CN117265004A - 一种跨膜蛋白41b(tmem41b)基因敲除小鼠动物模型的构建方法和应用 - Google Patents
一种跨膜蛋白41b(tmem41b)基因敲除小鼠动物模型的构建方法和应用 Download PDFInfo
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- CN117265004A CN117265004A CN202311207510.1A CN202311207510A CN117265004A CN 117265004 A CN117265004 A CN 117265004A CN 202311207510 A CN202311207510 A CN 202311207510A CN 117265004 A CN117265004 A CN 117265004A
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- transmembrane protein
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Abstract
本发明属于生物医药技术领域,具体涉及一种跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法以及通过该构件方法得到的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型在血管内膜新生或冠状动脉狭窄疾病研究中的应用,还涉及一种跨膜蛋白41B(TMEM41B)作为药物靶点在制备用于治疗血管内膜新生或冠状动脉狭窄的药物中的应用。本发明根据Cre‑loxP重组酶系统原理构建平滑肌细胞TMEM41B特异性敲除小鼠,成功获得转基因敲除鼠,并且发现TMEM41B平滑肌细胞缺失的转基因小鼠可自发性发生血管内膜新生。与传统的导丝损伤诱导方法相比,操作简便、疾病模型稳定,并为临床TMEM41B缺失病人提供治疗依据,这类人群可能更易发生血管内膜新生导致的冠状动脉狭窄疾病或者PCI后的再狭窄。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法以及通过该构件方法得到的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型在血管内膜新生或冠状动脉狭窄疾病研究中的应用,还涉及一种跨膜蛋白41B(TMEM41B)作为药物靶点在制备用于治疗血管内膜新生或冠状动脉狭窄的药物中的应用。
背景技术
心血管病是近十年来威胁人类健康的全球性疾病,心肌缺血导致的心力衰竭在心血管疾病中占有重要地位。冠状动脉狭窄是引起心肌缺血的主要原因,经皮冠状动脉介入治疗(PCI)、球囊血管成形术或冠状动脉搭桥术(CABG)是治疗狭窄性动脉粥样硬化病变最常见的血管重建治疗策略。冠状动脉狭窄患者及时送往医院手术治疗,实现血流再通,有着立竿见影的效果。但是,这些血管介入性手术(包括治疗血管狭窄和闭塞的经皮腔内血管成形术和血管支架植入术等)不可避免的会损伤血管内膜,引发血管内膜新生。内膜的异常新生是患者支架内再狭窄的重要原因,也是预后不良的关键因素。研究发现,接受PCI治疗的患者发生再狭窄的发生率最高可达50%,甚至部分患者需要在6个月内进一步接受血管重建手术。这无疑是对患者在精神、身体以及经济上再次打击。因此进一步探寻血管内膜新生机制并筛选干预靶点是一项具有积极意义的临床课题。
血管内膜新生在支架术后再狭窄的发生中扮演重要角色,同时也是动脉粥样硬化、静脉移植、高血压等疾病的共同病理学基础。血管平滑肌细胞具有很强的可塑性,在病理刺激下会发生表型转化,即由原来的收缩型细胞转化为合成型细胞(以高增殖、迁移和蛋白合成能力为特征)。平滑肌细胞的表型转化是血管内膜新生必经病理学过程。既往研究发现,内膜新生的主要原因是平滑肌细胞会发生过度增殖与迁移。因此,阐明血管平滑肌细胞过度增殖的分子机制,并有效抑制血管内膜新生,对研发干预血管再狭窄的药物、优化治疗措施、提高患者术后生存质量至关重要。
TMEM41B(跨膜蛋白41B,也称为Stasimon)最初发现是由于脊柱运动神经元(SMN)蛋白丧失而引起的剪接功能障碍的靶点。TMEM41B同时也是是一种位于内质网(ER)的跨膜蛋白,其位于ER与线粒体的接触部位。近年研究发现,TMEM41B是自噬形成的新型调节分子,同时具有磷脂翻转酶的活性,参与调控脂质稳态。此外,TMEM41B也参了黄病毒和冠状病毒(包括SARS-CoV-2)感染宿主的过程。但是,迄今为止TMEM41B血管内膜新生的作用以及机制尚未报道。因此揭示TMEM41B在内膜新生过程中的作用以及机制,为治疗内膜新生提供新的思路和靶点。
发明内容
鉴于此,本发明的目的在于提供一种跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法以及通过该构件方法得到的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型在血管内膜新生或冠状动脉狭窄疾病研究中的应用。
为实现上述目的,本发明所采取的解决方案如下:
第一个方面,本发明提供一种跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法,包括如下步骤:
步骤(1),小鼠胚胎细胞中在TMEM41B基因的3号和5号外显子插入floxp位点,通过胚胎打靶技术构建TMEM41B-floxp转基因小鼠;
步骤(2),根据Cre-flox敲除鼠构建系统,运用Sm22α-Cre工具鼠与TMEM41B-floxp转基因小鼠进行杂交繁育,获得平滑肌细胞特异性组成性敲除TMEM41B转基因小鼠(Tmem41bf/f,SM22α-Cre);
步骤(3),根据Cre-flox敲除鼠构建系统,运用Myh11-ERTCre工具鼠与TMEM41B-floxp转基因小鼠进行杂交繁育,获得平滑肌细胞特异性条件性敲除TMEM41B-floxp转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)。
优选地,步骤(2)获得的平滑肌细胞特异性组成性敲除TMEM41B转基因小鼠(Tmem41bf/f,SM22α-Cre)饲养至6-8周自发性发生血管内膜新生。
优选地,步骤(3)获得的平滑肌细胞特异性条件性敲除TMEM41B-floxp转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)饲养至6周,连续腹腔注射Tamoxifen(60-100mg/kg)5天,饲养至16周,自发性发生血管内膜新生。
第二个方面,本发明还提供一种通过如上所述的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法得到的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型。
第三个方面,本发明还提供一种如上所述的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型在血管内膜新生或冠状动脉狭窄疾病研究中的应用。
第四个方面,本发明提供一种跨膜蛋白41B(TMEM41B)作为药物靶点在制备用于治疗血管内膜新生或冠状动脉狭窄的药物中的应用。
优选地,血管平滑肌细胞的跨膜蛋白41B的缺失或突变会自发引起血管内膜新生或者由此导致的冠状动脉狭窄疾病、PCI后的再狭窄。
第五个方面,本发明还提供一种跨膜蛋白41B(TMEM41B)的促进剂在制备用于治疗血管内膜新生或冠状动脉狭窄的药物中的应用。
第六个方面,本发明还提供一种用于治疗结血管内膜新生或冠状动脉狭窄的药物组合物,所述药物组合物包括药学上可接受的载体和有效量的活性成分,所述活性成分包括如上所述的跨膜蛋白41B(TMEM41B)的促进剂。
优选地,所述药物组合物的剂型选自片剂、粉剂、注射剂、胶囊、混悬剂、糊剂、凝胶、涂布剂、药膜剂、缓释剂和微球中的任意一种。
在本发明中,本发明根据Cre-loxP重组酶系统原理构建平滑肌细胞TMEM41B特异性敲除小鼠,成功获得转基因敲除鼠,并且发现TMEM41B平滑肌细胞缺失的转基因小鼠可自发性发生血管内膜新生。与传统的导丝损伤诱导方法相比,操作简便、疾病模型稳定,并为临床TMEM41B缺失病人提供治疗依据,这类人群可能更易发生血管内膜新生导致的冠状动脉狭窄疾病或者PCI后的再狭窄。
附图说明
图1为本发明实施例中,雄性转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠饲养6周,主动脉部分进行HE染色的对比图。
图2为本发明实施例中,雌性转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠饲养6周,主动脉部分进行HE染色的对比图。
图3为本发明实施例中,雄性转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)与同窝对照(Tmem41bf/f),Tamoxifen注射后16周,主动脉部分进行HE染色的对比图。
图4为本发明实施例中,雄性转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)与同窝对照(Tmem41bf/f),Tamoxifen注射后16周,股动脉和颈动脉部分分别进行HE染色的对比图。
图5为本发明实施例中,分别分离转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠主动脉平滑肌细胞体外进行培养,进行Westernblot验证的对比图。
图6为本发明实施例中,分别分离转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠主动脉平滑肌细胞体外进行培养,进行EDU染色的对比图。
具体实施方式
本发明根据Cre-loxP重组酶系统原理构建平滑肌细胞TMEM41B特异性敲除小鼠,成功获得转基因敲除鼠,构建方法包括如下步骤:
1、小鼠胚胎细胞中在TMEM41B基因的3号和5号外显子插入floxp位点,通过胚胎打靶技术构建TMEM41B-floxp转基因小鼠。
2、根据Cre-flox敲除鼠构建系统,运用Sm22α-Cre工具鼠与TMEM41B-floxp转基因小鼠进行杂交繁育,获得平滑肌细胞特异性组成性敲除TMEM41B转基因小鼠(Tmem41bf/f,SM22α-Cre)。转基因小鼠饲养至6-8周自发性发生血管内膜新生。
3、根据Cre-flox敲除鼠构建系统,运用Myh11-ERTCre工具鼠与TMEM41B-floxp转基因小鼠进行杂交繁育,获得平滑肌细胞特异性条件性敲除TMEM41B转基因小鼠(Tmem41bf /f,Myh11-ERT2Cre)。转基因小鼠饲养至6周,连续腹腔注射Tamoxifen(80mg/kg)5天,饲养至16周,转基因小鼠自发性发生血管内膜新生。
平滑肌细胞的表型转化是血管内膜新生的主要原因。血管平滑肌细胞中的TMEM41B是否参与血管内膜新生至今尚未报道。本发明实施例根据Cre-loxP重组酶系统原理构建平滑肌细胞TMEM41B特异性敲除小鼠,方法真实可靠,并且已经成功获得转基因敲除鼠。
本发明将通过如上所述的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法得到的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型应用在血管内膜新生或冠状动脉狭窄疾病研究中。
因此,跨膜蛋白41B(TMEM41B)可以作为药物靶点用于制备治疗血管内膜新生或冠状动脉狭窄的药物。
本发明通过构建的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型发现,血管平滑肌细胞的跨膜蛋白41B的缺失或突变会自发引起血管内膜新生或者由此导致的冠状动脉狭窄疾病、PCI后的再狭窄。
因此,本发明还提供一种跨膜蛋白41B(TMEM41B)的促进剂在制备用于治疗血管内膜新生或冠状动脉狭窄的药物中的应用。
在本发明的一种实施方式中,所述跨膜蛋白41B(TMEM41B)的促进剂可以包括:
(1)能够特异性促进跨膜蛋白41B(TMEM41B)表达的siRNA、miRNA、IncRNA、gRNA和反义核苷酸中的任意一种;以及
(2)用于递送上述(1)的体内或体外递送载体。
作为本发明的一种具体实施方式,本发明的体内或体外递送载体例如为脂质纳米颗粒。本发明所述的“脂质纳米颗粒”指具有至少一个纳米量级尺寸的颗粒(如1-1000nm)。脂质纳米颗粒可被包含在药物组合物中,用于将活性剂或治疗剂(在本发明中为跨膜蛋白41B(TMEM41B)的促进剂)递送至目标靶部位(如细胞、组织(如肿瘤组织等患病组织)、器官)。在一些实施方案中,本发明的脂质纳米颗粒包含核酸。此类脂质纳米颗粒通常包含一种或多种辅助脂质分子、一种或多种胆固醇或胆固醇衍生物和/或一种或多种聚合物缀合的脂质分子。所述辅助脂质分子可以是一种或多种中性脂质分子。活性剂或治疗剂(在本发明中为跨膜蛋白41B(TMEM41B)的促进剂)被封装在脂质纳米颗粒的脂质部分中或者由脂质纳米颗粒的一些或所有脂质部分包封的水性空间中,从而保护其免于酶促降解,或不会产生由宿主有机体或细胞的机制诱导的其它不期望的作用,如不良免疫应答。
本领域周知,脂质纳米颗粒的平均直径可为约30nm至约40nm至约150nm、约50nm至约150nm、约60nm至约130nm、约70nm至约110nm、约70nm至约100nm、约80nm至约100nm、约90nm至约100nm、约70nm至约90nm、约80nm至约90nm、约70nm至约80nm、或约30nm、约35nm、约40nm、约45nm、约50nm、约55nm、约60nm、约65nm、约70nm、约75nm、约80nm、约85nm、约90nm、约95nm、约100nm、约105nm、约110nm、约115nm、约120nm、约125nm、约130nm、约135nm、约140nm、约145nm或约150nm,并且脂质纳米颗粒基本上是无毒的。
另,本发明的跨膜蛋白41B(TMEM41B)的促进剂还可以作为活性成分的组分制备用于治疗结血管内膜新生或冠状动脉狭窄的药物组合物。
一般来说,本发明的药物组合物可以在有效量下,通过任何可接受的用于其他类似用途的给药方式进行给药。举例来说,本发明的药物组合物可以口服、非肠道给药、透皮给药、局部给药、直肠给药或鼻内给药。
当用作药物时,本发明通常以药物组合物的形式给药。这些组合物可用制药学领域熟知的方法进行制备,这些组合物包含至少一种活性化合物,在本发明中,该活性化合物为所述的跨膜蛋白41B(TMEM41B)的促进剂。在配制本发明提供的组合物时,有效成分通常与医学上可接受的辅料或载体混合,被医学上可接受的辅料或载体稀释或被以胶囊,小袋,纸或其他形式的容器包裹。当所述医学上可接受的辅料或载体作为稀释剂时,可以是固体,半固体,或液体物质,可作为有效成分的运载体、载体或媒介。由此,所述组合物可以是药片、药丸、粉末、锭剂、小袋、胶囊、酏剂、混悬剂、乳剂、溶液、糖浆、喷雾(作为固体或在液体介质里),软膏,软和硬的明胶胶囊,栓剂,无菌注射溶液,和无菌包装粉末的形式。
一些典型的医学上可接受的辅料或载体包括乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、阿拉伯树胶、磷酸钙、海藻酸盐、黄芪胶、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、无菌水、糖浆和甲基纤维素。另外还可以包括润滑剂(如滑石粉、硬脂酸镁和矿物油)、浸润剂、乳化和悬浊剂、防腐剂(如对羟基苯甲酸甲酯和对羟基苯甲酸丙酯)、甜味剂和增味剂。本发明的药物组合物可以通过具体赋形方式在对病人进行给药之后达到药物活性成分的快速、持续或延时释放,这也是本领域中广泛应用的方法。
活性成分,即本发明中的跨膜蛋白41B(TMEM41B)的促进剂,在药物构成和单位剂型中的数量可根据具体应用情况、特定化合物的活性和预期浓度进行改变或有较大调整。
“治疗”意味着对哺乳动物体内疾病的任何治疗,包括:(1)防止疾病,即造成临床疾病的症状不发展;(2)抑制疾病,即阻止临床症状的发展;(3)减轻疾病,即造成临床症状的消退。
本发明通过构建的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型发现,血管平滑肌细胞的跨膜蛋白41B的缺失或突变会自发引起血管内膜新生或者由此导致的冠状动脉狭窄疾病、PCI后的再狭窄,表明跨膜蛋白41B(TMEM41B)可以成为治疗血管内膜新生或者由此导致的冠状动脉狭窄疾病、PCI后的再狭窄的药物靶点。
以下结合具体实施例,对本发明的技术方案做进一步的描述,但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
实施例:
以下实施例中涉及的小鼠代繁殖于上海塞业实验动物中心。
1、构建平滑肌细胞组成性敲除TMEM41B转基因小鼠(Tmem41bf/f,SM22α-Cre),人工饲养6周,安乐处死小鼠分离主动脉血管,进行病理学观察。
2、构建平滑肌细胞特异性条件性敲除TMEM41B转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)。转基因小鼠饲养至6周,连续腹腔注射Tamoxifen(80mg/kg)5天,继续饲养至16周,安乐处死小鼠分离主动脉血管,进行病理学观察。
动物实验结果:
图1为雄性转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠饲养6周,主动脉部分进行HE染色,与对照相比,转基因小鼠内膜层显著增厚,内膜新生明显。
图2为雌性转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠饲养6周,主动脉部分进行HE染色,与对照相比,转基因小鼠内膜层显著增厚,内膜新生明显。
图3为雄性转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)与同窝对照(Tmem41bf/f),Tamoxifen注射后16周,主动脉部分进行HE染色,与对照相比,转基因小鼠内膜层显著增厚,内膜新生明显。
图4为雄性转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)与同窝对照(Tmem41bf/f),Tamoxifen注射后16周,股动脉和颈动脉部分分别进行HE染色,与对照相比,转基因小鼠内膜层显著增厚,内膜新生明显。
图5为分别分离转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠主动脉平滑肌细胞体外进行培养,进行Westernblot验证。与对照细胞相比,KO小鼠的平滑肌细胞收缩型Markers(SMMHC、α-SMA、SM22α)显著下降,而合成型Marker(SPP1)明显升高。由此证明TMEM41B缺失的平滑肌细胞发生表型转化。
图6为分别分离转基因小鼠(Tmem41bf/f,SM22α-Cre)与同窝对照(Tmem41bf/f)小鼠主动脉平滑肌细胞体外进行培养,进行EDU染色。与对照相比,KO小鼠的细胞EDU信号显著增强,表明TMEM41B缺失的平滑肌细胞具有很强的增殖能力。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法,其特征在于,包括如下步骤:
步骤(1),小鼠胚胎细胞中在TMEM41B基因的3号和5号外显子插入floxp位点,通过胚胎打靶技术构建TMEM41B-floxp转基因小鼠;
步骤(2),根据Cre-flox敲除鼠构建系统,运用Sm22α-Cre工具鼠与TMEM41B-floxp转基因小鼠进行杂交繁育,获得平滑肌细胞特异性组成性敲除TMEM41B转基因小鼠(Tmem41bf/f,SM22α-Cre);
步骤(3),根据Cre-flox敲除鼠构建系统,运用Myh11-ERTCre工具鼠与TMEM41B-floxp转基因小鼠进行杂交繁育,获得平滑肌细胞特异性条件性敲除TMEM41B-floxp转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)。
2.根据权利要求1所述的方法,其特征在于,步骤(2)获得的平滑肌细胞特异性组成性敲除TMEM41B转基因小鼠(Tmem41bf/f,SM22α-Cre)饲养至6-8周自发性发生血管内膜新生。
3.根据权利要求1所述的方法,其特征在于,步骤(3)获得的平滑肌细胞特异性条件性敲除TMEM41B-floxp转基因小鼠(Tmem41bf/f,Myh11-ERT2Cre)饲养至6周,连续腹腔注射Tamoxifen(60-100mg/kg)5天,饲养至16周,自发性发生血管内膜新生。
4.一种通过权利要求1-3任一项所述的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型的构建方法得到的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型。
5.如权利要求4所述的跨膜蛋白41B(TMEM41B)基因敲除小鼠动物模型在血管内膜新生或冠状动脉狭窄疾病研究中的应用。
6.跨膜蛋白41B(TMEM41B)作为药物靶点在制备用于治疗血管内膜新生或冠状动脉狭窄的药物中的应用。
7.根据权利要求6所述的应用,其特征在于,血管平滑肌细胞的跨膜蛋白41B的缺失或突变会自发引起血管内膜新生或者由此导致的冠状动脉狭窄疾病、PCI后的再狭窄。
8.跨膜蛋白41B(TMEM41B)的促进剂在制备用于治疗血管内膜新生或冠状动脉狭窄的药物中的应用。
9.一种用于治疗结血管内膜新生或冠状动脉狭窄的药物组合物,其特征在于,所述药物组合物包括药学上可接受的载体和有效量的活性成分,所述活性成分包括如权利要求8所述的跨膜蛋白41B(TMEM41B)的促进剂。
10.根据权利要求7所述的药物组合物,其特征在于,所述药物组合物的剂型选自片剂、粉剂、注射剂、胶囊、混悬剂、糊剂、凝胶、涂布剂、药膜剂、缓释剂和微球中的任意一种。
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