CN107828878B - Fkbp11基因在防治主动脉夹层中的应用 - Google Patents
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Abstract
本发明属于基因的功能与应用领域，具体公开了FKBP11基因在防治主动脉夹层中的应用。以体外细胞实验表明，在血管内皮细胞SiRNA介导的FKBP11基因沉默可抑制NF‑kB信号通路的激活,降低促炎因子的表达，抑制巨噬细胞向主动脉中层的迁移，从而抑制中层基质的降解,结果表明FKBP11抑制剂可防治主动脉夹层发生,本发明将为寻找预防、缓解和/或治疗主动脉夹层的新靶点和新策略提供理论依据和临床基础。
Description
技术领域
本发明属于基因的功能与应用领域，具体涉及FKBP11基因在防治主动脉夹层中的应用，还涉及以FKBP11基因作为药物靶标在筛选治疗主动脉夹层的药物中应用，以及FKBP11基因抑制剂在制备治疗主动脉夹层疾病的药物中应用。
背景技术
主动脉夹层(Aortic dissection，AD)是指各种原因导致的主动脉内膜突然撕裂，循环血液进入血管壁中层并导致其分层，随着血流压力驱动，逐渐在主动脉中层内扩展形成夹层。急性起病，突然剧烈胸痛、休克并累及相应的主动脉分支血管导致脏器的急性缺血是该病的特点。在我国随着人口老龄化和饮食结构的改变，AD的发病率在迅速攀升。流行病学发现许多心血管危险因素例如吸烟、男性、年龄、高血压和动脉粥样硬化都与AD的发病密切相关[1-3]。尽管目前的各种治疗手段不断改进，但是AD的发病率和死亡率依然居高不下，并且如果不进行恰当和及时的治疗，AD死亡率极高。有报道称约有近20％的AD患者在到达医院之前死亡，而30％的患者在住院治疗期间死亡[4-7]。目前临床上对AD缺乏有效的药物干预手段，主要采用外科手术进行血管置换、介入手术进行主动脉腔内支架植入以及两者结合应用，但是手术风险大，费用高，并且远期的复发率和再手术率仍然较高[8-12]。因此我们迫切需要寻找新的靶点以探讨新的防治策略，这就需要进一步明确AD的发病机制[13]。
FKBPs是结合免疫抑制药物如FK506，环孢菌素和雷帕霉素的肽基-脯氨酰顺式/反式异构酶(PPIases)，也称为免疫亲和素[14]。FKBP12是该家族中最丰富的成员，可与雷帕霉素形成复合物，通过抑制雷帕霉素(TOR)激酶[15]的靶标阻断翻译过程。许多免疫亲和家族成员涉及蛋白质折叠，组装和转运等事件，分子复合物的共调节，转录和翻译监管和细胞-细胞相互作用等[16]。FKBP11是一个新的成员，最早由Rulten及其同事[14]描述。FKBP11mRNA在分泌组织如肝脏和胰腺中丰富。FKBP11基因编码具有25个残基的富含亮氨酸N-末端前导序列的22kDa前蛋白。前导肽的切割将留下19kDa的成熟蛋白质，因此FKBP11也被命名为FKBP19。该蛋白质的C-末端含有推定的跨膜结构域，随后是内质网(ER)定位基序。因此，FKBP11已经提出参与蛋白质折叠和分泌[14,17]。已有研究证实，FKBP11的过表达是狼疮B细胞的特征，导致B细胞耐受性破坏并引发浆细胞分化[18]。另外，FKBP11的表达在肝细胞癌的发展过程中逐步升高，具有成为肝细胞癌早期标志物的潜力[19]。但目前为止，尚未见FKBP11在心血管疾病，尤其是主动脉夹层中作用的相关报道。
参考文献：
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[12]Trimarchi S and Eagle KA.Thoracic Endovascular Aortic Repair inAcute and Chronic Type B Aortic Dissection.JACC Cardiovasc Interv 2016；9:192-194.
[13]Yang GF,Sheng LJ,Peng W,Peng ZY and Chai XP.Comorbidity:A novelinsight to aortic dissection.Int J Cardiol 2016；207:53-54.
[14]Rulten SL,Kinloch RA,Tateossian H,Robinson C,Gettins L and KayJE.The human FK506-binding proteins:characterization of human FKBP19.MammGenome 2006；17:322-331.
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发明内容
为解决临床防治主动脉夹层现有技术的缺陷和不足，本发明的目的是提供了FKBP11基因在防治主动脉夹层中的应用，申请人发现FKBP11基因在急性主动夹层患者主动脉组织内皮细胞中高表达，体外实验进一步证明，下调该基因的表达可以抑制NF-kB信号通路的激活,从而降低促炎因子等的表达，最终可对主动脉夹层的发生起到缓解和治疗的作用，因此可以以FKBP11基因或其编码的蛋白作为药物靶标来筛选防治主动脉夹层疾病的药物，或是构建主动脉夹层细胞模型。
为了达到上述目的，本发明采取以下技术措施：
1、FKBP11基因与急性主动脉夹层的关系的发现：
申请人以急性主动夹层患者主动脉组织的的临床样本，通过Western Blot、免疫组化实验研究FKBP11基因与急性主动脉夹层的关系，结果表明急性主动夹层患者主动脉组织中FKBP11基因的表达明显升高，同时运用免疫荧光双标技术发现，FKBP11的表达主要定位于内皮细胞，并且MMP-9的表达同FKBP11的表达类似，在急性主动夹层患者主动脉组织中的表达明显升高，主要定位于巨噬细胞。
2、体外细胞实验对FKBP11基因功能进行验证：
申请人对其功能进行了体外细胞实验，免疫共沉淀实验表明FKBP1可以与p-P65相互结合，Western Blot、细胞免疫荧光及细胞迁移实验表明SiRNA介导的FKBP11干扰可抑制血管紧张素转换酶II(Ang II)诱导的NF-kB P65入核的增加，抑制NF-kB信号通路的激活,从而降低促炎因子等的表达，进而抑制巨噬细胞向主动脉中层的迁移，抑制主动脉中层基质的降解,结果表明FKBP11与主动脉夹层发生发展相关，对其表达进行抑制，可对主动脉夹层的发生起到缓解和治疗的作用。
FKBP11基因在防治主动脉夹层中的应用，包括以FKBP11基因或其编码的蛋白作为药物靶标来筛选防治主动脉夹层疾病的药物，或是通过过表达FKBP11基因来构建主动脉夹层细胞模型或是促炎细胞模型，或是FKBP11基因或其编码的蛋白的抑制剂在制备治疗或预防主动脉夹层疾病的药物中的应用，或是FKBP11基因或其编码的蛋白的抑制剂在制备促炎因子抑制剂中的应用。
以上所述的方案，优选的，所述的防治主动脉夹层疾病的药物包括FKBP11基因抑制物，包括但不限于针对FKBP11基因设计的siRNA和RNA干扰载体，或是FKBP11蛋白的抗体及其他能够抑制FKBP11表达的抑制剂。
以上所述的方案，优选的，所述的siRNA为：
FKBP11-si2：GAGAAGCGAAGGGCAATCA；或FKBP11-si3：CTCACTTGGCCTATGGAAA。
以上所述的应用，优选的，所述的促炎因子包括但不限于：VCAM1，ICAM1，MCP1或IL-1β。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明首次发现FKBP11基因的新功能，申请人对其功能进行了体外细胞实验，免疫共沉淀实验表明FKBP1可以与p-P65相互结合，Western Blot、细胞免疫荧光及细胞迁移实验表明SiRNA介导的FKBP11干扰可抑制Ang II诱导的NF-kB P65入核的增加，抑制NF-kB信号通路的激活,从而降低促炎因子等的表达，进而抑制巨噬细胞向主动脉中层的迁移，抑制主动脉中层基质的降解,结果表明FKBP11与主动脉夹层发生发展相关，对其表达进行抑制，可对主动脉夹层的发生起到缓解和治疗的作用。因此,该基因将为研究预防、缓解和/或治疗主动脉夹层的新靶点和新策略提供理论依据和临床基础。
(2)基于FKBP11基因在主动脉夹层疾病中的功能，为研制预防、缓解和/或治疗主动脉夹层疾病的药物提供靶标。
(3)FKBP11基因可作为药物靶点，构建FKBP11基因过表达的体外细胞模型或动物模型，用于筛选预防、缓解和/或治疗主动脉夹层疾病的药物；FKBP11基因也可作为基因治疗中的靶基因，设计并制备预防、缓解和/或治疗主动脉夹层疾病的药物和/或生物学试剂，通过基因工程技术达到预防、缓解和/或治疗主动脉夹层的目的。例如以FKBP11为靶基因，设计可干扰FKBP11表达的双链siRNA，通过化学方法合成以后，注射入人体通过RNA干扰的方法使FKBP11基因沉默来治疗主动脉夹层疾病；还可以设计并构建FKBP11的突变体，注射后进入细胞，竞争FKBP11原形的作用底物，从而抑制FKBP11的功能，起到治疗目的；此外，还可以以FKBP11为靶点设计小分子化合物抑制剂，利用FKBP11基因过表达的体外细胞模型或动物模型，通过筛选，发现其中能够特异性抑制FKBP11的分子，从而为主动脉夹层疾病的治疗提供新的治疗策略。
附图说明
图1为FKBP11基因在临床主动脉标本中的表达结果图；
图1中：A图为代表性WB图片(上图)及柱形图(下图)显示AAD患者主动脉中FKBP11表达显著高于对照组；
B图为代表性EVG染色显示AAD患者主动脉中层弹力纤维较对照组断裂显著增多(弹力纤维为深蓝色)，标尺:100μm；
C图为FKPB11蛋白在AAD组表达显著增加，并且主要集中在内皮细胞(FKBP11为棕黄色)，标尺:100μm；
D图为免疫荧光双染显示FKBP11蛋白(红色)主要和内皮细胞标志CD31(绿色)重叠,进一步表明FKBP11的表达在AAD组内皮细胞显著增加,核染色DAPI(蓝色),标尺:100μm。
图2为在AAD情况下FKBP11基因同侵入主动脉中层的单核巨噬细胞MMP9表达相关结果图；
图2中：A图为代表性WB图片及柱状图显示AAD组MMP9的蛋白表达显著高于对照组；
B图为代表性MMP9免疫组化图片显示AAD组MMP9表达显著高于对照组，标尺:100μm；
C图为免疫荧光双染显示MMP9(红色)主要同单核巨噬细胞相重叠(Mac-2，绿色),核染色DAPI(蓝色),标尺:100μm；
图3为在内皮细胞中FKBP11基因的促炎作用是通过NF-kB p65活化介导；
图3中：A图为WB结果显示FKBP11siRNA、FKBP11-Si2及FKBP11-Si3能显著减低FKBP11基因在EA.hy926细胞中的表达。
B图为在EA.hy926细胞Ang II能促进p-p65的核转位,而调低FKBP11基因的表达可以削弱其转位；
C图为代表性WB图片；
D图柱状图显示FKBP11siRNA可以显著抑制p65的磷酸化及其下游促炎因子MCP1,VCAM1,ICAM1and IL1-β.p-p65染色(绿色)；核染色DAPI(蓝色),标尺:100μm；
E图免疫共沉淀结果显示FKBP11同p-p65相互作用，Ang II能促FKBP11同p-p65的相互作用。
图4为FKBP11基因促进单核细胞穿过内皮细胞结果图
图4中：A图为细胞迁移实验显示使用Ang II刺激下面小室EA.hy926内皮细胞可以显著促进上面小室中单核细胞向THP1细胞的迁移。而如果当FKBP11siRNA干预EA.hy926内皮细胞后，Ang II刺激THP1细胞的迁移显著减少。
B图为柱状图总结迁移实验的结果。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。本发明所述技术方案，如未特别说明，均为本领域的常规技术；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：FKBP11基因与急性主动脉夹层的关系的发现：：
申请人运用主动脉夹层患者整体主动脉组织RNA经过Illumina HumanHT-12V4.0基因芯片(Illumina Inc.,San Diego,CA,USA)分析得到了转录组数据库，并应用WGCNA法构建了差异表达谱矩阵数据，去除离群的样本后，进行基因模块分析，基因富集分析，枢纽基因筛选，筛选出的同主动脉夹层发生发展相关的基因经过独立样本及离体细胞实验验证，确立了FKBP11为目标靶基因。
申请人以急性主动夹层患者主动脉组织的的临床样本，以正常主动脉组织样本为对照，通过Western Blot、免疫组化实验研究FKBP11基因与急性主动脉夹层的关系，结果表明急性主动夹层患者主动脉组织中FKBP11蛋白的表达明显升高。
激光共聚焦免疫荧光多重染色技术(Confocal Microscopy):将准备好的组织切片按照标准流程进行至一抗孵育，此时我们同时孵育两种一抗(不同种属来源)4℃过夜，1XPBS洗净后孵育与一抗种属相对应的不同荧光素标记的二抗，30min后1XPBS洗净后，加入DAPI(细胞核染料)复染并封片，用激光共聚焦显微镜观察拍照。同样方法检测如下表不同一抗组合在人AD主动脉组织的染色，在血管内皮细胞(Endothelial,CD31)，血管平滑肌细胞(VSMC,α-SMA)，单核巨噬细胞
中的表达水平。
结果发现，FKBP11蛋白的表达主要定位于内皮细胞(图1)，并且MMP-9的表达同FKBP11的表达类似，在急性主动夹层患者主动脉组织中的表达明显升高，主要定位于巨噬细胞(图2)。
实施例2：FKBP11基因导致主动脉夹层疾病作用机理
内皮细胞受损是多种心血管疾病的始动环节，当内皮细胞受到外界刺激时，内皮细胞高表达的FKBP11通过促进p-P65进入细胞核激活NF-KB通路，增加内皮细胞炎症因子的表达，促使循环中的单核细胞发生迁移分化导致巨噬细胞通过活化的内皮细胞进入主动脉中膜，聚集的巨噬细胞等会通过分泌炎症因子(例如IL-6，MCP-1等)招募更多的炎症细胞向内皮细胞粘附及中膜的迁移从而加剧炎症细胞在局部的浸润。同时巨噬细胞等会分泌产生大量的基质金属蛋白酶(MMPs)和大量可以降解弹性蛋白、胶原蛋白和层粘连蛋白等结构蛋白的蛋白酶，这些蛋白水解酶都会引起血管壁的结构被破坏，导致主动脉夹层的发生和发展。
实施例3：FKBP11基因敲减显著降低促炎因子的表达及单核巨噬细胞的迁移
申请人在体外用血管紧张素转换酶II(Ang II)诱导EA.hy926内皮细胞模拟主动脉夹层的发生。
1、有效FKBP11SiRNA的筛选与验证：
(1)细胞培养：THP-1细胞(
tib-202TM)和EA.hy926细胞(Manassas,VA)，培养在含10％胎牛血清(Sigma-Aldrich,St.Louis,MO)的RPMI 1640(Gibco,CA,USA)，含有10mMHEPEs，0.1mM MEM非必需氨基酸，1mM丙酮酸钠，和100nM的青霉素/链霉素(LifeTechnologies)，保持在5％的CO2，37℃的细胞培养箱中。合成的FKBP11-siRNA购自Ruibo(广州，中国)，转染细胞具体操作根据制造商的协议。人工合成的三种FKBP11-siRNA，具体如下：
FKBP11-si1：GGGCAATCATTCCTTCTCA；
FKBP11-si2：GAGAAGCGAAGGGCAATCA；
FKBP11-si3：CTCACTTGGCCTATGGAAA。
(2)细胞转染：采用riboFECTTMCP Reagent转染siRNA于24孔板的EA.hy926细胞中，转染浓度为50nM；具体如下：
1)接种细胞：接种EA.hy926细胞1×105～5×105个细胞至含有适量完全培养基的24孔板培养孔中，使转染时的细胞密度能够达到30～50％。
2)转染：对于每个转染样品，请按以下步骤准备：
a.稀释siRNA：用30μl 1X riboFECTTMCP Buffer稀释1.25μl 20μM FKBP11-siRNA，轻轻混匀。
b.混合液制备：加入3μl riboFECTTMCP Reagent，轻轻吹打混匀，室温孵育0～15min，制备成转染复合物。
c.将riboFECTTMCP转染复合物加入步骤1)的24孔板的EA.hy926细胞中，轻轻混匀。
实验分为5组，具体如下：
(a).空白对照；
(b).转染NC-SiRNA；
(c).转染FKBP11-si 1RNA；
(d).转染FKBP11-si 2RNA；
(e).转染FKBP11-si 3RNA；
d.将培养板置于37℃的CO2培养箱中培养24h
孵育结束后，收取各组细胞蛋白进行Western Blot实验验证。实验结果表明：上述三种FKBP11-siRNA均能够抑制EA.hy926细胞FKBP11的表达，但FKBP11-si2对FKBP11基因的抑制效果最为显著。因此以下实验，选用FKBP11-si2进行实验(图3中A)，以下实验中，所述的FKBP11-siRNA均指得是FKBP11-si 2。
2、FKBP11基因敲减抑制Ang II诱导的p-P65的入核及促炎因子的表达：
(1)FKBP11基因敲减抑制Ang II诱导的p-P65的入核，采用细胞免疫荧光技术，实验分为5组，具体如下：
a.空白对照，即爬片的EA.hy926细胞；
b.向爬片的EA.hy926细胞添加1.0×10-6mol/LAng II；
c.对爬片的EA.hy926细胞转染NC-SiRNA，孵育24h后添加1.0×10-6mol/L Ang II；
d.对爬片的EA.hy926细胞转染FKBP11-siRNA；
e.对爬片的EA.hy926细胞转染FKBP11-siRNA，孵育24h后添加1.0×10-6mol/L AngII；
上述5组细胞爬片各继续孵育24h后，细胞被固定在3.7％多聚甲醛(PFA)并用含有0.2％Trition X-100的PBS缓冲液洗3次。盖玻片用封闭液封闭1小时后与p-p65抗体在4℃孵育过夜。用PBS洗3次后与相应的荧光标记的二抗孵育60分钟，PBS洗3次后，DAPI染细胞核并洗涤后用共聚焦显微镜观察分析。结果显示FKBP11-siRNA能够明显抑制Ang II诱导的EA.hy926细胞p-p65的入核增加(图3中B)。
(2)FKBP11基因敲减抑制Ang II诱导的促炎因子的表达，
采用Western Blot技术，实验分为5组，具体如下：
a.空白对照，即EA.hy926细胞；
b.EA.hy926细胞添加1.0×10-6mol/LAng II；
c.EA.hy926细胞转染NC-SiRNA，孵育24h后添加1.0×10-6mol/L Ang II；
d.EA.hy926细胞转染FKBP11-siRNA；
e.EA.hy926细胞转染FKBP11-siRNA，孵育24h后添加1.0×10-6mol/L Ang II；
上述5组细胞各继续孵育24h后，收取各组细胞蛋白进行Western Blot实验验证。实验结果表明：FKBP11-siRNA显著抑制Ang II诱导的促炎因子MCP1,VCAM1,ICAM1和IL1beta等的表达(图3中C和D)。
3、Ang II能够诱导FKBP11与p-p65的结合增加：
采用免疫共沉淀实验技术，对EA.hy926细胞用Ang II(1.0×10-6mol/L)干预,对照组细胞不做任何处理，孵育24h后，用含有磷酸酶抑制剂和蛋白酶抑制剂的蛋白裂解液提取细胞总蛋白。离心蛋白收取上清液留取部分作为input。剩余蛋白用3μg抗体孵育4摄氏度孵育2小时。蛋白A/G琼脂糖珠进行纯化并用含有磷酸酶抑制剂和蛋白酶抑制剂的PBS洗涤。免疫沉淀通过10％SDS-PAGE分离，转移到膜上，并进行免疫印迹实验。实验结果表明，Ang II能够诱导FKBP11与p-p65的结合增加(图3中E)。
4、FKBP11基因敲减抑制THP-1细胞向单层EA.hy926细胞的迁移：
采用细胞迁移实验技术，用
polycarbonate membraneinserts(6.5mm diameter,8μmpore size)进行实验。实验分为4组，如下：
a.EA.hy926单层细胞转染NC-SiRNA；
b.EA.hy926单层细胞转染FKBP11-siRNA；
c.EA.hy926单层细胞NC-SiRNA，孵育24h后添加1.0×10-6mol/L Ang II；
d.EA.hy926单层细胞转染FKBP11-siRNA,孵育24h后添加1.0×10-6mol/L Ang II；
以上四组实验处理完毕后，再继续孵育24小时，更换细胞培养液，5x 105THP-1细胞接种到含100μL无血清RPMI培养基的上室。在37℃，5％CO2培养箱中培养12h后上室被拆除并用结晶紫染色后计数移到下室THP-1细胞。每次实验至少重复三次。在五个随机选取的正方形中计数细胞，并以迁移细胞/视野的数目表示。实验结果表明，FKBP11-siRNA能够显著抑制THP-1细胞向单层EA.hy926细胞的迁移(图4)。
综合以上研究结果表明，FKBP11与主动脉夹层发生发展相关；SiRNA介导的FKBP11干扰可抑制Ang II诱导的NF-kB p-P65入核的增加，抑制NF-kB信号通路的激活,从而降低促炎因子等的表达，进而抑制巨噬细胞向主动脉中层的迁移，抑制主动脉中层基质的降解,结果表明对其表达进行抑制，可对主动脉夹层的发生起到缓解和治疗的作用。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 华中科技大学同济医学院附属同济医院
<120> FKBP11基因在防治主动脉夹层中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gagaagcgaa gggcaatca 19
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctcacttggc ctatggaaa 19
Claims (2)
1.以FKBP11基因或其编码的蛋白作为药物靶标在筛选防治主动脉夹层疾病的药物中的应用。
2.FKBP11基因或其编码的蛋白的抑制剂在制备治疗或预防主动脉夹层疾病的药物中的应用。
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