CN103702555B - Wdr13作为用于治疗糖尿病和癌症的新的生物标志物 - Google Patents
Wdr13作为用于治疗糖尿病和癌症的新的生物标志物 Download PDFInfo
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Abstract
WD‑重复蛋白种类繁多,但它们都是参与广泛的细胞功能的结构上关联的蛋白。该家族中的一个成员WDR13从鱼类到人类中都是保守的并定位在细胞核中。为了理解Wdr13基因的体内功能,我们创建并表征了缺乏该基因的突变小鼠品系。该突变小鼠由于β细胞增殖增强而具有较高的血清胰岛素水平和较高的胰岛质量。然而,一个已知的细胞周期抑制物p21在突变胰岛中被下调。在胰脏MIN6细胞系中过量表达WDR13导致p21的上调并伴随着细胞生长延迟。我们表明WDR13是胰脏β细胞增殖的新的负向调控者。免疫共沉淀实验显示该蛋白与雌激素受体和多种HDACs具有相互作用。我们提供了证据显示WDR13以HDAC依赖性和HDAC非依赖性两种方式来调控雌激素受体所介导的转录。鉴于Wdr13敲除小鼠中胰岛素水平较高,葡萄糖清除较好以及缺少胰岛素抵抗,我们提出该蛋白可能作为用于减轻糖尿病中受损的葡萄糖代谢的潜在候选药物靶标。
Description
发明领域
本发明涉及用作治疗糖尿病和癌症的药物靶标的生物标志物。进一步地,抑制Wdr13能够治疗精子减少症和精子缺乏症。本发明涉及缺失Wdr13基因的小鼠突变株的产生和表征,由于β细胞增殖增强,使得胰岛素水平较高和胰岛质量增加,这导致年龄依赖性轻度肥胖表型。进一步地,该蛋白的过量表达引起p21蛋白水平上调并且导致细胞生长延迟。
发明背景
WD重复蛋白属于结构上关联的蛋白的大家族,其成员具有多种多样的功能,例如细胞周期调控、转录、染色体组构和蛋白运输[1,2]。这些蛋白为蛋白-蛋白相互作用提供了平台。WDR13蛋白是该家族成员之一并且定位于细胞核[3]。Wdr13基因在脊椎动物中高度保守并且表达于大多数组织中[4],在胰脏、脑、睾丸和卵巢中表达水平最高。该基因在人和小鼠中分别定位于X染色体的Xp11.23和XA1.1位点处。在人中,包含该基因的X染色体缺失已经与精神发育迟缓、肥胖和干皮症相关[5,6,7,8]。已经鉴定出几种WD重复蛋白,其表达于胰脏β细胞中并且在β细胞的增殖中发挥作用[9,10]。
β细胞质量由新生/增殖和凋亡/坏死之间的平衡调控。在小鼠中,胰岛前体的分化和扩增是生命第一周前的细胞新生的原因[11,12]。其后,所存在的β细胞的扩增是新形成的β细胞的主要来源[13,14]。在病理情况下,可以存在α至β细胞的转分化[15]。已经鉴定出多种在胰脏β细胞增殖中发挥作用的细胞周期调控物[16]。胰岛中的细胞周期进程受到细胞周期蛋白、细胞周期蛋白依赖性激酶(CDKs)、细胞周期蛋白依赖性激酶抑制物以及激素即雄激素和雌激素的控制[16,17]。雌激素通过防止胰脏β细胞凋亡而提高胰脏β细胞的质量[17,18,19,20]。
胰岛质量、胰岛素的产生和体重是相互关联的[12,21]。在人[22]和啮齿目动物[23]中胰岛素水平与肥胖正相关。一般来讲,肥胖使得对胰岛素产生的需求较高,通过β细胞质量增加能够达到相同效果。肥胖还是发生外周胰岛素抗性的主要风险因素[24]。胰岛素抗性使得对来自β细胞的胰岛素的需求进一步提高,引发β细胞衰竭。这导致β细胞存活缺陷、β细胞质量不足、β细胞的关键功能(例如葡萄糖刺激下分泌胰岛素)退化以及最终导致2型糖尿病。因此,产生胰岛素的β细胞的质量根据代谢情况动态变化[25,26]。或者,肥胖可能是胰岛素水平较高的结果[27,28,29],因为胰岛素通过增加脂肪细胞中的脂质积累而对脂肪生成具有刺激作用[30,31,32]。胰岛素还参与脂肪细胞的存活[33]。敲除脂肪组织特异性胰岛素受体防止肥胖发生,这强调了胰岛素至脂肪细胞的信号对于肥胖的发生十分重要[31]。MOR-1阿片样受体敲除的小鼠中胰岛素超分泌导致随年龄体重增加较多[29],而CHOP敲除的小鼠通过增加胰岛素分泌而变得肥胖,尽管葡萄糖耐受未受影响[34]。
为了理解Wdr13基因的体内作用,我们已经制备了缺少该基因的小鼠株,并且显示这些小鼠由于β细胞增殖较多而具有较高的胰岛质量,发展为胰岛素高血症和轻度肥胖。我们还鉴定出WDR13蛋白的几种相互作用的伴侣,并且提供了该蛋白可能用作转录阻遏物的证据。
定义:
WD-重复蛋白-该蛋白含有结构上保守的基序,一般以C末端处的二肽色氨酸-天门冬氨酸结束。然而,给定的WD-重复蛋白中重复的数量可以从4-16变化。
嵌合体(Chimaeras)-嵌合体(chimera)是在多种组织中具有基因上两种不同的细胞类型的单个有机体。
敲除小鼠-经基因改造的小鼠,其中通过利用人为干预而引入的一段DNA将染色体上的内源基因替代或破坏而使其失活。
肥胖-肥胖是一种身体状况,其中身体积累过多脂肪,所述过多脂肪可能具有负面的健康结果。
胰脏β细胞-胰岛中产生胰岛素的细胞被称为胰脏β细胞。
发明目的
本发明的主要目的是提供一种生物标志物WDR13蛋白,其具有Seq Id no.1,能够用于治疗糖尿病和癌症。
本发明的另一个目的是提供一种WDR13基因靶向载体(Seq Id no.2)。
本发明的另一个目的是提供一种通过增强β细胞增殖从而治疗糖尿病的方法。
本发明的另一个目的是提供一种治疗癌症的方法。
本发明的另一个目的是提供WDR13作为β细胞增殖的新的负向调控物的用途及其应用。
本发明的另一个目的是提供WDR13作为雌激素受体所介导的转录阻遏物的用途。
本发明的另一个目的是提供WDR13蛋白及其下游靶标用于调节胰脏β细胞和睾丸精原细胞的用途。
本发明的另一个目的是提供WDR13敲除小鼠模型系统在癌症的多个时期研究癌症的演进的用途。
本发明的另一个目的是提供WDR13敲除小鼠模型系统在研究药物效果中的用途,所述药物选自包含具有抗增殖活性的组。
本发明的另一个目的是产生小鼠肿瘤模型系统,包括:
a.提供具有Seq Id no 1的WDR13基因
b.用步骤a.中获得的基因靶向ES。
c.由被靶向的ES细胞产生敲除小鼠
d.育种敲除小鼠
e.获得小鼠肿瘤模型
本发明的另一个目的是利用该方法制备小鼠肿瘤模型系统。
本发明的另一个目的是产生小鼠肿瘤模型系统。
总结
因此,本发明涉及WDR13蛋白用作治愈糖尿病和癌症的药物靶标的用途。为了阐述WD重复基因家族成员之一的Wdr13基因的体内功能,我们制备了缺失该基因的小鼠株。在本研究中,我们显示WDR13是β细胞增殖的新的负向调控物。突变小鼠显示胰岛质量显著增高、血液胰岛素水平升高、年龄依赖性轻度肥胖和较好的葡萄糖清除,而没有任何胰岛素抗性的迹象。突变小鼠中β细胞增殖增强可能是由于已知的细胞周期抑制物p21的下调。与这些发现一致,MIN6细胞中WDR13的过量表达使得p21上调和细胞增殖延迟。最后,我们显示WDR13是β细胞增殖的新的负向调控物。WDR13以HDAC依赖性和HDAC非依赖性两种方式充当雌激素受体所介导的转录阻遏物。
在本发明的一个实施方案中,具有Seq Id no.1的生物标志物用于治疗糖尿病和癌症。
在本发明的另一个实施方案中,具有Seq Id no.2的表达构建体用于靶向WDR13基因,其由以下组成:
a.具有polyA的新霉素作为阳性选择标志物。
b.HSV-tk作为阴性选择标志物。
c.用于5’重组的1.6kb 5’同源区。
d.用于3’重组的4.1kb 3’同源区。
在本发明的另一个实施方案中,一种制备小鼠肿瘤模型系统的方法,包括:
a.提供如权利要求1中所述的Seq Id no 1
b.制备WDR13靶向构建体
c.将如步骤b中所述的WDR13靶向构建体电穿孔入ES细胞中,
d.通过southern印迹对步骤c中获得的被靶向的ES细胞克隆进行选择,
e.通过已知方法由步骤d中获得的被靶向ES细胞克隆产生敲除小鼠,
f.通过已知方法育种步骤e中获得的敲除小鼠,以达到种系传递,
g.获得小鼠肿瘤模型
在本发明的另一个实施方案中,用于在癌症的多个时期研究癌症的演进的肿瘤模型系统。
在本发明的另一个实施方案中,一种治疗癌症的方法,包括在MIN6细胞中利用腺病毒系统(100MOI)过量表达如权利要求1中所述的基因序列。
在本发明的另一个实施方案中,一种通过破坏β细胞中的生物标志物治疗糖尿病和增强β细胞增殖的方法。
在本发明的另一个实施方案中,WDR13作为β细胞增殖的负向调控物的用途。
在本发明的另一个实施方案中,WDR13作为以HDAC依赖性(ERα)和HDAC非依赖性(ERβ)方式的雌激素受体阻遏物的用途。
在本发明的另一个实施方案中,WDR13蛋白及其下游靶标用于体内调节β细胞和睾丸精原细胞增殖的用途。
在本发明的另一个实施方案中,WDR13蛋白及其下游靶标在发现用于调节如权利要求9所述细胞增殖的药物中的用途。
在本发明的另一个实施方案中,WDR13敲除小鼠模型系统在研究药物效果中的用途,所述药物选自包含具有抗增殖活性的药物的组。
附图和表格简述:
图1.Wdr13敲除小鼠的产生。A)靶向方案:用新霉素抗性标志物基因替代Wdr13基因的内含子1(部分)、外显子2、内含子2和外显子3(部分)。B)利用来自5’末端的座位的700bp探针进行Southern印迹分析,其显示了来自野生型等位基因的9.3kb EcoRI片段和来自突变等位基因的3.8kb片段。C)利用Wdr13cDNA探针进行Northern印迹分析,其显示了Wdr13-/0小鼠中Wdr13转录子的缺失(上图)和溴化乙锭染色作为上样对照(下图)。D)利用抗WDR13兔多克隆抗体(上图)对来自睾丸、胰脏和经纯化胰岛进行Western印迹分析,其显示了Wdr13-/0小鼠中WDR13蛋白的缺失和抗β-肌动蛋白作为上样对照(下图)。
图2.以正常食物饲养的Wdr13敲除小鼠的体重、身体组成以及葡萄糖和胰岛素水平。A)正常食物下Wdr13+/0雄性、Wdr13-/0雄性、Wdr13+/+雌性、Wdr13+/-雌性和Wdr13-/-雌性的生长曲线(n=12)。B)Wdr13-/0小鼠中增加的脂肪组织质量。C)Wdr13-/0小鼠附睾脂肪垫切片的H&E染色,其显示脂肪组织肥大。D)12个月时雄性中附睾脂肪垫和雌性中卵巢脂肪垫的重量(n=6)。E)2个月和12个月时Wdr13敲除小鼠中的16小时空腹葡萄糖水平。F)2个月和12个月时Wdr13敲除小鼠中的随机餐后葡萄糖水平。G)2个月和12个月时Wdr13敲除小鼠中的空腹胰岛素水平。H)2个月和12个月时Wdr13敲除小鼠中的随机餐后胰岛素水平。
图3.以高脂饮食饲养的Wdr13敲除小鼠的体重以及葡萄糖和胰岛素水平。A)高脂饮食下Wdr13+/0雄性和Wdr13-/0雄性的生长曲线(n=10至12)。B)6个月时Wdr13-/0小鼠和其野生型同窝出生仔畜中的空腹和餐后胰岛素水平。C)6个月时的胰岛素耐受测试(n=8)。D)6个月时的葡萄糖耐受测试(n=8)。E)6个月时应答于葡萄糖的体内胰岛素分泌(n=4至6)。
图4.野生型和Wdr13敲除小鼠的GTT和ITT。A)正常食物下2月龄时的GTT。B)正常食物下12月龄时的GTT。C)正常食物下2月龄时的ITT。D)正常食物下12月龄时的ITT。E)高脂饮食下9月龄时的ITT。
图5.Wdr13敲除小鼠的胰岛组织学和体外胰岛素分泌。A)利用H&E染色的胰岛形态学,其显示胰岛质量增加。B)高脂饮食下6个月时胰岛的质量(mg),其显示Wdr13敲除小鼠中胰岛质量增加(n=4)。C)2.8mM和16mM葡萄糖浓度下野生型和Wdr13敲除小鼠由分离的胰岛的体外胰岛素分泌。
图6.Wdr13敲除小鼠中胰脏β细胞的增殖和细胞周期调控物p21的相对表达。A)利用BrdU标记的胰脏β细胞增殖分析。B)利用BrdU标记的胰岛中正在分裂的细胞百分比。C)通过western印迹的、高脂饮食下6个月时Wdr13敲除小鼠和其野生型同窝出生仔畜的胰岛中细胞周期抑制物p21的表达。在敲除小鼠的胰岛中p21的表达较少。
图7.Wdr13-/0小鼠中精原细胞的增殖和调亡。A)利用H&E染色的细精管形态。B)利用BrdU标记的细精管中精原细胞的增殖。C)细精管中BrdU标记指数。D)利用TUNEL染色的细精管中的凋亡细胞。E)细精管中的凋亡指数。
图8.MIN6和MCF7细胞系中WDR13蛋白的过量表达及其对细胞增殖速率的作用。A)用AdWdr13和AdGFP病毒的转染显示WDR13蛋白在MIN6细胞中过量表达,如利用抗WDR13抗体通过免疫印迹所见。下图显示肌动蛋白作为上样对照。100MOI下72h后,WDR13蛋白的过量表达导致细胞增殖速率延迟(通过MTT测定和细胞计数二者)。B)在不依赖于雌二醇的MCF7细胞中用100MOI转染72h后,WDR13蛋白的过量表达导致细胞增殖速率延迟(MTT测定)。C)WDR13蛋白的过量表达导致p21积累,而细胞周期蛋白D1、细胞周期蛋白D2、细胞周期蛋白E1和p27的水平保持未受影响。D)利用p21特异性引物和对照通过染色质免疫沉淀揭示WDR13占据p21的启动子处。
图9.WDR13蛋白中LxxLL基序的存在。经过球形结构域过滤、结构过滤和上下文过滤后的ELM基序检索结果显示WDR13蛋白中存在NRBOX[377-383]CLNKLLL。
图10.与WDR13相互作用的蛋白的鉴定。A)利用抗FLAG抗体进行的Western印迹显示经pCMV-FLAG对照载体和具有Seq.ID No.3A的pCMV-FLAG-Wdr13载体转染后,HELA细胞中WDR13蛋白过量表达。下图显示β肌动蛋白作为上样对照。B)利用抗FLAG抗体对经pCMV-FLAG-Wdr13(Seq.ID No.3A)转染的HELA细胞中的WDR13蛋白进行的免疫定位。C)利用抗FLAG抗体来自用pCMV-FLAG对照和具有Seq.ID No.3A的pCMV-FLAG-Wdr13载体转染的HELA细胞中免疫沉淀的蛋白的银染凝胶。D)FLAG-NR2E1(Seq.ID No.6中的第922-2196位)/Myc-Wdr13共转染细胞的HEK293细胞裂解物与抗FLAG抗体免疫沉淀,随后用抗Myc抗体进行免疫印迹,其未显示NR2E1与WDR13蛋白之间的相互作用。通过FLAG或Myc特异性抗体,5%的上样量显示细胞裂解物中的蛋白表达。
图11.WDR13与ERs、HDACs和PHIP1的相互作用。A)HEK293细胞被用FLAG-Wdr13(Seq.ID No.3A中的第922至2443位)和GFP-ERα共转染,在存在或不存在雌二醇下生长。细胞裂解物用抗FLAG抗体进行免疫沉淀,并用抗GFP抗体进行免疫印迹。5%的上样量显示用FLAG或GFP特异性抗体的蛋白表达。B)HEK293细胞被用FLAG-Wdr13(Seq.ID No.3A中的第922至2443位)和Myc-ErβI(Seq.ID No.5中的第900-2673位)共转染,在存在或不存在雌二醇下生长。细胞裂解物用抗FLAG抗体进行免疫沉淀,并用抗Myc抗体进行免疫印迹。5%的上样量显示用FLAG或Myc特异性抗体的蛋白表达。C)HEK293细胞被用Myc-Wdr13(Seq.IDNo.3B中的第900至2448位)/多种FLAG-HDACs共转染,细胞裂解物用抗FLAG抗体进行免疫沉淀,并用抗Myc抗体进行免疫印迹。使用FLAG或Myc特异性抗体,5%的上样量显示蛋白表达。D)HEK293细胞被用FLAG-Wdr13/Myc-PHIP1共转染,细胞裂解物用抗FLAG抗体进行免疫沉淀,并用抗Myc抗体进行免疫印迹。使用FLAG或Myc特异性抗体,5%的上样量显示蛋白表达。E)在ERα或ERβ存在下HEK293细胞中WDR13对ERE-TATA-LUC报告基因的阻遏。TSA,一种HADCs抑制物,在ERα的情况下缓解这种阻遏。细胞在24孔板中用ERE-TATA-LUC(250ng)、βgal(50ng)、ERα或ERβ(50ng)、Wdr13(250ng)转染,一式3份,并在转染后30h测定荧光素酶和βgal的活性。用βgal的数值标准化荧光素酶数值。
发明详述
实施例
以下实施例以阐述本发明的方式给出,因此其不应当被视为限制本发明的范围。
实施例1:
Wdr13敲除小鼠的产生
Wdr13是位于X染色体上的单拷贝基因[4]。靶向策略设计为用含有polyA的新霉素基因盒取代内源性基因的内含子1(部分)、外显子2、内含子2和外显子3(部分)(图1A)。为了构建Wdr13基因靶向载体(Seq ID no.1),将来自该基因的包含外显子1至外显子7的7.1kbHindIII片段亚克隆入pBluescrip II KS载体(Stratagene)中。用pMC1neo Poly A(Stratagene)的XhoI-SalI片段替代来自该片段的跨越外显子2和外显子3(部分)的1.35kb区域。为了进一步富集靶向事件,在Wdr13基因同源序列的5’末端前置入负向选择性HSV-tk基因。所得载体在5’和3’末端分别具有1.6kb和4.1kb的同源序列。将40微克线性化靶向载体DNA电穿孔入R1ES细胞中。用G418(0.25mg/ml)和更昔洛韦(2μM)对ES细胞进行选择。为了鉴定被靶向的克隆,从ES细胞中分离出基因组DNA,并利用来自该5’末端的座位的700bpEcoRV-BamHI片段作为探针进行southern杂交(图1B)。将被靶向的克隆之一注射入3.5-dpcC57BL/6的胚泡中,并将后者转移入CD1假妊娠雌性的子宫中。为了获得突变等位基因的种系传递,使嵌合雄性小鼠与CD1雌性配对。通过southern分析证实种系传递。使用PCR利用来自Wdr13座位的引物对(表1)和来自新霉素的另一个引物对对突变小鼠进行常规筛选。
表1-引物列表
为了进一步证实Wdr13基因的靶向,进行northern印迹和western印迹。在液氮中将多种组织瞬间冷冻并在进一步使用前储存于-80℃。利用Rneasy Mini Kit(Qiagen)分离总RNA。对于Northern分析,使RNA在含有2.2M甲醛的1%琼脂糖凝胶上电泳,并用50mM NaOH印迹于N+尼龙膜上。利用随机引物试剂盒用αdATP对Wdr13cDNA进行放射性标记。在0.5M磷酸盐缓冲液/7%SDS/1mM EDTA中于65℃杂交过夜,接着在40mM磷酸盐缓冲液/1%SDS/1mMEDTA中于65℃清洗膜3次。使膜暴露于X射线板(Fuji胶片)中并显影。利用Wdr13 cDNA探针进行的Northern印迹分析,其显示与野生型组织相比,来自敲除小鼠的脑和睾丸分别缺少4kb和2kb转录体(图1C)。从多种组织中提取蛋白,在10%SDS-PAGE上分离,印迹于PVDF膜上并进行western印迹。用经纯化的抗WDR13抗体(Sigma)使蛋白可视。利用抗WDR13抗体进行的Western印迹,其显示敲除小鼠的多种组织中缺失WDR13蛋白。这些结果证实了Wdr13基因的靶向已经导致产生无效等位基因。
实施例2:
Wdr13敲除小鼠是有活力且可育的。Wdr13-/0雄性和Wdr13-/-雌性敲除小鼠是有活力且可育的。鉴于Wdr13基因在精原细胞和精母细胞中表达水平相对较高以及睾丸中存在不同大小的转录体[4],我们对来自包括突变和野生型小鼠在内的多种配对的幼崽量进行分析。亲本的基因型对幼崽量没有影响(表2)。来自Wdr13-/0雄性的精子数量(40.6±5.49万/ml)与Wdr13+/0雄性类似(37.9±3.05万/ml)。
表2-Wdr13基因型对幼崽量的影响
配对类型 | 配对数量 | 平均幼崽量 |
Wdr13-/0X Wdr13-/+ | 6 | 11.7 |
Wdr13+/0X Wdr13-/+ | 7 | 10.6 |
Wdr13-/0X Wdr13+/+ | 5 | 10.4 |
Wdr13-/0X Wdr13-/- | 6 | 09.8 |
实施例3:
Wdr13敲除小鼠的体重增加。将小鼠饲养于温度、湿度和日光/黑暗循环(12小时,早6时-晚6时)可控的动物房中。经高压蒸汽灭菌的正常饮食或高脂饮食随意采食,并每周测定饲料摄取量。每两周对小鼠进行称重。当饲喂正常食物时,9月龄左右,Wdr13缺失小鼠的体重不同于其野生型同窝出生仔畜。11个月时,突变雄性和雌性小鼠分别具有高于其同窝出生仔畜13%和11%的体重(P≤0.05)(图2A、B)。为了确定身体组成,将小鼠解剖并测量多种器官的重量。Wdr13-/0小鼠中附睾脂肪垫的重量与野生型相比高2.5倍,而Wdr13-/-中卵巢脂肪垫的重量与野生型相比高2倍(图2D)。附睾脂肪垫的组织学检查显示Wdr13敲除小鼠中脂肪细胞肥大(图2C)。在突变小鼠的皮肤切片中也观察到了脂肪细胞肥大。12个月时对突变和野生型小鼠的多种器官,包括脑、肺、心、肝、胰脏、脾脏和睾丸进行称重。突变小鼠具有显著较高的胰脏重量。甚至在突变小鼠中相对于体重的胰脏重量较高。其他器官的重量无差别(表3)。
表3-12月时来自Wdr13突变小鼠和其野生型同窝出生仔畜的多种器官的重量(克)
*数值是统计学上有差异的(P<0.05)
为了了解饮食对突变小鼠较高体重的影响,以高脂(60%)饮食饲养1月龄小鼠。有趣的是,5个月时Wdr13-/0称重显著较高(图3A),表明突变小鼠轻度肥胖表型加重。
实施例4:
Wdr13敲除小鼠中的葡萄糖和胰岛素水平。鉴于年龄依赖性肥胖表型,我们对以正常食物饲喂的小鼠中不同年龄点时的空腹和随机血糖水平进行测定(图2E、F)。利用优越血糖测试带(Accu-Check)用血糖仪测定血糖。Wdr13基因型对空腹葡萄糖水平没有影响。类似地,2个月时的随机葡萄糖水平在突变和野生型小鼠之间没有差异。然而,在正常食物下12个月时的突变小鼠显示随机葡萄糖较低。鉴于12个月时的随机葡萄糖较低,我们利用ELISA试剂盒(Linco Research)对血清胰岛素水平进行评估。在两个年龄点,空腹胰岛素水平在野生型和突变小鼠中类似(图2G)。2个月时随机胰岛素水平在Wdr13缺失和野生型小鼠中类似,有趣的是,在正常食物下12个月时,突变小鼠具有2.13倍多的随机胰岛素水平(图2H)。类似地,当饲喂高脂饮食时,6个月时突变小鼠具有1.6倍多的随机胰岛素水平(图3B)。
实施例5:
Wdr13敲除小鼠中葡萄糖清除增加但是胰岛素敏感性不变。鉴于Wdr13敲除小鼠中随机胰岛素水平较高,我们用葡萄糖对这些小鼠进行刺激,以确定在正常食物下2个月和12个月时以及在高脂饮食下6个月时其葡萄糖清除情况。为了比较葡萄糖清除和胰岛素分泌,小鼠停止饲喂16小时,腹腔内注射葡萄糖(2.0克葡萄糖/Kg体重)并以0″、30″、60″和120″的间隔收集血液,以用于葡萄糖和胰岛素测定。突变和野生型小鼠在2个月时显示类似的葡萄糖清除(图4A、B)。12个月时,突变小鼠看似具有更好的葡萄糖清除;然而,在统计学上没有显著差异。在高脂饮食下6个月时,突变小鼠显示一致地较好的葡萄糖清除(图3D)。腹腔内注射人胰岛素(1U/kg)后,通过以0″、30″、60″和120″的间隔测定葡萄糖水平以对胰岛素耐受进行评估。Wdr13-/0小鼠以与野生型类似的方式对外来胰岛素产生应答,其表明在正常食物下,胰岛素敏感性在2个月时(图4C)和12个月时(图4D)没有不同。类似地,没有证据表明在高脂饮食下6个月时(图3C)和9个月时(图4E)突变小鼠中产生胰岛素抗性,尽管这些小鼠中有显著较高的随机胰岛素水平(图3B)。
实施例6:
Wdr13敲除小鼠中胰岛质量增加以及β细胞的增殖。由于Wdr13缺失小鼠是血胰岛素高的和轻度肥胖的,我们对饲喂高脂饮食6个月时这些小鼠中的胰岛组织学进行分析。对于组织学检查,将组织在缓冲的4%多聚甲醛中固定过夜,包埋于石蜡中并进行切片(厚度4μm)。将切片置于正电荷玻片(Fisher Scientific)上并用苏木精-伊红进行染色。利用Axioskop(Axivision软件)从来自于野生型和突变基因型每种4只小鼠的胰脏切片中对胰脏和胰岛区域进行测定。用相对胰岛区域乘以胰脏湿重从而计算每个胰脏的胰岛质量。有趣的是,6个月时Wdr13敲除小鼠中胰岛的总质量显著较高(图5A、B)。为了理解增加的胰岛质量,我们通过原位BrdU标记对β细胞的增殖进行测定。简而言之,将1月龄小鼠在高脂饮食下饲喂3周,随后灌入BrdU。为了进行β细胞增殖的测定,BrdU在饮用水中(1mg/ml)给予小鼠7天,进行胰脏切片,并用抗胰岛素(R&D System)和抗Brdu(BD Biosciences)抗体进行免疫染色。通过cy3或FITC偶联的抗体检测一抗。我们观察到Wdr13突变小鼠与其野生型同窝出生仔畜相比2倍多的β细胞的增殖(图6A、B)。类似地,Wdr13敲除小鼠的睾丸中存在精原细胞增殖较多的趋势(图7)。
实施例7:
敲除小鼠中胰岛素水平较高是由于胰岛质量增加而非每单位胰岛的胰岛素分泌增加。鉴于Wdr13-/0小鼠中葡萄糖清除较好,将葡萄糖注射入6月龄(高脂饮食下)空腹小鼠中,并在不同的时间点对胰岛素分泌进行监测(图3E)。葡萄糖注射后30分钟,Wdr13-/0小鼠中的胰岛素分的分泌为1.8倍多。突变小鼠中胰岛素水平较高可能是我们观察到的这些小鼠中胰岛质量增加的结果(图5A、B),或者可能是由于应答于葡萄糖刺激,每单位β细胞的胰岛素分泌较高。为了排除后一种可能性,我们从3月龄Wdr13-/0和其野生型同窝出生仔畜(n=3)中如Ting Wen等人所描述[29]的分离胰岛。简而言之,将溶解于Hanks’平衡盐溶液(HBSS)中的2mg/ml IV型胶原酶(Invitrogen)注射入胆总管中并使胰脏于37℃孵育30分钟。经胶原酶消化后,胰脏组织用冰冷HBSS冲洗2次,并通过聚蔗糖梯度纯化胰岛。经纯化的胰岛用HBSS清洗2次,并转移至添加了10%胎牛血清、100U/ml青霉素和50μg/ml链霉素的RPMI1640培养基中过夜复苏。第二天早晨,将胰岛转移至添加了10mM HEES、2.8mM葡萄糖、0.2%BSA的Krebs-Ringer碳酸氢盐缓冲液(111mM NaCl、4.8mM KCl、2.3mM CaCl2、1.2mMMgSO4、25mM NaHCO3)中并在5%CO2下于37℃孵育90分钟。为了测定体内的胰岛素分泌,将5份相等大小的胰岛放置(一式3份)于含有250μl Krebs-Ringer碳酸氢盐缓冲液/2.8mM葡萄糖或250μl Krebs-Ringer碳酸氢盐缓冲液/16.0mM葡萄糖的96孔板单个孔中,并在5%CO2下于37℃孵育1h。收集上清液并储存于-80℃,以进行胰岛素测定。突变和野生型胰岛之间的体内胰岛素分泌没有差异(图5C)。
实施例8:
Wdr13基因的过量表达导致细胞生长延迟。为了进一步理解Wdr13基因在β细胞增殖中的作用,利用pAd Easy系统在MIN6细胞系中过量表达WDR13蛋白。为了过量表达WDR13蛋白,制备Wdr13腺病毒构建体。简而言之,利用T7正向引物(Seq.ID No.16)和反向引物从pCMV-FLAG-Wdr13(Seq.ID No.3A)载体中扩增Wdr13 cDNA,用PmeI消化并克隆到pAdTrack-CMV载体的EcoRV处,其中反向引物为5’GCTCTAGAGCAGCACAGGGTGACAGAACC3’。依照He等人[35]在HEK293包装细胞系中制备AdGFP或Ad Wdr13。MIN6细胞、HEK293细胞和MCF7细胞从印度浦那的国立细胞科学中心的细胞库获得。24孔板的每个孔接种10,000个Min6细胞[36],并用AdGFP或Ad Wdr13以20或100MOI的效价进行转染。通过MTT测定以及通过每间隔24h进行细胞计数监测细胞数量。对于western印迹分析,用AdGFP或Ad Wdr13病毒以100MOI转染MIN6细胞,并在感染后48小时裂解细胞。在24孔板中用AdGFP和Ad Wdr13以每种100MOI转染MIN6细胞。转染48小时后,利用抗WDR13抗体通过western印迹证实Wdr13的过量表达(图8A)。进一步地,在100MOI下过量表达WDR13后,通过细胞计数制作MIN6细胞生长曲线。在100MOI下,转染48h后细胞生长显著降低(图8A)。在MCF7细胞中进一步观察到生长延迟表型(图8B)。为了理解WDR13的过量表达所致生长延迟,通过western印迹对多种细胞周期调控物进行分析。WDR13蛋白过量表达后,细胞周期蛋白D1、细胞周期蛋白D2、细胞周期蛋白E和p27的蛋白水平没有变化,而p21高度上调(图8C)。相反,在Wdr13敲除胰岛中,western印迹显示p21水平降低(图6C)。为了理解WDR13对p21的调控性质,对WDR13蛋白在p21启动子处的启动子占据进行分析。用AdGFP和Ad Wdr13以20MOI转染MIN6细胞,并使其生长48小时。细胞在1%甲醛中于室温交联10分钟,并刮入含有蛋白酶抑制剂的PBS中。利用密理博的染色质免疫沉淀(ChIP)分析试剂盒(Catalogue Number:17-295)根据厂商说明进行染色质免疫沉淀。简而言之,通过在琼脂珠中于室温孵育1h对细胞裂解物进行预清洗,随后利用抗FLAG琼脂珠免疫沉淀3h。清洗珠,并洗脱出基因组DNA片段,以通过PCR对多个启动子区域进行鉴定。染色质免疫沉淀显示WDR13与p21启动子相互作用,表明WDR13在p21调控中直接发挥作用(图8D)。
实施例9:
与WDR13相互作用的蛋白的鉴定。真核生物蛋白功能位点数据库显示WDR13蛋白含有5个WD重复和第378-382位处的核受体盒(LNKLL)(图9)以及多个酪蛋白激酶和GSK3磷酸化位点。一般WD重复蛋白提供蛋白-蛋白相互作用的平台。LxxLL基序的存在进一步扩大了WDR13以配体依赖性或非依赖性方式与核受体相互作用的可能性。为了鉴定其相互作用的伴侣,在表达WDR13的Hela细胞中进行免疫沉淀(图10C)。为了过量表达FLAG-WDR13融合蛋白,通过将Wdr13 cDNA克隆到pCMV-FLAG质粒的EcoRI和XbaI位点处,构建pCMV-FLAG-Wdr13(Seq.ID No.3A)质粒[4]。通过western印迹和免疫定位证实WDR13的过量表达(图10A、B)。对于免疫沉淀,利用Xfect试剂[37]将pCMV-FLAG-Wdr13(Seq.ID No.3A)质粒和对照pEF1GFP质粒DNAs转染入HELA细胞(对于每种质粒,放置于10个100mm的盘中)中。48h后,在裂解缓冲液(50mM-Tris.HCl,150mM-NaCl,1mM-EDTA,1%-Triton X-100和蛋白酶抑制剂混合物)或(50mM-Tris.HCl,150mM-NaCl,1mM-EDTA,1%-Triton X-100,0.1%SDS和蛋白酶抑制剂混合物)中裂解细胞。对细胞裂解物进行离心,并用蛋白A预清洗。将抗FLAG琼脂珠(Sigma)加入到经预清洗的裂解物中,并使裂解物于室温孵育4h。免疫复合体用清洗缓冲液(50mM-Tris.HCl,150mM-NaCl,1mM-EDTA,0.5%-Triton X-100和蛋白酶抑制剂混合物)清洗4次。利用FLAG肽(Sigma)洗脱出免疫复合体,并在12%SDS PAGE上分离。凝胶用0.1%考马斯亮蓝R-250(40%甲醇和10%乙酸中)进行染色或者印迹于PVDF膜上。将凝胶切割成大致相等大小的3片,并根据厂商说明书(Sigma)用于胰蛋白酶消化。利用与在线偶联到MALDI靶向测位仪(Probot,LC Packings,Dionex)的反相柱(ZORBAX 300SB-C18,75μm×150mm,3.5μm,Agilent Technologies)相连的毛细液相色谱系统(Agilent HPLC 1100Series,Agilent Technologies)分离肽。在4800MALDI TOF/TOF分析仪(Applied Biosystems)上进行质谱分析。针对蛋白序列数据库2009智人蛋白序列数据库对所有MS/MS谱进行检索。联合使用MASCOT Server(2.0版本)和GPS-ExplorerTM 3.6软件(Applied Biosystems)对蛋白进行鉴定。肽离子得分≥15的肽被认为是显著的ID。从3个独立实验中鉴定出的蛋白列表总结于表4中。
表4-纯化WDR13免疫复合体后通过LC-MS/MS鉴定出的相互作用的蛋白候选物列表
离子得分大于15被认为是显著的。
实施例10:
WDR13与HDAC 1、3和7相互作用。LC-MS/MS结果(表4)表明,HDAC7与WDR13相互作用。HDAC7与WDR13的相互作用通过在HEK293细胞中进行免疫共沉淀实验进一步证实(图11C)。鉴于多种HDACs在结构和功能上的相似性[38],HDAC7与WDR13的相互作用扩大了该蛋白可能与其他HDACs相互作用的可能性。为了测试这一假设,使I类和II类HDACs二者单独地与WDR13共沉淀。为了过量表达多种HDACs,使用pCMX-hHDAC1-FLAG、pCMX-mHDAC2-FLAG、pCMX-hHDAC3-FLAG、pCMX-hHDAC4-FLAG和pCMX-mHDAC7-FLAG,并且为了过量表达Myc WDR13融合蛋白,通过将Wdr13cDNA克隆到N末端含有Myc肽的pCMV-Myc载体的EcoRI和XbaI位点处,构建pCMV-Myc-Wdr13(Seq ID No.3B)质粒。这些实验显示,WDR13与HDAC1和HDAC3而不与HDAC2和HDAC4相互作用(图11C)。
实施例11:
WDR13与核受体ERα、ERβ和PHIP1而不与NR2E1相互作用。LC-MS/MS结果(表4)表明,WDR13与PHIP1、NR2E1和ERβ相互作用(尽管离子得分较低)。WDR13蛋白中LxxLL基序(图9)的存在鼓励我们通过免疫共沉淀进一步检查WDR13与核受体即ERα、ERβ、NR2E1和PHIP1的相互作用。构建多个过量表达载体。对于Myc NR2E1融合蛋白的过量表达,利用
5’CCGGAATTCCGGATGAGCAAGCCCGCCGGATC3’和
5’GCTCTAGAGCTTTTGAGGCTTGACCTGCAT3’引物对从脑中克隆核受体2E1 cDNA(Seq.ID No.17和18),并克隆到pCMV-Myc质粒的EcoRI和XbaI位点处。对于Myc PHIP1(与普列克底物蛋白同源结构域相互作用蛋白1)融合蛋白的过量表达,利用针对N末端WD结构域的正向引物(Seq.ID No.19)
5’CTCGTGAGCACACACTGACA3’和反向引物(Seq.ID No.20)
5’TTCCATATCCCAAGGACTCA3’以及针对C末端溴结构域(bromodomain)的正向引物(Seq.ID No.21)5’CCCAGGAGATGTCCATTTGT3’和反向引物(Seq.ID No.22)5’AATCCGTCATAGCAGCAAGG3’从睾丸中扩增PHIP1 cDNA。通过连接这两个片段,产生pCMV-Myc-PHIP1(Seq.ID No.4)质粒。对于ERα的过量表达,使用pCI-nGL1-HEGO质粒[39]。为了构建带有Myc标签的雌激素受体β,利用
5’GCAGGAATTCATGTCCATCTGTGCCTCTTCT3’和
5’CAGTCTAGATCACTGTGACTGGAGGTTC4TG 3’引物对从睾丸中扩增ERβ cDNA(Seq.IDNo.23和Seq.ID No.24),并克隆到pCMV-Myc载体的EcoRI和XbaI位点处。免疫共沉淀实验显示,ERα和ERβ以配体非依赖性方式与WDR13相互作用(图11A、B)。进一步地,免疫共沉淀实验显示PHIP1与WDR13相互作用(图11D)。然而,在我们的实验条件下,我们不能检测到WDR13与NR2E1的任何相互作用(图10D)。
实施例12:
ERα和ERβ被WDR13所阻遏。为了理解WDR13与ERα、ERβ和HDACs相互作用的功能重要性,进行瞬时荧光素酶测定(图11E)。通过将雌激素应答元件和TATA序列克隆到Pg13载体的SmaI、BglII位点处,构建3XERE-TATA-LUC载体(Seq.ID No.7)[40]。对于雌二醇(10nM)和曲古抑霉素A(50nM)处理,将HEK293细胞培养于不含酚红的DMEM、5%经活性炭清除的血清、50μg/ml青霉素和50μg/ml链霉素中。利用脂质体2000(Invitrogen)根据厂商说明对细胞进行转染。WDR13的过量表达导致由ERα或ERβ介导的ERE驱动的荧光素酶活性降低~2倍。
在ERα的情况下,报告子活性的这种阻遏被一种HDACs抑制剂-曲古抑霉素A(TSA)所缓解,而TSA对ERβ介导的活性没有影响,表明WDR13可能通过HDACs阻遏ERα介导的转录。
统计学分析。利用非配对双尾t测试进行统计学分析。利用Microsoft Excel软件计数P值。P值<0.05被认为是显著的。数据以平均值±SEM表示。
伦理陈述。所有的小鼠实验在印度海得拉巴的细胞和分子生物学动物伦理委员会机构中心的批准下进行。
讨论
在本研究中,通过在小鼠中敲除该基因我们检查了WD重复蛋白中的一种即WDR13的体内作用。突变小鼠是有活力且可育的,无任何明显的表型,除了当饲喂正常食物时,小鼠在大约9个月时显著重于其野生型同窝出生仔畜并且直至12个月生长实验结束时持续增重更多。当饲喂这些小鼠高脂饮食时,突变小鼠的这种年龄依赖性的较高的体重达到5个月(图3A)。解剖学和组织学检查表明,突变小鼠体重增加主要是由于由脂肪细胞肥大导致的脂肪组织体积/重量的增加,其中无脂肪细胞数量改变的任何迹象(图2C)。
正常饮食下12个月时和高脂饮食下6个月时,Wdr13敲除小鼠轻度肥胖并且血胰岛素高,而空腹胰岛素水平是正常的。产生胰岛素的胰脏β细胞的质量根据代谢情形的动态变化以及体重、胰岛素产生和胰岛质量之间的正相关性已被详细记载[12,41]。多项研究已经显示,胰岛素水平增加是应答于肥胖的外周胰岛素敏感性降低的补偿机制,最终导致胰岛衰竭和II型糖尿病[42]。另一方面,还已知较高的胰岛素水平导致脂肪组织的葡萄糖摄取较高,这将转而改变脂质代谢和脂肪生成[43]。与后面的发现一致,胰岛素受体敲除小鼠表现出脂肪组织减少[44]。在Wdr13敲除小鼠中,高脂饮食下6个月时,胰岛质量、胰岛素水平和葡萄糖刺激的胰岛素分泌较多。从ITT结果显而易见的是,尽管6个月时以及先前体重较高,这些小鼠具有较好的葡萄糖清除,而胰岛素敏感性是正常的。我们表明,如我们在Wdr13小鼠中所观察到的,Wdr13敲除小鼠的体重增加与较高的胰岛素的总体生长刺激作用相关,而非较高的胰岛素水平是轻度肥胖的反馈。胰岛素刺激肝脏的脂肪生成以及脂肪细胞的脂质吸收,使得脂肪组织的形成增加[30]。此外,胰岛素受体葡萄糖转运蛋白-4途径帮助将脂肪组织中的葡萄糖转化为脂质[43]。除了胰岛素在脂肪生成中的作用之外,胰岛素分泌在人[22]、啮齿目动物[23]和非哺乳动物禽类模型中[45]与肥胖正相关。至少长达12个月时,我们未观察到突变小鼠的胰岛素敏感性与其野生型同窝出生仔畜有任何差异。如上面所讨论,Wdr13敲除小鼠中的葡萄糖清除较好,表明这些小鼠中较高的胰岛素分泌是本研究中观察到的低水平葡萄糖的原因。因此,鉴于胰岛素敏感性在Wdr13敲除小鼠中是正常的,胰岛素超分泌看似不太可能是这些小鼠轻度肥胖导致的胰岛补偿反应。Wdr13敲除小鼠中的高胰岛素血症以及轻度肥胖使得联想到MOR1[29]和chop[28,34]敲除小鼠,在该小鼠中较高的胰岛素分泌了加重肥胖症。Wdr13敲除小鼠中的食物摄取与野生型同窝出生仔畜相比稍高但是在统计学上不显著。由于Wdr13表达于下丘脑和脑的其他区域[7],基于我们目前的研究,在其他组织中不能排除Wdr13基因在饲喂行为和/或在系统代谢中发挥作用的可能性。
之前我们已经报道了Wdr13在胰脏中表达水平相对较高[3],并且与在腺泡细胞中可见的极低水平相比,该基因在胰岛中的表达要多得多(图1D)。当1月龄断乳后饲喂高脂饮食5个月时,Wdr13敲除小鼠具有较高的胰岛质量。为了理解这种胰岛质量增加的现象,对1月龄小鼠饲喂高脂饮食3周后,我们对其体内β细胞增殖进行测定。Wdr13敲除小鼠与其野生型同窝出生仔畜相比具有增强的β细胞增殖(图6A、B)。此处可以回想到成年突变小鼠中胰脏质量的相对重量显著较高。看似Wdr13敲除小鼠中β细胞质量的增加可能是由于β细胞增殖增强。通过使这种现象逆转而进一步加强了该结论,即过量表达Wdr13基因时,我们在胰脏MIN6细胞中观察到生长延迟(图8)。野生型和敲除胰岛的体外胰岛素分泌能力之间没有差异(图5C)为上述结论提供了进一步的间接支持。
已经报道了造成β细胞增殖的多种外在和内在因素[12,16]。存在许多β细胞的正向调控物,包括肠促胰岛素[46]、EGF[47]、催乳激素和生长激素[48]、HNF-4[49]、钙调磷酸酶/NFAT[50]、Wnt3a[51]和整合素[52]。已经鉴定出了多种细胞周期抑制物(p15、p16、p18、p19、p21、p27和p57),其靶向多种细胞周期蛋白或细胞周期蛋白依赖性激酶,以在细胞周期的多个时期抑制进程[16]。在本研究中,胰脏MIN6细胞系中WDR13的过量表达导致p21蛋白水平显著较高,而细胞周期蛋白D1、细胞周期蛋白D2、细胞周期蛋白E和p27的水平保持不变(图8C)。与这些结果一致,Wdr13基因的敲除导致敲除小鼠的胰岛中p21的下调[53](图6C)。
Wdr13是WD重复蛋白家族的成员,已知这些蛋白在蛋白-蛋白相互作用中发挥作用[1]。我们的免疫共沉淀实验显示,WDR13与ERα、ERβ、PHIP1(与普列克底物蛋白同源结构域相互作用蛋白1)、HDAC1、HDAC3和HDAC7相互作用。WDR13与PHIP1(含有溴结构域)、ERα、ERβ和组蛋白去乙酰化酶的相互作用表明WDR13在染色质调控中发挥作用。
PHIP1含有N末端的WD结构域和C末端的溴结构域以及使该蛋白核定位的双向核定位信号[9]。已经报道了含有溴结构域的蛋白发挥双向功能(共激活因子和协阻抑物)[54]。已经显示MIN6细胞系中PHIP1的过量表达通过增强细胞周期蛋白D2的水平而刺激细胞增殖,而PHIP1的基因沉默具有相反的效果[9]。然而,这些研究未提及其他细胞周期蛋白和CDKIs的水平。在我们的实验中,胰脏MIN6细胞系中Wdr13基因的过量表达显示出p21上调,并伴有生长延迟。基于这些发现和WDR13与PHIP1相互作用的证据,我们提出这两种蛋白可能共同作用以调节β细胞增殖。将需要进一步的实验,以理解这种相互作用在β细胞增殖中的功能重要性及其分子机制。
我们已经显示WDR13与雌激素受体(α、β)以配体非依赖性方式相互作用,并且在ERE荧光素酶报告基因测定中,WDR13以HDAC依赖性(ERα)和非依赖性(ERβ)方式阻遏雌激素受体的活性(图11)。WDR13与ERα/ERβ的相互作用可以通过存在于WDR13中的LxxLL基序介导(图9),然而这需要通过定点突变分析证实。其他研究已经显示,ERα和ERβ在不同组织中功能不同[17,55,56]。已经记载了在胰脏β细胞的情况下雌激素和雌激素受体的保护性作用[17]。Wdr13敲除小鼠中胰岛质量和β细胞增殖增加可能是由于一些基因组座位处Wdr13对雌激素受体阻遏作用的移除。在本研究中,Wdr13敲除胰岛中p21的下调、Wdr13基因过量表达所致胰脏MIN6细胞中p21的上调以及CHIP实验中p21启动子被WDR13占据的证据强烈地表明,p21基因是WDR13的靶标。有趣的是,已经报道了ERα和HDACi(HDAC抑制蛋白)对p21的调控作用[57,58]。在雌激素存在下,ERα通过以p53依赖性方式招募共阻遏物而阻止p21的表达[59];使用HDAC抑制因子导致p21上调和细胞增殖延迟[57]。然而,我们对使用ERE元件的报告基因测定的发现表明,ERα与WDR13的相互作用以HDAC依赖性方式抑制ERα。调和这些似乎矛盾的数据的一种方法是假设WDR13与ERα在p21启动子处的相互作用以HDAC非依赖性方式抑制ERα。这种设想将解释我们的研究中观察到的WDR13对p21的上调(图8C)。已知几种阻遏蛋白与ERα相互作用,并且可以在不同组织/靶基因中以HDAC依赖性或HDAC非依赖性方式发挥作用[60]。在本文中,有趣的是,注意到WDR13与ERβ在ERE元件处的相互作用导致以HDAC非依赖性方式的阻遏(图11E)。
可以回想到Wdr13基因在大多数组织中表达,并且可能该蛋白与其伴侣的相互作用中的一些甚至是组织特异性的,因此作用的精确机制在不同组织中和/或对不同的靶基因可能是不同的。大量的CHIP实验将是必需的,用以阐明多种细胞类型中WDR13的多种靶基因以及在这些座位处的作用方式。
总地来讲,我们提供了小鼠中Wdr13基因的缺陷使得β细胞质量增加、胰岛素高血症、葡萄糖清除较好以及轻度肥胖的证据。我们表明,我们在突变小鼠中观察到的这些胰脏相关的表型变化是由这些细胞中WDR13缺失下p21下调所致β细胞增殖增强的结果。然而,进一步的研究是必需的,用以揭示与该突变相关的任何其他表型变化。进一步地,我们的数据提供了WDR13与ERα、ERβ、PHIP1和多种HDACs即1、3和7相互作用的证据。如ERE报告基因测定中所见,WDR13与ERs的相互作用是雌二醇非依赖性的,并且导致其阻遏。有趣的是,WDR13的这种阻遏是HDAC依赖性(ERα)和非依赖性(ERβ)。最后,我们提出鉴于Wdr13敲除小鼠中胰岛素水平较高,葡萄糖清除较好以及缺少胰岛素抵抗证据,该蛋白可能被探索作为用于减轻糖尿病中受损的葡萄糖代谢的潜在候选药物靶标。
优势:
1.已经鉴定出在葡萄糖稳态和细胞增殖中发挥作用的新蛋白。
2.已经鉴定出雌激素受体α和雌激素受体β的新的阻遏蛋白。
3.已经鉴定出以HDAC依赖性方式调控雌激素受体α和以HDAC非依赖性方式调控雌激素受体β的新的调控蛋白。
4.已经鉴定出一种新的p21表达调控蛋白。
5.WDR13是在糖尿病和癌症的治疗中可用作药物靶标的新蛋白。
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Claims (9)
1.一种表达构建体,其具有用于靶向Seq Id No.1所示的WDR13基因的Seq Id No.2,包括:
a.作为阳性选择标志物的具有polyA的新霉素,
b.作为阴性选择标志物的HSV-tk,
c.用于5’重组的1.6kb 5’同源区,
d.用于3’重组的4.1kb 3’同源区。
2.一种制备小鼠肿瘤模型系统的方法,其包括:
a.提供Seq Id no 1,
b.制备如权利要求1中所述的表达构建体,
c.将如步骤b中所述的表达构建体电穿孔入ES细胞中,
d.通过southern印迹对步骤c中获得的被靶向的ES细胞克隆进行选择,
e.通过已知方法由步骤d中获得的被靶向的ES细胞克隆产生敲除小鼠,
f.通过已知方法育种步骤e中获得的敲除小鼠,以达到种系传递,
g.获得小鼠肿瘤模型。
3.通过如权利要求2中所述的方法制备的肿瘤模型系统,其用于在小鼠的多个时期研究癌症的演进。
4.具有Seq Id no.1的WDR13基因作为生物标志物用于治疗糖尿病和癌症的用途。
5.WDR13作为β细胞增殖的负向调控物的用途。
6.WDR13作为以HDAC依赖性和HDAC非依赖性方式的雌激素受体阻遏物的用途。
7.WDR13蛋白及其下游靶标用于体内调节β细胞和睾丸精原细胞增殖的用途。
8.WDR13蛋白及其下游靶标在发现用于体内调节β细胞和睾丸精原细胞增殖的药物中的用途。
9.WDR13敲除小鼠模型系统在研究能够体内调节β细胞和睾丸精原细胞增殖的药物的效果中的用途。
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A Mouse Gene Encoding a Novel Member of the WD Family of Proteins Is Highly Conserved and Predominantly Expressed in the Testis (Wdr13);AMRITHA SURESH等;《Molecular reproduction and development》;20051231;第72卷;第299-310页 * |
Essay:Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century;MARIO R. CAPECCHI;《NATURE REVIEWS GENETICS》;20050601;第6卷(第6期);第507-512页 * |
Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags;Nam-Soon Kim等;《Genomics》;20040601;第83卷(第6期);第1024-1045页 * |
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