CN112472690B - 一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病 - Google Patents
一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病 Download PDFInfo
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Abstract
本发明涉及一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病。化学方法涉及使用用贝壳杉烷类化合物上调CNPase的表达和活性。生物治疗药涉及使用重组腺相关病毒表达CNPase酶的制备及局部介入的疗法。在大鼠心肌肥大和心衰的模型中，上述方法可以有效改善心肌肥大、心肌重构、抑制心肌肥大和纤维化增加，增加心脏功能。
Description
背景技术
2’,3’-环核苷酸-3’-磷酸二酯酶(2',3'-cyclic nucleotide3'-phosphodiesterase，CNPase)在1960年代早期被发现，具有催化2’，3’-cAMP和2’，3’-cGMP降解的功能，在中枢神经系统具有高量表达。细胞外2’，3’-cAMP的释放与损伤有关，而2’，3’-cAMP可激活线粒体通透性转换孔(mPTPs)导致细胞凋亡。目前发现2’，3’-cAMP及CNPase与线粒体膜通透性相关，Jackson EK等人报道CNPase敲除可保护缺血/再灌注的肾脏功能(J Am Soc Nephrol.2016)。至今，没有人报道CNPase在心肌病的靶点及功能。
心衰是各种心脏疾病的严重表现或晚期阶段，死亡率和再住院率居高不下。心衰是多种原因导致心脏结构和/或功能的异常改变，使心室收缩和/或舒张功能发生障碍，从而引起的一组复杂临床综合征，主要表现为呼吸困难、疲乏和液体潴留(肺淤血、体循环淤血及外周水肿)等。心脏重构是心脏损伤或在血液动力学的应激反应时，由于分子和基因表达的变化，导致心脏的大小、形状和功能发生变化。
左室心肌肥大是心脏对于压力或容量超负荷、肌节蛋白基因突变或心肌梗死导致收缩力下降等病理情况的适应性反应,其伴随着多种心脏疾病,如高血压、瓣膜病及缺血性心脏病。在这些疾病的病理过程中,压力负荷过重会导致向心性心肌肥大,而向心性心肌肥大被认为是一种具有代偿作用的病理改变因其可以增加左室收缩力、降低室壁压力和心肌耗氧量。然而,左室心肌肥大同样可以成为严重心力衰竭以及恶性心律失常的危险因素。因此,在不引起循环功能障碍的前提条件下有效的抑制左室心肌肥大被认为具有重要的临床意义。
在众多心肌肥大所带来的不利因素中,能量代谢异常显得尤为突出：在左室心肌肥大的条件下,心脏的能量代谢方式由主要依靠长链脂肪酸有氧氧化转变为葡萄糖有氧氧化。能量代谢底物的改变一方面可以减少生成每摩尔ATP所需的耗氧量,即能够减少细胞内活性氧簇(Reactive Oxygen Species,ROS)的生成；但另一方面,这一改变不可避免的存在着大量的不利因素,如慢性脂肪酸有氧氧化障碍导致心肌细胞内脂质累积、乳酸堆积、以及糖酵解增多所带来的总ATP水平下降。
本团队采用腹主动脉结扎构建的压力引起的慢性心肌肥大大鼠模型，发现CNPase蛋白表达量没有变化，而酶活下降，这提示CNPase蛋白酶活不足可能是心肌肥大发生的原因，本团队直接以CNPase作为治疗靶点，在心脏过表达CNPase则可以逆转心肌肥大的病程，证明过表达CNPase可以逆转心肌肥大及慢性缺血过程中的代偿性心功能损伤为治疗靶点。
本专利申请人通过免疫荧光技术确定了CNPase在线粒体中的亚定位，探索其影响线粒体能量供应的功能；通过分子克隆技术，构建了表达CNPase蛋白的重组AAV包装质粒，使用病毒包装以及纯化技术，制备了重组AAV2/9-CNPase病毒，并通过了大鼠心肌肥大以及慢性缺血过程中的代偿性心功能损伤模型，确定了过表达CNPase对心肌病的保护功能以及临床上治疗潜力。同时，本发明揭示贝壳杉烷类化合物，如异甜菊醇钠可以增强CNPase的酶活。
发明内容
本发明旨是为提供一种在心脏中增强CNPase活性或表达的新工具，包括使用可以增强CNPase活性的腺相关病毒的生物药物及化合物异甜菊醇钠，用于心衰和心肌重构的保护药物。其中前临床心衰阶段的患者没有任何心衰的症状或体征，却以发展为结构性心脏病，例如左心室肥厚、无症状瓣膜性心脏病等。本发明揭示贝壳杉烷类化合物，如异甜菊醇及其衍生物，可以增强CNPase的活性，用于治疗心衰和心肌重构。
本发明的生物治疗的方法包括重组载体构建、重组病毒包装、浓缩和纯化以及相应的鉴定。具体地，采用AAV过表达载体，结合分子克隆技术，构建AAV-CNPase载体，其表达活性通过瞬时转染与western blot技术进行鉴定。进一步地，重组AAV2/9-CNPase病毒基于病毒包被、病毒颗粒收集以及浓缩纯化、滴度鉴定等步骤。本发明重组AAV2/9-CNPase病毒用于预防/或治疗心肌肥大及慢性缺血过程中的代偿性心功能损伤相关疾病。进一步地，通过AAV2/9-CNPase病毒实现心肌特异性CNPase上调，针对性的预防和/或治疗大鼠心肌肥大及慢、性缺血过程中的代偿性心功能损伤相关疾病的用途。
具体地，通过腹腔主动脉结扎术构建大鼠的慢性心肌肥大疾病模型，采用超声引导的方式，每只大鼠注射1×1012病毒，在第二周处理部分大鼠，取心脏，分别采用荧光定量PCR以及western blot技术，证实重组AAV2/9-CNPase病毒的定点注射可以提高CNPasemRNA、蛋白的表达。进一步地，分别在注射病毒之后的第二周、第四周、第六周、第八周，使用超声检测，检测参数包括射血分数、缩短分数、舒张压、收缩压、LVSP、max pressure、MaxdP/dt等动力学参数。进一步地，分别在注射病毒之后的第八周，处死大鼠，收集心脏，使用电镜固定液进行固定，经过电镜片子的制备之后，使用扫描电镜进行检测。通过对线粒体形态的观察，确认了CNPase注射可以促进线粒体自噬，通过促进受损线粒体的清除，改变心脏能量稳态的变化。分别在注射病毒之后的第八周，处死大鼠，收集心脏，提取总蛋白，使用western blot检测TGF-β1/2信号通路的变化，CNPase可以抑制该信号通路的激活。
具体地，采用冠状动脉结扎法对大鼠进行心肌缺血造模，采用超声引导的方式，每只大鼠注射1×1012病毒，在注射重组病毒的第四周，使用超声检测，检测参数包括射血分数、缩短分数等心功能指标。具体地，CNPase酶在心肌缺血损伤相关疾病的保护药物中的用途，其中所述的心肌缺血损伤相关疾病为心力衰竭，心律失常，缺血性心肌病，心脏破裂。本发明第一次揭示了CNPase与心功能的直接联系，首次发现了CNPase在心肌肥大及慢性缺血过程中的代偿性心功能发挥了保护作用，上调CNPase可以预防/保护心肌肥大及慢性缺血过程中的代偿性心功能的双重功效，揭示了CNPase在心肌疾病治疗中的全新领域，并为预防和治疗心肌肥大、心肌成纤维化及心肌缺血损伤相关的心力衰竭、心律失常、缺血性心肌病、心脏破裂等疾病提供了新的方案，具有广阔的临床应用前景。
以上是本发明整体的介绍。为了更好地说明本发明的方法和技术，以下将给出实施案例，以便可以由本领域技术人员执行。
在以下实施例中详细提供了本发明的方法和实施方式。
具体实施方式
为了进一步说明用于实现本发明目的的技术，下文描述了关于确定和鉴定本发明中化合物的药物和治疗用途的详细方法，技术，流程和特点。案例提供了用于支持及验证本发明所使用的动物模型的实验方法和结果。涉及的案例均使用了适当的对照组实验及统计分析方法。以下的案例均用于描述而非限制本发明的应用。这些案例所涉及的方法及技术可用于制备增强CNPase活性的化学或生物药物的方法。其他此类化合物制剂的治疗效果评价可使用相同的方法。
本发明中列举的案例用于支持本发明的实验方法和结果，并验证本发明中使用的动物模型。本发明的所有实验均采用了适当的对照和统计检验。提供以下实施例来说明而非限制本发明。这些例子说明了一种制备增强CNPase活性的化学或生物药物的方法用于治疗心衰和心肌重构。
实验材料实验动物：成年雄性Sprague Dawley大鼠，体重230g±20g，6-8周龄。饲养环境包括恒定的温度、湿度以及严格的黑暗光照周期，自由采食。
化学试剂：异甜菊醇(ent-17-norkaurane-16-oxo-18-oic acid，分子式，C20H40O3，分子量：318.5)是由甜菊糖通过酸性水解、结晶纯化而得到。异甜菊醇的钠盐可以通过加入NaOH或其他含钠碱获得；用高效液相色谱法测得异甜菊醇钠盐的纯度大于99％。
AAV-CNPase重组质粒的构建：采用断颈的方法处死SD大鼠，取心脏，称取适当重量的心脏组织，采用TRIZOL的方法提取总RNA；取5μg RNA作为模板，采用QIAGEN公司的RNA逆转录试剂盒制备cDNA；以cDNA作为模板，采用Thermo ScientificTMPhusionTM高保真DNA聚合酶扩增CNPase编码序列(coding sequence，CDS)，其中引物为带有酶切位点BamHI与HindIII；PCR产物采用琼脂糖电泳割胶纯化、BamHI与HindIII双酶切、纯化，制备出带有酶切位点黏性末端的CNPase CDS产物，pAAV-MCS质粒采用采用BamHI与HindIII双酶切、纯化，制备带有黏性末端的线性化质粒；采用T4连接酶，连接酶切质粒与CNPase CDS产物，转入大肠杆菌感受态DH5α细胞中，在LB培养基中37℃孵育一个小时；离心，取沉淀，重悬之后，均匀涂抹到带有氨苄抗生素的琼脂糖固体平板上，37℃培养过夜；提取单克隆，采用菌落PCR的方法筛选阳性克隆，并通过测序的方法确认重组质粒的构建成功。
重组AAV2/9-CNPase病毒的包装、纯化与浓缩以及滴度的鉴定：HEK293细胞系作为包装重组病毒的工具，病毒包装的具体步骤如下：采用质粒大提试剂盒，提取无内毒素的pAAV-MCS-CNPase质粒、pHELPER质粒、pAAV-RC9质粒，采用脂质体转染的方法转染；分别收取培养基上清和细胞，对上清采用PEG6000进行沉淀和浓缩，对细胞中的病毒颗粒采用反复冻融的方法获取，并使用PEG6000进行浓缩；浓缩后的病毒采用超高速离心以及透析的方法进行纯化，分装，冻存在-80℃备用；采用荧光定量PCR的方法检测病毒滴度。
实验方法
腹主动脉狭窄(TAAC)后压力诱导的大鼠肥厚心肌的实验方案
用10％的水合氯醛0.3ml/100g腹腔注射麻醉。麻醉后，将大鼠仰卧，四肢固定。行腹部备皮。沿剑突下腹正中，左肾上方处为手术部位，行2.0～2.5cm纵行切口，逐层打开腹腔。暴露腹后壁及左侧肾脏，找到左、右肾动脉分支，在左肾动脉以上钝性游离腹主动脉。在左、右肾动脉分支之间腹主动脉段下面穿入1-0号手术丝线，沿血管走行方向放置磨钝的7号注射器针头。在左、右肾动脉分支之间将腹主动脉和尼龙线一同结扎。观察左肾发白(表明结扎可靠)，然后迅速抽出注射器针头，观察左肾充血变红，使大鼠腹主动脉横截面积减少50％左右。手术后逐层缝合。术后腹腔注射庆大霉素3日，预防感染。假手术组开腹后将手术丝线穿过腹主动脉，除不缩窄腹主动脉外，其它操作均与正式手术组完全相同。观察期结束，测定体内血流动力学后，将所有动物处死，并把心脏取出做进一步分析。
心脏血流动力学参数测量
造模后的第2周，然后给AAV2/9-CNPase的第6周后，将大鼠麻醉后将心电图针电极插入四肢皮下(20％氨基甲酸乙酯，12g/kg，腹腔注射)。心电图和体温(37℃)稳定后，分离右侧颈总动脉，结扎颈总动脉末端进行动脉插管。压力传感器与多通道生理信号采集处理系统相连。稳定后15分钟记录收缩压(sbp)、舒张压(dbp)和心率(bpm)。然后，将套管插入左心室，直到出现心室压力波形、左心室收缩压(LVSP)和左心室舒张末期压(LVEDP)。稳定后15分钟记录所有(-dp/dtmax)。所有变量均由Power Lab软件(Power Lab 8/30ADInstruments，澳大利亚)监测。
组织学分析
利用4％的中性福尔马林固定大鼠心肌组织，进行石蜡包埋，切成3毫米的切片，然后用苏木精-伊红(H&E)及马松染色。采用蔡司共聚焦显微镜拍照。用H&E染色结果检测细胞形态大小，使用马松染色检测纤维化。使用计算机辅助图像分析(图像处理软件)来确定细胞横截面面积和间隙胶原含量。样本量至少为四个或五个不同的心脏组织。
统计分析
依次通过方差分析(单因素方差分析)，Fisher检验比较多组间的差异。所有检验的P值均为双尾，以P<0.05被认为是具有统计学差异。
实施例1
本案例主要说明异甜菊醇钠增强采用在血管紧张素II诱导的心肌肥厚细胞中的CNPase活性。采用小牛肠碱性磷酸酶活性的检测方法，在给予异甜菊醇后,检测肥厚心肌样本中CNPase的酶活。
表1.异甜菊醇钠可以增强采用血管紧张素II诱导的心肌肥厚中的CNPase活性(n＝3)
实施例2
本案例主要说明过表达CNPase改善TAAC诱导的心肌肥厚的作用。
成年的SD大鼠经过TAAC诱导2周后，分别使AAV-CNPase治疗。心脏体重比(HW/BW)是反映心肌肥大的指数。在8周TAAC模型组中，我们发现TAAC组大鼠的心脏体重比(HW/BW)显著上升；而过表达CNPase可以有效减低心脏体重比。
表2.过表达CNPase对TAAC模型大鼠心重与体重的影响(n＝8-14)
实施例3
本案例主要说明了AAV-CNPase在抑制心肌纤维化形成中的作用。
为了确定过表达CNPase是否可以减弱TAAC诱导的心肌纤维化，我们使用马松染色检测左心室心肌间质胶原的改变。我们发现TAAC组大鼠心肌纤维化增强及胶原沉积增多；而过表达CNPase可以有效减少心肌纤维化及胶原的沉积。
表3.过表达CNPase抑制TAAC模型大鼠心肌纤维化形成中(n＝8-14)
实施例4
为了评估CNPase是否也可以改善TAAC大鼠的心功能，我们发现在TAAC引起的心肌肥厚大鼠模型中，过表达CNPase大鼠的左心室射血分数(EF)、分数缩短(FS)和心输出量(CO)均得到了改善。
表4.心脏特异性过表达CNPase有效改善TAAC大鼠的心功能等指标(n＝8-14)
Claims (6)
1.一种过表达CNPase或其编码mRNA的重组质粒在制备提高心肌CNPase的活性或表达的药物中的应用，其特征在于，所述药物用于治疗和/或预防心肌肥大及慢性缺血过程中的代偿性心功能损伤相关疾病。
2.根据权利要求1所述的应用，其特征在于，所述的重组质粒的制备使用慢病毒，腺病毒及腺相关病毒，以及细菌或酵母。
3.根据权利要求1所述的应用，其特征在于，所述的重组质粒可生产具有CNPase功能的蛋白。
4.根据权利要求1所述的应用，其特征在于，所述心肌肥大及慢性缺血过程中的代偿性心功能损伤相关疾病包括心力衰竭，心律失常，缺血性心肌病中的一种或多种。
5.根据权利要求1所述的应用，其特征在于，所述的代偿性相关疾病包括心肌重构及心肌纤维化。
6.根据权利要求1所述的应用,其特征在于，以不同的溶剂或脂质体载体，通过肌肉，静脉、或心导管注入到心脏或心包膜腔中上调CNPase活性或表达。
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| CN202210882975.6A CN115137718A (zh) | 2020-11-15 | 2020-11-15 | 一种制备恢复心脏肥厚病中减低的CNPase活性的药物的方法 |
| PCT/CN2021/130496 WO2022100720A1 (zh) | 2020-11-15 | 2021-11-14 | 一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病 |
| US18/036,404 US20240016769A1 (en) | 2020-11-15 | 2021-11-14 | Method for preparing compound or biological drug enhancing CNPase activity for treating heart diseases |
| AU2021380634A AU2021380634A1 (en) | 2020-11-15 | 2021-11-14 | Method for preparing compound or biological drug enhancing cnpase activity for treating heart diseases |
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