CN114437179B - 一种建立哺乳动物慢性磷脂代谢异常模型的多肽tac及其应用 - Google Patents
一种建立哺乳动物慢性磷脂代谢异常模型的多肽tac及其应用 Download PDFInfo
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Abstract
本发明公开了一种建立哺乳动物慢性磷脂代谢异常模型的多肽TAC及其应用。一种建立哺乳动物慢性磷脂代谢异常模型的多肽TAC,其全序列为CYGRKKRRQRRRATALYSAASKEKKL。它既能穿过细胞膜,又能特异抑制内源ATP5O的巴豆酰化而降低其总蛋白水平,用多肽TAC腹腔注射小鼠一个月,发现小鼠磷脂变化与慢性应激小鼠的磷脂变化高度一致。因此我们提出TAC腹腔注射可以成功建立磷脂代谢异常的模型,由于此方法不会使任何基因彻底丧失表达或功能,因此更接近慢性疾病(比如慢性应激)中的磷脂代谢异常。
Description
技术领域
本发明属于医学领域,具体涉及一种建立哺乳动物慢性磷脂代谢异常模型的多肽TAC及其应用。
背景技术
脂类是细胞膜或细胞器膜的主要组成部分,是许多重要生化反应的必需物质,也是重要的能量储存和提供者。脂类中的磷脂包括磷脂酰胆碱(Phosphatidylcholine,PC)、磷脂酰乙醇胺(Phosphatidylethanolamine,PA)和磷脂酰肌醇(Phosphatidylinositol,PI),是细胞膜或细胞器膜的主要成分,且在细胞增殖、凋亡、细胞器膜融合、氧化磷酸化、线粒体合成和自噬中发挥重要作用。它们的含量和动态变化与各种退化性疾病和代谢紊乱密切相关。比如,apoe4诱导的磷脂调节异常是导致AD(阿尔茨海默病)相关认知缺陷的关键因素。在糖尿病小鼠模型中,磷脂对调节脂质代谢的PPARα(过氧化物酶体增殖物激活受体α)的激活、介导磷脂生成、减少缺血后肢灌注和血管生成的CEPT1(胆碱-乙醇胺磷酸转移酶1)的缺失至关重要。
值得指出的是,虽然已有多项研究在药物处理小鼠或基因敲除小鼠中发现有磷脂代谢的异常,但目前还没有一种主要影响磷脂代谢的合适的动物模型。主要原因是这些模型都有复杂的多类代谢物的异常。比如,四氯化碳(CCl4)、双酚S(bisphenol S)及油酸盐(Oleate)等处理的小鼠模型除磷脂代谢异常外,还有大量其他类代谢物如脂肪酸、性激素、氨基酸、氧甾酮及胆汁酸的异常(1-3);另外,已有的多种基因敲除(KO)小鼠模型如ASCL4-KO、BLOC-1-KO及LRH-1-KO小鼠也存在类似问题(4-6)。因此,在磷脂代谢研究方面还没有一种大家公认的模型小鼠。
而本研究中的TAC多肽处理小鼠可以作为磷脂代谢的更好模型。体现在以下几点:1)、TAC处理小鼠血液生化指标除CREA、SOD及HDL-C外,大部分与对照组没有差异,提示其机体大部分生化过程没有受到影响;2)、定量脂代谢组差异脂类中,磷脂类是占比第二大的脂类(26.03%)。虽然甘油三酯(TG)和甘油二脂(DG)占比最大,但游离脂肪酸几乎没有差异,所以并不会造成显著的生理影响,这可以从大部分血液生化正常间接反映出来。
发明内容
本发明的目的是针对现有技术的上述不足,提供一种建立哺乳动物慢性磷脂代谢异常模型的多肽TAC。
本发明的另一目的是提供该多肽的应用。
本发明的又一目的是提供一种一种建立哺乳动物慢性磷脂代谢异常模型的方法。
本发明的目的可通过以下技术方案实现:
一种用于建立哺乳动物慢性磷脂代谢异常模型的多肽TAC,包含ATALYSAASKEKKL(SEQ ID NO.1)所示序列。这段序列腹腔注射到小鼠体内后,可以引起磷脂代谢异常,而无其他生理功能的显著异常。
作为本发明的一种优选,所述的多肽TAC全序列如CYGRKKRRQRRRATALYSAASKEKKL(SEQ ID NO.2)所示。SEQ ID NO.2所示多肽中穿膜肽TAT融合了能特异抑制内源ATP5O的巴豆酰化的多肽序列,既能穿过细胞膜,又能特异抑制内源ATP5O的巴豆酰化而降低其总蛋白水平,从而可以通过简单的腹腔注射建立哺乳动物磷脂代谢异常模型。
我们在一项慢性应激模型小鼠翻译后修饰组学的研究发现,慢性应激小鼠卵巢中ATP合成酶的亚基ATP5O在K51位点巴豆酰化水平降低最显著,相应地总ATP5O水平及ATP水平也显著降低。接着我们发现血清中总ATP5O水平及ATP水平也显著降低。接下来我们通过定量代谢组发现,慢性应激小鼠的有益磷脂类包括PC、PA及PI显著下调,而有害磷脂类包括溶血性PC、PA及PI(LPC、LPA及LPI)显著上调。因此我们推测ATP5O在K51位点巴豆酰化水平的下调导致的ATP5O总蛋白水平下调是导致磷脂代谢异常的主要原因。因此我们用K51位点附近序列ATALYSAASKEKKL融合穿膜肽序列TAT序列CYGRKKRRQRRR,命名为TAC(TAT-fusedATP5Ocrotonylation sequence),推测如将其注入动物体内,TAC可以与内源ATP5O竞争被巴豆酰化从而降低内源ATP5O巴豆酰化的水平,从而复现慢性应激小鼠血清磷脂代谢异常的表型。实验结果证实TAC腹腔注射处理小鼠血清的磷脂代谢的变化确实与慢性应激小鼠相似。
本发明所述的多肽TAC在建立哺乳动物慢性磷脂代谢异常模型中的应用。
一种建立哺乳动物慢性磷脂代谢异常模型的方法,向哺乳动物腹腔注射本发明所述的多肽TAC。
作为本发明的一种优选,所述的方法包括对实验哺乳动物腹腔注射6mg/Kg TAC,每日一次,持续4周,结束时可以取血清进行ATP5O blot,如果ATP5O水平显著降低,则说明造模成功。
作为本发明的进一步优选,所述的实验哺乳动物选自小鼠、大鼠、恒河猴或食蟹猴。
有益效果:
已知机体ATP水平降低可以导致脂代谢异常,而我们在一项慢性应激模型小鼠翻译后修饰组学的研究发现,慢性应激小鼠卵巢中ATP合成酶的亚基ATP5O巴豆酰化水平降低最显著,相应地总ATP5O水平及ATP水平也显著降低。接着我们发现血清中总ATP5O水平及ATP水平也显著降低。接下来我们通过定量代谢组发现,慢性应激小鼠的有益磷脂类包括PC、PA及PI显著下调,而有害磷脂类包括溶血性PC、PA及PI(LPC、LPA及LPI)显著上调。本发明提供一种高效建立哺乳动物慢性磷脂代谢异常模型的多肽TAC,该多肽能够通过简单的腹腔注射而降低ATP合成酶亚基ATP5O的水平而造成机体能量水平显著降低,从而引起模型动物的磷脂代谢异常,模型动物磷脂变化与慢性应激动物的磷脂变化高度一致。由于本发明方法不会使任何基因彻底丧失表达或功能,因此更接近慢性疾病(比如慢性应激)中的磷脂代谢异常。同时,ATP5O在不同哺乳动物物种中的高保守性,TAC腹腔注射可能广泛用在小鼠、大鼠甚至灵长类建立慢性磷脂代谢异常模型,为进一步全面研究慢性磷脂代谢对机体各方面的影响提供更合适的模型。
附图说明
图1.慢性应激小鼠卵巢的ATP5O-K51巴豆酰化及水平及总蛋白水平显著降低
A-D.我们通过束缚成功建立了慢性应激小鼠模型。E-K.卵巢泛修饰显示慢性应激导致蛋白泛巴豆酰化水平显著降低。L-P.卵巢定量巴豆酰化组显示,慢性应激导致ATP5O-K51巴豆酰化水平降低最显著,达10倍。Q-U.Blot验证了慢性应激导致ATP5O在K51的巴豆酰化和总蛋白水平显著降低,而ATP5O mRNA水平保持不变。
图2.慢性应激小鼠血清的磷脂代谢发生显著异常
A-F.定量广靶全谱代谢组显示,应激小鼠血清中有益的PC、PA及PI显著下调,而有害磷脂类包括溶血性PC、PA及PI(LPC、LPA及LPI)显著上调。G和H.应激小鼠血清中脂代谢相关酶STAT5A、FAM126A及PTDSS1显著下调。I和J.血液生化指标测定显示,应激小鼠血清中多项代谢相关指标发生异常。
图3.TAC特异下调性多肽ATP5O-K51巴豆酰化而致血清磷脂代谢异常
A-E.K51位点是ATP5O巴豆酰化的关键,失活性突变体K51A(不能发生巴豆酰化)转染的细胞相比野生ATP5O转染的细胞有显著低的ATP水平。F-I.与对照小鼠相比,TAC处理导致小鼠血清ATP5O-K51巴豆酰化和总蛋白水平均显著降低。J和K.与对照小鼠相比,TAC处理导致小鼠血清中代谢关键酶STAT5A显著降低。L-O.定量脂代谢组显示,TAC处理导致有益的PC、PA及PI显著下调,与应激小鼠血清中PC、PA及PI变化一致。P-R.血液生化指标测定显示,TAC处理小鼠血清中CREA、SOD及HDL-C水平异常。
图4.高应激评分女性的总ATP5O与磷脂水平变化趋势与stress小鼠及TAC处理小鼠一致
A-E.与低应激评分女性相比,高应激评分女性血清中总ATP5O水平显著降低。F-I.定量代谢组显示,高应激评分女性血清中有益的PC、PA及PI显著下调,LPC、LPA及LPI显著上调。
图5.人和小鼠ATP5O氨基酸序列对比
氨基酸序列对比显示人和小鼠TAC序列高度保守。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明,但不以任何方式限制本发明。
动物/个体样本纳入、实验分组、数据收集和数据分析见附件1方法1.18,统计分析见附件1方法1.19。
实施例1
束缚造模,如图1A所示(方法详见附1方法1.1),称重,结果见图1B;用ellisa试剂盒检测血清corticosterone水平(方法详见附1方法1.3);Q-PCR检测应激相关基因RFRP、GnRH、GPR147的mRNA水平(方法详见附1方法1.4,引物详见附2表1),结果如图1C和D,可见,本实施例通过束缚成功建立了慢性应激小鼠模型(图A-D)。用泛修饰抗体(各种泛修饰抗体均购自杭州景杰生物公司,各种抗体信息详见附1方法1.2)进行western blot检测(方法详见附1方法1.14)应激卵巢的泛修饰水平及量化,如图1E-K所示,卵巢泛修饰显示慢性应激导致蛋白泛巴豆酰化水平显著降低;用质谱(方法详见附1方法1.9)进行卵巢定量巴豆酰化组,结果如图1L-P所示,可见慢性应激导致ATP5O-K51巴豆酰化水平降低最显著,达10倍。用western blot验证应激小鼠卵巢的ATP5O总蛋白水平及巴豆酰化水平,结果如图1Q-U,可见慢性应激导致ATP5O在K51的巴豆酰化和总蛋白水平显著降低,而ATP5O mRNA水平保持不变。
实施例2
用质谱进行广靶全谱代谢组测定(方法详见附1方法1.10)并进行PCA(方法详见附1方法1.11)、热图分析(方法详见附1方法1.12)及代表性代谢物量化,结果显示,应激小鼠血清中有益的PC、PA及PI显著下调,而有害磷脂类包括溶血性PC、PA及PI(LPC、LPA及LPI)显著上调(图2A-F);用Western blot检测代谢相关酶的水平,结果显示应激小鼠血清中脂代谢相关酶STAT5A、FAM126A及PTDSS1显著下调(图2G和H);用生化仪进行血液生化检测,血液生化指标测定显示,应激小鼠血清中多项代谢相关指标发生异常(图2I和J)。
实施例3
用western blot检测ATP5O-WT和ATP5O-K51A突变体(质粒构建详见附1方法1.5,突变方法详见附1方法1.6)的巴豆酰化水平及量化;用lipo3000脂质体将ATP5O-WT和ATP5O-K51A质粒转染到293T细胞中(转染方法详见附1方法1.7),用ATP检测试剂盒测定ATP水平(方法详见附1方法1.17);用lipo3000脂质体将不同剂量(0、0.25、0.5、0.75、1、1.25μg)ATP5O-WT和ATP5O-K51A质粒转染到293T细胞中,加定量巴豆酸钠(2.5mM),用westernblot检测ATP5O-WT和ATP5O-K51A的巴豆酰化水平;失活性突变体K51A(不能发生巴豆酰化)转染的细胞相比野生ATP5O转染的细胞有显著低的ATP水平(图3A-E)。
对小鼠腹腔注射6mg/Kg ATP5O巴豆酰化竞争性多肽TAC(方法详见附1方法1.15),每日一次,持续4周。结束时取血清,用western blot检测血清ATP5O总蛋白及巴豆酰化水平,动物实验显示,与对照小鼠相比,TAC处理导致小鼠血清ATP5O-K51巴豆酰化和总蛋白水平均显著降低(图3F-I),说明造模成功;用western blot检测代谢酶STAT5A的水平,结果显示,与对照小鼠相比,TAC处理导致小鼠血清中代谢关键酶STAT5A显著降低(图3J-K);用质谱进行对照小鼠和TAC处理小鼠血清定量脂代谢测定,结果显示,TAC处理导致有益的PC、PA及PI显著下调,与应激小鼠血清中PC、PA及PI变化一致(图3L-O)。用生化仪进行血液生化检测,量化展示有差异的CREA,SOD及HDL-C,血液生化指标测定显示,TAC处理小鼠血清中CREA、SOD及HDL-C水平异常(图3P-R)。
实施例4
以上小鼠上的实验显示,ATP5O巴豆酰化及总蛋白水平与慢性应激导致的磷脂代谢异常密切相关。接下来我们想检验在慢性应激女性中ATP5O水平是否与血清磷脂代谢异常也相关。我们发现,用汉密尔顿焦虑量表对临床女性进行焦虑评分(HAMA),HAMA≦7作为对照组,HAMA≥14为焦虑组,14>HAMA>7为焦虑倾向组(方法详见附1方法1.8)。共纳入对照组女性23例,焦虑倾向女性15例及焦虑女性14例。用western blot对三组每个对象的血清的ATP5O进行检测及量化;结果显示与低应激评分女性相比,高应激评分女性血清中总ATP5O水平显著降低(图4A-E)。用质谱进行人血清定量脂代谢组测定并进行差异代谢物PCA、饼图、热图分析及代表性代谢物量化,定量脂代谢组显示,高应激评分女性血清中有益的PC、PA及PI显著下调,LPC、LPA及LPI显著上调(图4F-I)。
附1.实验方法
1.1、动物模型
本研究中的动物实验程序均由南京医科大学(NJMU)机构动物关爱和使用委员会(IACUC)批准,批准号为IACUC-2005003。所有小鼠均在ACF(核心动物部)的标准化无特定病原体(SPF)条件下饲养。如需获得卵巢或其他组织,首先用CO2麻醉小鼠,然后通过颈椎脱位法处死。
制作慢性应激(CS)模型雌性小鼠时,用不锈钢圆柱体(长15cm,直径2.5cm)(一个圆柱体对应一只小鼠),将雌鼠在圆筒中禁锢6小时/每天,然后解禁放回标准笼子中。每天上午9点开始处理。
对于所有生殖力测定,使用10只对照和10只慢性应激ICR雌鼠。用于交配的野生雄鼠根据随机分配表每月在笼间轮换,交配开始于2月龄。
1.2、抗体
一抗:
鼠单克隆GAPDH抗体(货号:30201ES60;上海翊圣生物,上海,中国);
鼠单克隆β-Actin抗体(货号:A5316-100;Sigma,MS,美国);
鼠单克隆β-Tubulin抗体(货号:sc-5274;Santa Cruz,TX,美国);
鼠单克隆alpha Tubulin抗体(Acetyl Lys40)(货号:bsm-33235M;Bioss,北京,中国);
鼠单克隆泛巴豆酰化修饰抗体(货号:PTM-502;杭州景杰生物,杭州,中国);
鼠单克隆泛乙酰化修饰抗体(货号:PTM-101;杭州景杰生物,杭州,中国);
兔多克隆泛丙二酰化抗体(货号:PTM-901;杭州景杰生物,杭州,中国);
鼠单克隆泛苯甲酰化修饰抗体(货号:PTM-762;杭州景杰生物,杭州,中国);
鼠单克隆泛羟基丁酰化修饰抗体(货号:PTM-802;杭州景杰生物,杭州,中国);
兔多克隆泛乳酸化修饰抗体(货号:PTM-1401;杭州景杰生物,杭州,中国);
兔多克隆泛琥珀酰化修饰抗体(货号:PTM-401;杭州景杰生物,杭州,中国);
兔多克隆Transferrin抗体(货号:17435-1-ap;Proteintech,Chicago,美国);
兔多克隆ATP5O抗体(货号:D126152;上海生工生物,上海,中国);
兔多克隆ATP5A1抗体(货号:D154243;上海生工生物,上海,中国);
兔多克隆ATPB抗体(货号:A5286;Selleckchem,上海,中国);
鼠单克隆COX4L1抗体(货号:D190618;上海生工生物,上海,中国);
兔多克隆AKT(Ab-129)抗体(货号:D151616-0100;上海生工生物,上海,中国);
兔多克隆磷酸化AKT(Ser473)抗体(货号:4060,Cell Signaling Technology);
兔多克隆磷酸化RPS6(Ser235/236)抗体,(货号:D155178;上海生工生物,上海,中国);
兔多克隆STAT5A抗体(货号:D220085;上海生工生物,上海,中国);
兔多克隆FAM126A抗体(货号:bs-11554R;Bioss,北京,中国);
兔多克隆PTDSS1抗体(货号:BS-19583R;Bioss,北京,中国);
兔多克隆HDAC2抗体(货号:12922-3-ap,Proteintech,芝加哥,美国);
鼠单克隆strep II标签抗体(货号:YFMA0054,上海翼飞雪生物,南京,中国);
鼠单克隆flag标签抗体(货号:D190828,上海生工生物,上海,中国);
兔多克隆巴豆酰化ATP5O(K51)抗体由武汉普健生物公司针对抗原多肽“ASK(crotonyl)EKKLDQVEKELLC”制备并纯化;兔多克隆磷酸化HDAC2(S424)抗体由钟鼎生物公司针对抗原多肽“SDS(phospho)EDEGEGGRRC”制备并纯化。
二抗:
辣根过氧化物酶标记的兔抗羊IgG和羊抗鼠IgG购自诺唯赞生物(南京,江苏,中国)。Cy2偶联驴抗兔IgG(货号:711-225-152)、Cy2偶联驴抗小鼠IgG(货号:I715-225-150)和Rhodamine(TRITC)偶联驴抗兔IgG(货号:711-025-152)购自Jackson ImmunoResearchLaboratory(West Grove,PA,美国)。
1.3、Corticosterone水平ellisa检测
肾上腺酮elisa试剂盒购自cayman公司(美国,货号:501320)。根据试剂盒操作手册,操作步骤简述如下:首先,用超纯水提前稀释试剂盒中Elisa缓冲液浓储和洗涤液浓储并配好肾上腺酮Elisa标准品,标准品浓度梯度分别为50ng/ml、20ng/ml、8ng/ml、3.2ng/ml、1.28ng/ml、0.5ng/ml、0.2ng/ml、0.08ng/ml,标准品使用缓冲液稀释。同时用6mlellisa缓冲液稀释肾上腺酮乙酰胆碱酯酶示踪剂(CORT AChE Tracer)和肾上腺酮抗血清(Corticosterone ELISA Antiserum)。接下来,向每个96孔酶标板中各孔将肾上腺酮标准品(或血清样品)、肾上腺酮乙酰胆碱酯酶示踪剂及抗肾上腺酮血清依次加入标准品孔(或血清样品测定孔),塑封膜封酶标板并4℃孵育过夜。第二天早上,吸出各测定孔中液体并用wash buffer重复洗五次。配制新鲜的Ellman试剂(Ellman试剂不稳定,现用现配)。每个孔中加入Ellman试剂200ul,用塑封膜封酶标板,在避光条件下使用恒温微孔震荡仪震动120分钟。最后,将酶标板在酶标仪上读数。波长设定为412nm。根据酶标仪对标准品的读数绘制标准曲线并换算每个血清样本对应的肾上腺酮浓度。
1.4、实时RT-PCR
使用RNAprep Pure组织试剂盒(北京天根生物)从组织中分离总RNA,并使用分光光度计(美国Thermo,NanoDrop 2000c)定量。使用FastQuant RT试剂盒(天根生物科技)进行RNA反转录(500ng RNA/反应)以合成cDNA。然后在ABI Step One Plus平台(ThermoFisher Scientific)上使用Eva Green qPCR主混合物(Applied Biological MaterialsInc.,Richmond,BC,Canada)进行实时定量PCR。通过熔融曲线分析评估PCR产物的特异性,并通过2%琼脂糖凝胶电泳确定扩增产物大小。使用actin扩增产物作为内参,对各种mRNAs进行定量。所使用的特定引物如附2表1所示。
1.5、质粒构建
对于图3D和图3E中的ATP5O表达质粒,用高保真反转录酶SSRT VI(ThermoFisher,美国)由小鼠mRNA反转录得到cDNA,然后用高保真DNA聚合酶(诺唯赞生物)扩增所有待克隆构建的基因片段,然后用高保真限制性内切酶(NEB)消化、纯化并插入连接到pcDNA3.1+。构建的所有引物序列见附表2。在构建中ATP5O(WT或突变体)与EGFP-strepII融合,这样可使用strepII标签抗体进行快速检测外源表达蛋白。
1.6、DNA定点突变
对于图3D和图3E中的ATP5O-K51A失活型突变体(不能发生巴豆酰化,图中简写为ATP5O-toA),采用南京诺唯赞公司的Mut Express-II Fast Mutagenesis Kit V2,以ATP5O-WT(野生型)为模板进行构建,简言之,设计一对引物(表3),这对引物有15个碱基的互补区,但K51突变位点对应的三个碱基不同。然后用试剂盒中的高保真DNA聚合酶Max Super-Fidelity DNA Polymerase扩增整个质粒、用限制性内切酶DpnI消化ATP5O-WT模板、用重组酶/>II进行重组获得ATP5O-K51A。最后将突变产物转化到感受态细胞中送公司测序确定突变成功。
1.7、质粒转染
用碧云天生物(北京)的Lipo6000TM转染试剂(货号C0526)对293T细胞系进行转染。以24孔板为例,每孔铺细胞至密度60%,约10小时后(细胞状态完全恢复)开始转染操作。每个孔转染先分别设两个组份管,一管加1μl lipo6000和25μl无血清DMEM;另一管加500ng质粒DNA及25μl无血清DMEM。将两管用枪头温和混匀,室温静置5分钟。接着,将两组份混合到一管中温和混匀,然后分滴散加于细胞孔中并轻微混匀。4-6小时后换液(培养液为10%FBS+DMEM),约10小时后通过荧光判断质粒表达情况并开始下一步实验。
1.8、人类受试者问卷调查和分析
所有与人类相关的问卷调查和分析均由南京医科大学附属江苏省人民医院人类医学伦理委员会(HMEC)批准,批准号为2019-SR-227。
为了对女性受试者的应激水平进行评分,我们使用汉密尔顿焦虑评定量表(HARC),每个受试者由两位独立的心理学家进行评估,最终得分是两位心理学家的平均得分。
受试者血清用于定量脂质组和生化指标测定。
1.9、非标定量巴豆酰化组学
对照和慢性应激组均重复两次,每个重复约包括60个2月龄雌鼠的卵巢(共约300毫克),样本被送往杭州景杰有限公司(中国浙江杭州)进行质谱鉴定。程序简述如下:卵巢首先被裂解,上清液被胰蛋白酶消化成肽段。肽段通过巴豆酰化树脂富集(景杰公司,目录号PTM503),然后将肽段组分置于NSI源中,然后在Q ExactiveTM Plus质谱仪(Thermo)中进行串联质谱(MS/MS)并在线耦合到UPLC以进行蛋白质组学肽段鉴定。对产生的MS/MS数据使用Maxquant搜索引擎(v.1.5.2.8)鉴定到的肽段(定量总蛋白组)或巴豆酰化位点(定量巴豆酰化组)。定量总蛋白数据用于标准化定量巴豆酰化数据。
1.10、无标记定量代谢组学
代谢组学均由武汉迈威生物技术有限公司(中国湖北武汉)完成。对人女性血清或雌鼠血清的代谢组学进行5至12次重复。利用覆盖各种代谢物的全谱代谢组学初步筛选对照组和CS雌性小鼠之间存在的显著差异代谢物。我们发现差异代谢物中脂类占比最大,因此,之后用脂代谢组测定对照组与ATP5O巴豆酰化抑制组之间血清的脂代谢物差异、慢性应激组与HDAC2磷酸化抑制之间的差异以及人对照组女性与中等应激评分女性及高应激评分女性之间的差异。
分析样品提取物使用LC-ESI-MS/MS系统(UPLC,ExionLC AD′https://sciex.com.cn/;MS,系统,https://sciex.com/)分析。在LC/MS实验中用三重TOF质谱仪基于IDA(信息相关基础)获得MS/MS质谱。在此模式下,采集软件(TripleTOF 6600,ABSCIEX)根据预选标准采集和触发MS/MS光谱采集过程中对全扫描测量MS数据进行持续评估。在每个循环中,在30V的碰撞能量(CE)下进行代谢物碎裂(12MS/MS事件,每个事件的产物离子累积时间为50毫秒),选择强度大于100的12个前体离子。ESI源条件设置如下:离子源气体1为50Psi,离子源气体2为50Psi,幕帘气体为25Psi,源温度500℃,离子喷射电压浮动(ISVF)5500V或-4500V,分别处于正模式或负模式。
1.11、主成分分析(PCA)
主成分分析(Principal Component Analysis,PCA)是一种多维数据统计分析方法,通过正交变换将一组可能存在相关性的变量转换为一组线性不相关的变量,转换后的这组变量叫主成分。通过将原始数据压缩成n个主成分来描述原始数据集的特征,PC1表示能描述多维数据矩阵中最明显的特征,PC2表示除PC1之外的所能描述数据矩阵中最显著的特征,PC3……PCn以此类推。PCA用R软件(www.r-project.org/)的内置统计prcomp函数,设置prcomp函数参数scale=True,表示对代谢组数据进行unit variance scaling(UV)标准化。PCA结果显示各组之间代谢组分离趋势,提示样品组间代谢组是否存在差异。
1.12、热图
对代谢组数据进行标准化处理,设定差异倍数和p值后筛选出差异代谢物,对差异代谢物在所有样品中的表达量进行聚类热图分析,并使用R程序中R包heatmaply,ComplexHeatmap绘制聚类热图。
1.13、ATP5O-K51巴豆酰化特异抗体制备
抗体对应的ATP5O巴豆酰化多肽ASK(crotonyl)EKKLDQVEKELLC由上海波泰(BioTech)公司合成,然后交给由武汉普健公司注射四只兔子进行三轮免疫,共三个月。之后取兔子血清,用结合巴豆酰化多肽ASK(crotonyl)EKKLDQVEKELLC的树脂进行巴豆酰化抗体纯化,然后用对应的非巴豆酰化多肽ASKEKKLDQVEKELLC(由上海波泰合成)结合树脂与巴豆酰化抗体孵育去除非巴豆酰化抗体,得到最终的ATP5O-K51巴豆酰化特异抗体。
1.14、聚丙烯酰胺凝胶电泳(SDS-PAGE)和western blot
将细胞或组织样本在SDS样品缓冲液中煮沸4分钟,冰上冷却,12000rpm转速离心4分钟后取上清,用4%积层胶和12.5%分离胶在120伏下电泳分离蛋白质2.5h,然后在100伏下电泳转印到硝酸纤维素膜上(需2.5h)。转印好的膜在TBS(20mmTris,137mmNaCl,pH7.4)中洗涤三次(每次10分)后,用含有5%脱脂奶粉的TBST(含0.1%tween-20的TBS)缓冲液中室温封闭1小时,然后用一抗(稀释在含5%脱脂奶粉的TBST中培养1:1000)孵育膜过夜(4℃)。在TBST中洗涤三次(每次10分钟)后,在室温下用HRP(辣根过氧化物酶)偶联的山羊抗鼠IgG二抗(1:3000稀释于TBST/5%脱脂奶粉)孵育膜1小时。膜在TBST中洗涤三次(每次10分钟)后,加增强化学发光试剂(ECL)到膜上显色,在化学发光成像仪上收集信号。
1.15、竞争性多肽与动物注射
对于所有竞争性肽,细胞穿透肽TAT序列CYGRKKRQRRR与蛋白质特异性序列融合,由上海波泰生物公司合成。
为了抑制内源ATP5O在K51的巴豆酰化,序列为CYGRKKRRRRRATALYSASKEKKL,命名为TAC(TAT融合ATP5O巴豆酰化序列)。
将每种肽溶解在含有10%二甲基亚砜(西格玛)的无菌超纯水中配成5mg/ml的浓储液。小鼠注射时,用0.9%的NaCl溶液将浓储肽稀释至0.5mg/ml最终浓度,注射剂量为6mg/Kg。对照仅注射TAT多肽。
1.16、体内剂量依赖性ATP5O巴豆酰化
对于图3D和图3E中的分析,通过lipo3000(Thermo)将增加量(0.25、0.5、0.75、1和1.25μg)的ATP5O-WT-strepII质粒或ATP5O-K51A-strepII质粒(在pcDNA3.1中)转染到293T细胞中,同时加2.5mM固定量的巴豆酸钠并培养3天。然后裂解细胞并进行蛋白质印迹检测ATP5O的巴豆酰化水平。
1.17、线粒体染色和ATP测定
对于线粒体染色,卵母细胞在含有100nM Mito Tracker(Cat#:M7521,Invitrogen,Carlsbad,CA,USA)和10μg/ml Hochest 33342(Sigma)的Hepes中培养30分钟。使用Andor Revolution转盘式激光共聚焦工作站拍摄图像。
对于ATP测量,首先用100μl ATP裂解液(Cat#:S0026,Beyotime)在冰上裂解卵母细胞。然后用酶标仪Synergy2(BioTek,Winooski,VT,USA)检测样本ATP水平。
1.18、动物/个体样本纳入、实验分组、数据收集和数据分析
任何选定的卵母细胞必须具有正常质量(来自有腔卵泡的成熟卵母细胞、正常直径、透明带和卵母细胞膜之间的紧密连接等)。任何选定的雌性老鼠必须身体健康(正常体重、正常进食、正常活动等)。任何质量差或不健康的卵母细胞或小鼠将被排除在外。
对于所有的实验分组、数据收集和数据分析,我们都试图遵循盲选原则。数据收集、数据分析和数据输入(excel文件)由不同的作者完成。
对于所有其他实验分组和数据收集,必须清楚标记对照或处理过的样品。但在图像拍摄、卵泡计数或强度量化过程中,每个样本的组标签都被一个黑色胶带覆盖,并重新标记为数字或字母。实验过程结束后,除去黑色胶带,第一作者之一可以很容易地找到分析数据和样本信息之间的相关性,并将数据输入到相应的原始excel文件中。
在实验操作之前,在一个独立重复或组中的个体(卵母细胞、卵巢或小鼠)都是通过随机和盲选法选择和分配。对于独立重复的数据收集,随机选择每个数据点。
1.19、统计分析
对western blot或DNA凝胶的所有统计图均来自三个独立重复,血液生化指标的所有统计图均来自五个独立重复。图中的每个点代表一个重复。如果一组中所有随机收集的单个数据点的标准误差明显小于平均值,则相应的样本量是适当且可信的。数据以平均值±SEM表示。两组之间的统计比较采用Excel的Student’s t-test。进行多重比较时采用Kruskal-Wallis的one-way nonparametric ANOVA。P<0.05被认为具有统计学意义。
附2.专利中各种相关引物
表1.Q-PCR引物(用于图1D)
表2.质粒构建引物(用于图3D中pcDNA3.1-ATP5O-EGFP-strep II构建)
表3.质粒定点突变引物(用于图3D中将ATP5O-WT突变为ATP5O-K51A)
Name | Sequence |
ATP5O(K to A)-F | TGCTGCATCTGCGGAGAAGAAGCTGGACCAGGTGGAGAAGGAG |
ATP5O(K to A)-R | GCTTCTTCTCCGCAGATGCAGCAGAGTACAGGGCGGTTGCATA |
附3.参考文献
1.An H,Yu H,Wei Y,Liu F,Ye J.Disrupted metabolic pathways andpotential human diseases induced by bisphenol S.Environ ToxicolPharmacol.2021Nov;88:103751.doi:10.1016/j.etap.2021.103751.Epub 2021Oct5.PMID:34624477
2.Ye G,Yang BC,Gao H,Wu Z,Chen J,Ai XY,Huang Q.Metabolomics Insightsinto Oleate-Induced Disorders of Phospholipid Metabolism in Macrophages.JNutr.2021Mar 11;151(3):503-512.doi:10.1093/jn/nxaa411.PMID:33571370.
3.Jia M,Peng Z,Yang K,Su C,Wang Y,Yan C.A high-throughput targetedmetabolomics method for the quantification of 104non-polar metabolites incholesterol,eicosanoid,and phospholipid metabolism:application in the studyof a CCl4-induced liver injury mouse model.Analyst.2020May 18;145(10):3575-3591.doi:10.1039/d0an00385a.PMID:32329491.
4.Singh AB,Kan CFK,Kraemer FB,Sobel RA,Liu J.Liver-specific knockdownof long-chain acyl-CoA synthetase 4 reveals its key role in VLDL-TGmetabolism and phospholipid synthesis in mice fed a high-fat diet.Am JPhysiol Endocrinol Metab.2019 May 1;316(5):E880-E894.doi:10.1152/ajpendo.00503.2018.Epub 2019 Feb 5.PMID:30721098;PMCID:PMC6580179.
5.van Liempd SM,Cabrera D,Lee FY,González E,Dell'Angelica EC,GhianiCA,Falcon-Perez JM.BLOC-1 deficiency causes alterations in amino acid profileand in phospholipid and adenosine metabolism in the postnatal mousehippocampus.Sci Rep.2017 Jul 12;7(1):5231.doi:10.1038/s41598-017-05465-z.PMID:28701731;PMCID:PMC5507893.
6.Miranda DA,Krause WC,Cazenave-Gassiot A,Suzawa M,Escusa H,Foo JC,Shihadih DS,Stahl A,Fitch M,Nyangau E,Hellerstein M,Wenk MR,Silver DL,Ingraham HA.LRH-1 regulates hepatic lipid homeostasis and maintainsarachidonoyl phospholipid pools critical for phospholipid diversity.JCIInsight.2018 Mar 8;3(5):e96151.doi:10.1172/jci.insight.96151.PMID:29515023;PMCID:PMC5922282.
序列表
<110> 南京医科大学
<120> 一种建立哺乳动物慢性磷脂代谢异常模型的多肽TAC及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ala Thr Ala Leu Tyr Ser Ala Ala Ser Lys Glu Lys Lys Leu
1 5 10
<210> 2
<211> 26
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Cys Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Thr Ala Leu
1 5 10 15
Tyr Ser Ala Ala Ser Lys Glu Lys Lys Leu
20 25
Claims (6)
1.一种用于建立哺乳动物慢性磷脂代谢异常模型的多肽TAC,其特征在于如SEQ IDNO.1所示。
2. 根据权利要求1所述的多肽TAC,其特征在于全序列如SEQ ID NO.2所示。
3.权利要求1或2所述的多肽TAC在建立哺乳动物慢性磷脂代谢异常模型中的应用。
4.一种建立哺乳动物慢性磷脂代谢异常模型的方法,其特征在于向哺乳动物腹腔注射权利要求1或2所述的多肽TAC。
5.根据权利要求4所述的方法,其特征在于对实验哺乳动物腹腔注射6mg / Kg TAC,每日一次,持续4周,结束时可以取血清进行ATP5O blot,如果ATP5O水平显著降低,则说明造模成功。
6.根据权利要求5所述的方法,其特征在于所述的实验哺乳动物选自小鼠、大鼠、恒河猴或食蟹猴。
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