2h后,加入Adra1a基因的激动剂HY,培养24h后收样并检测相关蛋白的变化情况,结果显示,同时加入Adra1a基因激动剂(HY)和AKT信号通路抑制剂(DIH),Pgc1α基因的表达量相对于只加HY组和空白对照组有所下降,但是Fndc5基因的表达量并没有下降,说明Adra1a基因通过“PI3K-AKT”信号通路影响Pgc1α基因的表达,而Pgc1α基因为白色脂肪棕色化的关键转录因子,因此Adra1a基因可能通过PI3K-AKT-Pgc1α影响白色脂肪的棕色化。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 内蒙古大学
<120> 一种调控白色脂肪棕色化的基因Adra1a的应用
<130> KHP191115301.2
<160> 51
<170> SIPOSequenceListing 1.0
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<211> 466
<212> PRT
<213> 人工序列(Artificial Sequence)
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Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asn Phe Lys Pro Pro
290 295 300
Glu Thr Val Phe Lys Ile Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys
305 310 315 320
Ile Asn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala
325 330 335
Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Arg Arg Arg Gln Ser Ser
340 345 350
Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu
355 360 365
Gly Gln His Arg Gly Met Val Arg Ile Pro Val Gly Ser Gly Glu Thr
370 375 380
Phe Tyr Lys Ile Ser Lys Thr Asp Gly Val Arg Glu Trp Lys Phe Phe
385 390 395 400
Ser Ser Met Pro Gln Gly Ser Ala Arg Ile Thr Met Pro Lys Asp Gln
405 410 415
Ser Ala Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gln Val
420 425 430
Cys Cys Cys Val Gly Ser Ser Thr Pro Arg Pro Glu Glu Asn His Gln
435 440 445
Val Pro Thr Ile Lys Ile His Thr Ile Ser Leu Gly Glu Asn Gly Glu
450 455 460
Glu Val
465
<210> 2
<211> 1401
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggtgcttc tttctgaaaa tgcttctgaa ggctccaact gcacccaccc gccagcacag 60
gtgaacattt ctaaggccat tctacttggg gtgatcttgg ggggcctcat cattttcggg 120
gtcttgggga atattttagt gatcctctcg gtggcctgtc atcggcatct gcactcggtg 180
actcactact acattgtcaa cctggctgtg gcagacctcc tcctcacctc caccgtgctg 240
cccttctctg ccatctttga gatcctgggc tactgggcct ttggcagggt gttctgcaac 300
atctgggcgg cggtggacgt cttatgctgc acagcgtcca tcatgggcct ctgcatcatc 360
tccatcgacc gatacattgg tgtgagctac ccgctgcgct accccaccat tgtcacccag 420
aggaggggcg tcagggctct gctctgcgtc tgggcgcttt ccttggtcat ctccatcgga 480
cccctgttcg gctggaggca gcaggctccg gaggatgaga ccatctgcca aatcaatgag 540
gagccaggat acgtgctgtt ctcagcgctg ggctctttct acgtgccact gaccatcatc 600
ctggttatgt actgtcgagt ctacgtggta gccaagagag aaagccgagg cctcaagtcc 660
ggcctcaaga ccgacaagtc agactcagag caagtgacgc tccgtatcca ccgtaaaaat 720
gtccctgcag aaggcagcgg agtaagcagt gccaagaata agactcactt ctccgtgagg 780
ctgctcaagt tttcccgaga gaagaaagcc gccaagacgc tgggcattgt ggtgggatgc 840
ttcgtcctct gctggctgcc attcttcctc gtgatgccca ttgggtcctt cttcccgaat 900
ttcaagccac cggaaacagt tttcaaaata gtattttggc ttgggtacct aaatagttgc 960
atcaacccta tcatataccc atgctccagc caggagttca agaaagcctt tcagaatgtg 1020
ctgcgaatcc agtgtcttcg cagaaggcag tcttccaagc atgccctggg ctacactctg 1080
cacccaccca gccaggctgt agaggggcag cacagaggca tggtgcgtat cccggtgggc 1140
tcaggagaga ctttctataa gatctccaag acagatggag tccgtgaatg gaagtttttc 1200
tcttccatgc cccagggatc ggccaggatt accatgccga aggaccaatc cgcctgtacc 1260
acagcccggg tgagaagtaa aagctttttg caggtctgct gctgtgtggg gtcgtcgacc 1320
ccacgccctg aagaaaatca ccaagttcca accattaaga tccacaccat ctccctcggt 1380
gaaaacgggg aggaagtcta g 1401
<210> 3
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccaagaauaa gacucacuu 19
<210> 4
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcucugcaca agaucaauu 19
<210> 5
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccaucgaccu uaauaccuu 19
<210> 6
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccggaattcc atggtgcttc tttctgaaaa t 31
<210> 7
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgcggatccc tagacttcct ccccgtttt 29
<210> 8
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tgacattcat gggattgcag actaa 25
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tccagcacca gcgtaaccag 20
<210> 10
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccaggatcaa tgacatttca cacac 25
<210> 11
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aggtcattgg ctatctgcag cac 23
<210> 12
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cactcaggat tggcctctac gac 23
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gctctgggct tgcattctga c 21
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ttctgtctgt acgattgtca gtgg 24
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gtcatcttcg gcatgactgg 20
<210> 16
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tgctgatcat tgttgtggtc ctc 23
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gctccggtgt gctggtttct 20
<210> 18
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cctcgccatg tgtcagatca a 21
<210> 19
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ctttcacatg caccaacagt tcc 23
<210> 20
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tcgtttgtga gcaggagggt tc 22
<210> 21
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tgggtccttc ggatactcgt tc 22
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ctgaccacaa acgatgaccc tc 22
<210> 23
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gactgcggtt gtgtatggga ct 22
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gtgatccaca cgaaccagtg 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tcccgctttt tcttgtccta 20
<210> 26
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ggctgaaggc aaagtcagtg t 21
<210> 27
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tggaatctgt cctgctgtcc t 21
<210> 28
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ctgtccagtc tatccttgca cac 23
<210> 29
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
cagaaggcac agcagtcttg a 21
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
taccgccttg tcaagaaacc 20
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
agtggagcgc cagaatagaa 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
agagccccat ctgtcctctc 20
<210> 33
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
actggtagtc tgcaaaacca aa 22
<210> 34
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
aagtgggagt gggctttgc 19
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
tggtgaccaa atccccattt 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
caagtgtcca ccaacaagcg 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ggagcgcagg atagactcac 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
cgctggcaca tcaacttcac 20
<210> 39
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
aggaactcag aagcccaaag c 21
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gtccgcactg actgtaacca 20
<210> 41
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gccagactcg tttgtcagga 20
<210> 42
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
cacgatggag gggccggact catc 24
<210> 43
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
taaagacctc tatgccaaca cagt 24
<210> 44
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
cgagtgcaga tgccgatgac agg 23
<210> 45
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ggggggcctc atcattttcg ggg 23
<210> 46
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
tattttagtg atcctctcgg tgg 23
<210> 47
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
gccgatgaca ggccaccgag agg 23
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
cacgacttct tcaagtccgc 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
tgctcaggta gtggttgtcg 20
<210> 50
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
cgtatgttcc catagtaacg cc 22
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
tgctcaggta gtggttgtcg 20
Claims (6)
1.Adra1a基因或其编码的蛋白在非疾病治疗目的的调控白色脂肪棕色化中的应用;Adra1a基因的编码蛋白的氨基酸序列为如SEQ ID NO.1所示的氨基酸序列;或Adra1a基因的CDS序列为SEQ ID NO.2所示的核苷酸序列。
2.Adra1a基因或其编码的蛋白、或Adra1a基因表达促进剂在非疾病治疗目的的提高或促进棕色脂肪标记基因表达量、或在非疾病治疗目的的降低或抑制白色脂肪标记基因表达量中的应用;Adra1a基因的编码蛋白的氨基酸序列为如SEQ ID NO.1所示的氨基酸序列;或Adra1a基因的CDS序列为SEQ ID NO.2所示的核苷酸序列。
3. Adra1a基因或其编码的蛋白在非疾病治疗目的的提高或促进Pgc1α基因表达中的应用;Adra1a基因的编码蛋白的氨基酸序列为如SEQ ID NO.1所示的氨基酸序列;或Adra1a基因的CDS序列为SEQ ID NO.2所示的核苷酸序列。
4.Adra1a基因的表达抑制剂在非疾病治疗目的的抑制体内棕色脂肪细胞数量增多、体积增大、或在非疾病治疗目的的制备抑制棕色脂肪细胞增殖和分化的药物中的应用;Adra1a基因的编码蛋白的氨基酸序列为如SEQ ID NO.1所示的氨基酸序列;或Adra1a基因的CDS序列为SEQ ID NO.2所示的核苷酸序列。
5. Adra1a基因或其编码的蛋白、或Adra1a基因表达促进剂在非疾病治疗目的的抑制白色脂肪细胞增多、体积增大、增殖、分化中的应用;Adra1a基因的编码蛋白的氨基酸序列为如SEQ ID NO.1所示的氨基酸序列;或Adra1a基因的CDS序列为SEQ ID NO.2所示的核苷酸序列。
6.Adra1a基因、或其编码的蛋白、或Adra1a基因的表达促进剂在制备缓解或治疗肥胖的药物中的应用;Adra1a基因的编码蛋白的氨基酸序列为如SEQ ID NO.1所示的氨基酸序列;或Adra1a基因的CDS序列为SEQ ID NO.2所示的核苷酸序列。
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