CN104109670B - 一种双链siRNA分子及其应用 - Google Patents
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Abstract
本发明涉及生物医药技术领域,具体涉及一种双链siRNA分子及其应用,更具体地,本发明针对大鼠垂体瘤细胞MMQ细胞中D2R基因的核苷酸序列设计合成siRNA,所述siRNA转入MMQ细胞后能明显促进催乳素的合成和分泌。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种双链siRNA分子及其应用,更具体地,本发明针对大鼠垂体瘤细胞MMQ细胞中D2R基因的核苷酸序列设计合成siRNA,所述siRNA转入MMQ细胞后能明显促进催乳素的合成和分泌。
背景技术
RNA干扰(siRNA)是机体内广泛存在的一种双链RNA介导的序列特异性基因沉默现象[Fire A.,et al.Potent and specific genetic interference by double-strandedRNAin Caenorhabditis elegans.Nature1998;391:806-811],因其具有沉默效率高、特异性强以及操作简便等优于传统基因敲除手段的优点而得到迅速发展,现成为生命科学领域中重要的研究工具[Arziman Z.,et al.E-RNAi:a web application to designoptimized RNAi constructs.Nucleic Acids Res2005;33:W582-W588]。siRNA与蛋白质结合形成RNA诱导沉默复合物(RNA-induced silencing complex,RISC)。该复合物能够结合到同源的mRNA上并诱导其降解。
药源性高催乳素血症是临床常见的不良反应,发生率高达42%-66%[HalbreichU.,et al..Hyperprolactinemia and schizophrenia:mechanisms and clinicalaspects.Journal ofpsychiatric practice2003;9:344-353],短期内引起泌乳、闭经,长期会造成体重增加、骨质疏松及增加乳腺癌、子宫内膜癌的发病率,严重影响患者的身心健康及治疗的依从性。
催乳素的调节是一个很复杂的系统,涉及多种神经递质、神经肽、代谢产物、机体的激素信号改变等。这些因素大体可以分为两类:催乳素释放因子(PRF)和催乳素抑制因子(PIF)[Madhusoodanan S,.et al.Hyperprolactinemia associated withpsychotropics--a review.Human psychopharmacology2010;25:281-297]。多巴胺是最主要的催乳素抑制因子(PIF),它通过垂体催乳素细胞膜上的多巴胺D2受体(D2R),抑制催乳素的合成和释放[Cookson J.,et al.Prolactin,hyperprolactinaemia andantipsychotic treatment:a review and lessons for treatment of earlypsychosis.J Psychopharmacol2012;26:42-45]。
MMQ细胞是一种来源于大鼠垂体瘤细胞的典型高催乳素血症细胞模型[Judd AM.,et al.Characterization of the MMQ cell,a prolactin-secreting clonal cell linethat is responsive to dopamine.Endocrinology1988;123:2341-2350]。MMQ细胞的细胞膜上含有多巴胺D2受体(D2R),能够对多巴胺及多巴胺类药物反应。溴隐亭等多巴胺受体激动剂能够激动MMQ细胞上的D2R,抑制其胞内催乳素的分泌。临床中,甘草作为一种中草药,经常被用来治疗高催乳素血症。甘草苷是一种从甘草中提取的重要天然化合物,尽管现有文献已报道甘草苷具有治疗咳嗽、炎症、咽炎、抗心律失常、胃溃疡、肿瘤、脂肪肝、心肌缺血、脑血栓、脑缺氧等作用,还未见关于甘草苷治疗高催乳素血症的报道,但是甘草苷作为甘草中的活性成分,其作为候选药物在治疗高催乳素血症方面的疗效值得期待。为了研究候选药物对高催乳素血症的疗效及其作用机制,通常采用siRNA干扰的技术方案:利用针对D2R的siRNA,干扰D2R的功能,给研究对象施用候选药物,通过检测催乳素的合成和分泌情况,验证该候选药物是否具有治疗高催乳素血症的功效。
发明内容
本发明的目的在于公开一种高效的针对D2R基因的广谱小分子干扰RNA(siRNA)。因而,根据本发明的一个方面,本发明提供了一种双链siRNA分子,根据本发明的实施例,该双链siRNA分子由具有下述核苷酸序列的正义链和反义链组成:
siRNA:
正义链:5’-caGCAGGATTCACTGTGACAT-3’(SEQ ID NO:1),
反义链:5’-ATGTCACAGTGAATCCTGCTG-3’(SEQ ID NO:2);
本发明提供了siRNA分子在制备降低细胞D2R基因表达试剂中的应用。
本发明提供了siRNA分子在制备促进细胞催乳素分泌试剂中的应用。
本发明提供了siRNA分子在制备促进细胞催乳素合成试剂中的应用。
优选地,本发明使用的细胞是MMQ细胞。
根据本发明的实施例,siRNA能明显降低D2R基因的表达水平。
根据本发明的实施例,siRNA能明显缓解甘草苷介导的催乳素分泌的抑制。
根据本发明的实施例,siRNA能明显缓解甘草苷介导的催乳素合成的抑制。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
图1显示了利用免疫印迹的方法检测siRNA对D2R基因的沉默效果;
图2显示了siRNA对催乳素分泌的影响;
图3显示了siRNA对催乳素表达的影响,其中:
图3a显示了利用QPCR检测siRNA对催乳素基因转录水平的影响;
图3b显示了利用免疫印迹检测siRNA对催乳素基因表达水平的影响。
具体实施方式
下面详细描述本发明的实施例。下面通过参考附图描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
实施例1 siRNA对D2R基因沉默效果的检测实验
1.1主要仪器:细胞培养箱(Thermo)。
1.2主要材料和试剂:脂质体2000转染试剂(Invitrogen),DMEM培养基(Gibco)。
1.合成siRNA
在Genebank中找到D2R的基因序列(GenBank-ID:NM_012547)。由上海吉凯基因公司设计及化学合成,共设计三对siRNA序列,2.0OD(5nmol)*2只,如下:
siRNA1:
正义链:5’-caGCAGGATTCACTGTGACAT-3’(SEQ ID NO:1),
反义链:5’-ATGTCACAGTGAATCCTGCTG-3’(SEQ ID NO:2);
siRNA2:
正义链:5’-caGGATTCACTGTGACATCTT-3’,
反义链:5’-AAGATGTCACAGTGAATCCTG-3’;
siRNA3:
正义链::5’-aaCCTGTGTGCCATCAGCATT-3’,
反义链:5’-AATGCTGATGGCACACAGGTT-3’;
空的靶基因siRNA(siNT)
正义链:5’-UUAAGUAGCUUGGCCUUGAtt-3’,
反义链:5’-UCAAGGCCAAGCUACUUAAtt-3’。
2.沉默效率验证
2.1细胞培养:MMQ细胞在含10%FBS的DMEM培养基中,37℃、5%CO2培养箱培养。
2.2细胞铺板并转染:将细胞按1×104/孔接种到24孔细胞培养板中,在37℃、5%CO2培养箱细胞培养24小时,在无双抗含10%FBS的DMEM培养基中,转染按照脂质体转染试剂2000(购自于Invitrogen公司)的说明书转染,实验分为未转染组、阴性对照组和实验组(20nM),其中阴性对照组siRNA为通用对照siRNA,即siNT,与D2R基因的序列无同源性,浓度为20nM/孔。同时分别转染。
2.3免疫印迹(western blot)检测D2R基因蛋白表达水平:按照3.2的方法转染MMQ细胞后48小时,用细胞裂解液(150mM氯化钠+1%NP-40(去垢剂)+0.1%SDS(去垢剂)+2μg/ml Aprotinin(蛋白酶抑制剂)(使用前加入)+2μg/ml Leupeptin(蛋白酶抑制剂)(使用前加入)或1mM PMSF(蛋白酶抑制剂)+1.5mM EDTA(蛋白酶抑制剂)+1mM NaVanadate(磷酸脂酶抑制剂))收集细胞,用BCA蛋白检测试剂盒进行蛋白浓度的测定,各实验组取50μg蛋白进行SDS-PAGE,转膜,5%脱脂奶粉封闭,接着加入D2R抗体在4℃孵育过夜,膜用TBST液洗涤3次,然后再用辣根过氧化物酶(HRP)标记的山羊抗小鼠(中杉金桥有限公司,1:5000)室温孵育1h。用ECL免疫印迹化学发光试剂孵育5min后曝光、显影、定影。用凝胶成像分析系统对X-光片进行扫描并计算光密度值。蛋白相对表达量=待测蛋白的光密度值/内参的光密度值。管家蛋白β-actin为内参。
2.4统计处理:所有数据采用均数±标准差的方式,采用SPSS17.0统计软件对实验结果进行独立样本t检验和单因素方差分析,P<0.05视为差异有显著性(*代表P<0.05;#代表P<0.01)。
结果分析:siRNA与siNT瞬时转染MMQ细胞48小时后,siRNA1组MMQ细胞D2R蛋白的表达水平(0.23±0.12)与siNT组(0.81±0.24)、空白对照组(CTRL)(0.94±0.21)相比有了显著减少,siRNA对MMQ细胞的D2R蛋白表达抑制率为73.7%(F=23.89,P=0.00)。siRNA2组MMQ细胞D2R蛋白的表达水平(0.68±0.19),siRNA3组MMQ细胞D2R蛋白的表达水平(0.57±0.24)与siNT阴性对照组(0.81±0.24)、空白对照组(0.94±0.21)相比,对MMQ细胞的D2R蛋白表达抑制率分别为22%和35%。这些数据表明siRNA1能在蛋白水平强力抑制D2R的表达(图1)。每组三个平行样。因此在下面的实验中均使用siRNA1进行基因的沉默,即下文中的siRNA即代表siRNA1。
实施例2 D2R基因沉默对MMQ细胞催乳素分泌的影响
ELISA检测试剂盒检测细胞培养基中催乳素的含量:首先将标准品和待检测样本各50μl加入到相应的反应孔中。每孔加入100μl酶连物(大连泛邦公司),轻轻混匀30s,室温60min。用洗涤液洗板5次,加入100μl显色液,轻轻混匀10s,室温20min。每孔加入50μl终止液,轻轻混匀30s,30min内在ELISA检测仪上测定各孔的吸光度(A450nm值)。根据标准品的浓度和吸光度做标准曲线,计算待测样本的催乳素含量。每次实验重复3次,取平均值。
细胞培养和细胞转染:方法同实施例1
细胞转染48小时后,实验组、阳性对照组、空白对照组每组中细胞分别进行下列处理:不加药物(CTRL)处理、甘草苷100μM(Liq100)处理、溴隐亭1μM(BMT)处理,24小时后,收集细胞培养基利用ELISA检测试剂盒检测催乳素的分泌情况。
统计处理:所有数据采用均数±标准差的方式,采用SPSS17.0统计软件对实验结果进行独立样本t检验和单因素方差分析,P<0.05视为差异有显著性(*代表P<0.05;#代表P<0.01)。
结果分析:甘草苷处理后与同组的阴性对照(siNT)和空白对照(CTRL)相比,MMQ细胞催乳素的分泌减少了,但是经过siRNA干扰后,甘草苷抑制MMQ细胞催乳素分泌的结果消失了,表明甘草苷通过D2R的介导,抑制了催乳素的分泌(图2)。
实施例3 D2R基因沉默对MMQ细胞催乳素基因表达的影响
1.QPCR检测催乳素基因转录水平的变化
1.1仪器:PCR仪(ABI公司);实时定量PCR仪(Bio-Rad);细胞培养箱(Thermo),等。
1.2材料和试剂:mRNA提取纯化试剂盒(CWbio.Co.Ltd),脂质体2000转染试剂(Invitrogen),DMEM培养基(Gibco),HiFi-MMLVcDNA第一链合成试剂盒
1.3细胞培养:MMQ细胞在含10%FBS的DMEM培养基中,37℃、5%CO2培养箱培养。
1.4细胞铺板并转染:将细胞按1×104/孔接种到24孔细胞培养板中,在37℃、5%CO2培养箱细胞培养24小时,在无双抗含10%FBS的DMEM培养基中,转染按照脂质体转染试剂2000(购自于Invitrogen公司)的说明书转染,实验分为未转染组、阴性对照组和实验组(20nM),其中阴性对照组siRNA为通用对照siRNA,即siNT,与D2R基因的序列无同源性,浓度为20nM/孔。同时分别转染。
1.5荧光实时定量PCR(QPCR)检测催乳素基因mRNA水平:总RNA提取根据总RNA提取试剂盒(CWbio.Co.Ltd,Cat#CW0581)说明书进行。用HiFi-MMLVcDNA第一链合成试剂盒(CWbio.Co.Ltd,Cat#CW0744)进行反转录,实验操作按产品说明书进行。GAPDH基因(内参)的引物为5’-TGGAGTCTACTGGCGTCTT-3’(上游引物)和5’-TGTCATATTTCTCGTGGTTCA-3’(下游引物)。催乳素基因的引物为5’-TCAATGACTGCCCCACTTCT-3’(上游引物)和5’-AAACAGAGGGTCATTCCAGG-3’(下游引物)。反应体系:用UltraSYBR Mixture(With ROX)(CWbio.Co.Ltd,Cat#CW0956)进行扩增,实验操作按产品说明书进行。扩增程序为:95℃10min,(95℃15s,60℃60s)*45个循环。RealTime反应体系为:
1.6统计处理:所有数据采用均数±标准差的方式,采用SPSS17.0统计软件对实验结果进行独立样本t检验和单因素方差分析,P<0.05视为差异有显著性(*代表P<0.05;#代表P<0.01)。
结果分析:QPCR结果显示,甘草苷处理后与同组的阴性对照(siNT)和空白对照(CTRL)相比,MMQ细胞催乳素mRNA的水平下降,表明甘草苷抑制了催乳素基因的转录,但是siRNA干扰后,甘草苷抑制MMQ细胞催乳素基因转录的趋势消失了,表明甘草苷通过D2R的介导,在转录水平上抑制了催乳素的合成(图3a)。Liq100为甘草苷(100μM),BMT为溴隐亭(1μM)。
2.Western blot检测催乳素蛋白表达的变化
Western blot的步骤同实施例1,利用抗体为催乳素抗体。
统计处理:所有数据采用均数±标准差的方式,采用SPSS17.0统计软件对实验结果进行独立样本t检验和单因素方差分析,P<0.05视为差异有显著性(*代表P<0.05;#代表P<0.01)。
结果分析:western Blot结果显示,甘草苷处理后与同组的阴性对照(siNT)和空白对照(CTRL)相比,催乳素蛋白量降低,表明甘草苷抑制了催乳素的表达,但是siRNA干扰后,甘草苷降低MMQ细胞催乳素表达的趋势消失了,表明甘草苷通过D2R的介导,抑制了催乳素的合成(图3b)。Liq100为甘草苷(100μM),BMT为溴隐亭(1μM)。
尽管已经示出和描述了本发明的实施例,本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。
Claims (2)
1.一种双链siRNA分子在制备促进细胞催乳素蛋白分泌试剂中的应用,其特征在于,所述双链siRNA分子由具有下述核苷酸序列的正义链和反义链组成:
正义链:5’-caGCAGGATTCACTGTGACAT-3’,
反义链:5’-ATGTCACAGTGAATCCTGCTG-3’。
2.一种双链siRNA分子在制备促进细胞催乳素蛋白表达试剂中的应用,其特征在于,所述双链siRNA分子由具有下述核苷酸序列的正义链和反义链组成:
正义链:5’-caGCAGGATTCACTGTGACAT-3’,
反义链:5’-ATGTCACAGTGAATCCTGCTG-3’。
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