CN117327703B - 一种靶向平滑肌细胞的Agrin-shRNA及其在制备抗动脉粥样硬化的药物中的应用 - Google Patents
一种靶向平滑肌细胞的Agrin-shRNA及其在制备抗动脉粥样硬化的药物中的应用 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种靶向平滑肌细胞的Agrin‑shRNA及其在制备抗动脉粥样硬化的药物中的应用,属于生物医药领域。所述Agrin‑shRNA包括正义链和反义链;所述正义链的核苷酸序列如SEQ ID NO.1所示;所述反义链的核苷酸序列如SEQ ID NO.3所示。本发明制备了负载Agrin‑shRNA的腺相关病毒,通过小鼠尾静脉注射Agrin‑shRNA腺相关病毒,能够显著缓解动脉粥样硬化,从而为制备或筛选抑制平滑肌细胞Agrin基因表达的药物或制剂作为抗动脉粥样硬化的备选药物或制剂提供了理论依据;同时对动脉粥样硬化的治疗具有重要意义。
Description
技术领域
本发明涉及生物医药领域,特别是涉及一种靶向平滑肌细胞的Agrin-shRNA及其在制备抗动脉粥样硬化的药物中的应用。
背景技术
动脉粥样硬化(Atherosclerosis,AS)是一种动脉管壁的慢性炎症性疾病。世界卫生组织2019年公布的数据显示,AS所导致的缺血性心脏病(Ischemic heart disease,IHD)和脑卒中等心血管疾病仍是全世界最大的健康杀手,是过去15年间全球第一位死因,预测至2030年占全部疾病死因的26%。目前AS的发病机制尚未完全清楚,进一步阐明AS的发生机制并寻找有效的干预措施是当前心血管病领域中一个亟需解决的重大课题。
血管平滑肌细胞(VSMCs)的表型转化是AS斑块发生发展的重要病理过程。正常血管组织中的VSMCs处于收缩表型(分化状态),表达一系列收缩蛋白,维持动脉壁结构及调节血管张力。在炎症因子及氧化修饰的脂蛋白等病理性刺激作用下,VSMCs可由收缩表型向合成表型(去分化状态)转化。Notch1/Hes1通路激活可以抑制VSMCs中一系列收缩蛋白的转录和表达,导致VSMCs收缩性能下降,而分泌、迁移及成骨性能增强,参与粥样斑块纤维帽和潜在的坏死核心的形成。
集聚蛋白(Agrin)是一种硫酸肝素蛋白多糖,分子量约为400kDa,由三个结构域组成:分别为羧基片段、氨基片段及中间区域。作为一种细胞外基质(Extracellular matrix,ECM)蛋白,Agrin在神经肌肉接头、心肌、肾脏、血管等组织中均有表达。研究报道,小鼠心脏中的Agrin与心肌细胞受体肌营养不良蛋白聚糖(α-dystroglycan,DAG)结合,激活Hippo-Yap通路促进心肌细胞增殖。研究发现Agrin在刚出生小鼠心脏ECM内含量较高,而在7天时表达明显下调,从而心肌细胞再生修复能力减弱,在成年小鼠心梗模型中外源性注射Agrin能够促进心肌细胞增殖和血管新生形成。此外,Agrin在调控肿瘤微环境中的血管生成中扮演着关键的角色。Agrin通过与其受体复合物,包括脂蛋白相关受体4(Lrp4)和整合素β1,稳定了内皮细胞血管内皮生长因子受体2(VEGFR2),同时激活了细胞粘附激酶(FAK)。VEGFR2的稳定进一步激活了内皮细胞一氧化氮合酶(eNOS)-Akt-ERK1/2(细胞外信号调节激酶1/2)信号通路,从而在肿瘤组织中持续促进血管发芽和血管生成。总之,这些发现表明Agrin在心血管***中具有重要的功能。然而,Agrin与动脉粥样硬化的关系及机制尚不清楚。
发明内容
本发明的目的是提供一种靶向平滑肌细胞的Agrin-shRNA及其在制备抗动脉粥样硬化的药物中的应用,以解决上述现有技术存在的问题。本发明提供的Agrin-shRNA能够有效沉默平滑肌细胞中的Agrin基因,进而缓解动脉粥样硬化现象。
为实现上述目的,本发明提供了如下方案:
本发明提供一种靶向平滑肌细胞的Agrin-shRNA,所述Agrin-shRNA包括正义链和反义链;所述正义链的核苷酸序列如SEQ ID NO.1所示;所述反义链的核苷酸序列如SEQ IDNO.2所示。
本发明还提供一种包含所述Agrin-shRNA的载体。
进一步地,所述载体包括腺相关病毒载体。
本发明还提供一种包含所述载体的宿主。
进一步地,所述宿主包括腺相关病毒。
本发明还提供所述的Agrin-shRNA、所述的载体或所述的宿主在制备和/或筛选抗动脉粥样硬化的药物中的应用。
进一步地,所述Agrin-shRNA通过抑制平滑肌细胞中Agrin基因的表达,减少Notch-1和Hes-1蛋白的表达量,进而缓解动脉粥样硬化。
进一步地,所述Agrin-shRNA的靶点序列如SEQ ID NO.3所示。
进一步地,所述动脉粥样硬化由高脂饮食引起。
进一步地,所述药物包括核酸分子、脂类、小分子化合物、抗体、多肽、蛋白质或腺相关病毒。
本发明公开了以下技术效果:
本发明以小鼠Agrin基因为基础,合成了能够沉默平滑肌细胞中Agrin基因的shRNA,通过慢病毒包装,制备出了以Agrin基因沉默为作用靶标的腺相关病毒(AAV-Agrin-shRNA)。通过连续12周高脂喂养ApoE-/-小鼠来构建动脉粥样硬化模型,发现注射腺相关病毒的小鼠能够显著缓解动脉粥样硬化,从而为制备或筛选抑制平滑肌细胞Agrin基因表达的药物或制剂作为抗动脉粥样硬化的备选药物或制剂提供了理论依据。
通过对本发明实验结果的分析可知,可以以Agrin基因沉默为作用靶标,制备或筛选抗动脉粥样硬化的药物。药物筛选主要是针对未知药物,将药物作用于靶标基因,根据该药物是否可以抑制靶标基因的表达,进而筛选得到抗动脉粥样硬化的药物;药物制备主要是以靶标基因为基础,有针对性地制备或构建抗动脉粥样硬化的药物以抑制靶标基因的表达,对动脉粥样硬化的治疗具有重要意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为腺相关病毒载体图谱;
图2为对照组(Control)和实验组(AAV-agrin-shRNA)小鼠主动脉根部粥样斑块负荷情况;
图3为Western blot验证对照组(Control)和实验组(AAV-agrin-shRNA)小鼠Agrin、Notch-1和Hes1蛋白的表达情况。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1小鼠Agrin-shRNA腺相关病毒包装
一、腺相关病毒重组载体构建
1.引物设计
设计合成Agrin基因的Agrin-shRNA序列。
Agrin-shRNA序列如下:
正义链:
AGCGAGCTCAGATGCTTCCACCTATATAGTGAAGCCACAGATGTATATAGGTGGAAGCATCTGAGCC(SEQ ID NO.1);
反义链:
GGCAGGCTCAGATGCTTCCACCTATATACATCTGTGGCTTCACTATATAGGTGGAAGCATCTGAGCT(SEQ ID NO.2);
shRNA识别的靶点序列如下:
5’-GCTCAGATGCTTCCACCTATA-3’(SEQ ID NO.3)
2.载体酶切
腺相关病毒载体pAV-Sm22a-GFP-miR30-shRNA质粒(购自山东维真生物科技有限公司,质粒图谱如图1所示)用表1所示酶切体系酶切,胶回收载体。
表1酶切体系
反应液成分 | 体积 |
质粒(1μg/μL) | 1μL |
10×Buffer | 5μL |
BpiI酶 | 1μL |
双蒸水 | 42μL |
总体积 | 50μL |
加样混匀后置于37℃酶切2h,载体去磷酸化20min,反应结束后用1%琼脂糖凝胶电泳检测酶切,并用胶回收试剂盒回收载体。
3.退火
引物稀释成100μM的母液。退火反应体系如表2所示。
表2退火反应体系
反应液成分 | 体积 |
正义链 | 1μL |
反义链 | 1μL |
Buffer | 2μL |
双蒸水 | 15.5μL |
PNK酶 | 0.5 |
总体积 | 20μL |
退火反应程序如表3所示。
表3退火反应程序
步骤 | 温度/℃ | 时间 |
1 | 37 | 30min |
2 | 98 | 3min |
3 | 98ramp25 | 0.1℃/s |
4 | 25 | 20min |
4.连接
将退火产物稀释100倍,与酶切好的载体连接,连接体系如表4:
表4连接体系
成分 | 体积 |
稀释后的退火产物 | 2μL |
酶切载体 | 1μL |
10×T4 Buffer | 0.5μL |
T4 DNA连接酶(10U/μL) | 0.5μL |
双蒸水 | 1μL |
总体积 | 5μL |
混匀后瞬时离心,22℃连接1h,获得连接产物。
5.转化
连接产物转化大肠杆菌DH5α感受态细胞,涂布于具有卡那霉素抗性的LB平板上进行筛选。
转化的具体步骤:
从-80℃取出提前制备好的DH5α感受态置于冰浴中;
待DH5α感受态细胞融化后,取5μL连接产物于20μL DH5α感受态细胞中,充分混匀,冰浴中静置15min;
将离心管放入42℃水浴锅中40s(期间不要摇动离心管),然后快速移至冰浴中,静置2min;
向离心管中加入200μL的无菌的LB培养基(不加抗生素),混匀后置于摇床中37℃,220rpm,振摇1h。目的是使质粒上相关的抗性标记基因表达,使菌体复苏;
涂布到具有卡那霉素抗性的固体培养基平皿中;
37℃培养箱中培养过夜。
6.测序
挑取单菌落培养后提取质粒进行酶切鉴定阳性克隆,测序验证,验证正确的质粒大提后用于后续实验。
二、腺相关病毒包装:
1.细胞复苏
1)直径10cm的培养皿中加10mL新鲜DMEM培养基,放培养箱预热至37℃。
2)从液氮中取出冷冻管,迅速投入37℃水浴中,2min使其融化。
3)冷冻管中溶液融化后,200×g离心5min,弃掉液体并吸取1mL预热的培养基将细胞沉淀轻轻吹起,转移到直径10cm的培养皿中。
4)摇晃均匀,置于培养箱培养。
5)培养过夜,更换新鲜培养基(除去冻存液中DMSO对细胞的毒害作用)。
2.细胞传代(以直径10cm的培养皿为例)
1)生物安全柜紫外灭菌0.5h。
2)灭菌期间,将DMEM培养基(含10%FBS,1%青链霉素混合液)和PBS置于37℃水浴锅中预热;0.25%胰酶放置室温,不可水浴加热。
3)取汇合度接近100%且活性较好的HEK293T细胞,吸弃培养盘中培养基,加入5mL1×PBS,摇晃几下,吸弃PBS。
4)加1mL0.25%胰酶在生物安全柜内消化约1min,室温低时可放置于培养箱中消化。消化时间不宣过长,否则影响细胞再次贴壁效率与活性。
5)加入约5mL预热的培养基,终止消化。
6)用移液管吹打均匀(吹打过程中不可用力过大,否则会吹瞄胞),按照1:3的比例传代。各取2mL培养基至新的直径10cm的培养皿中,再加入8mL预热的DMEM培养基。
注意:传代盘数较多时,要先将预热的DMEM培养基加入到直径10cm的培养皿中,再加入含细胞的培养基,以避免细胞分布不均匀。置于培养箱前轻轻混匀培养皿中的培养基,使细胞均匀分散于培养基中。
3.AAV病毒包装(以直径10cm的培养皿为例)
第一天:汇合度90%以上的HEK293T细胞按1:3比例传盘(每盘大约2.5×106),培养基为Hydone高糖DMEM培养基(含10%FBS,购自Hydone公司)。
第二天:转染前2h,换成无血清培养基。
按照表4比例配制转染试剂:
表5转染试剂配制体系
Mix1和Mix2配制完成后,室温静置10min,将Mix1和Mix2混合,室温静置20min,逐滴加入至直径10cm的培养皿中。
第三天:质粒转染24h后,换新的无血清培养基。
第五天:转染72h收毒,将产毒的细胞连同培养基一起收集至50mL离心管中,离心,分别收获培养基上清与细胞沉淀,PEG8000沉淀培养基上清中的病毒;裂解细胞沉淀收毒;合并从细胞沉淀和上清中得到的AAV。
4.AAV病毒纯化与浓缩
4.1纯化-一碘克沙醇密度梯度离心
①配制不同浓度的碘克沙醇;
②取一个超离管,用电动移液器逐层、缓慢加入不同浓度的碘克沙醇;
③将处理好的病毒液加入到最上层;
④配平后超速,18℃、48000rpm、离心2.5h。
4.2浓缩
①离心完毕后,将超滤管底用针头刺破,收集腺相关病毒所在层至15mL管中;
②将收集的病毒液注入浓缩柱中,加PBS+0.001%PF68(泊洛沙姆188)至满,混匀;
③4000rpm,10℃,离心1h;
④将超滤管中剩下的液体反复吹打后吸至病毒储存管中,最后加入病毒储存液;
⑤将收集起来的病毒涡旋震荡混匀后离心,吸10μL病毒液进行滴度检测。
5.病毒滴度检测方法
实时定量PCR法是一种简单的、高通量的测定纯化病毒样本中腺相关病毒颗粒数量的方法。每个模板的Ct值与该模板起始拷贝数的对数存在线性关系,利用已知起始拷贝数的标准品可作出标准曲线,最后通过标准曲线对未知模板进行定量分析。
5.1去除游离DNA分子
先将病毒稀释10倍,以保证样品中游离的DNA充分降解:取5μL病毒至45μLPBS缓冲液中,充分混匀。按表5体系配制Mixture:
表6Mixture配制体系
37℃孵育30min,95℃加热5min使DNA酶失活。
5.2去除病毒蛋白外壳
向上述体系中再加入1μL蛋白酶K(5μg/μL),37℃孵育30min;再加30μL超纯水稀释至40μL(至此病毒原液稀释100倍),95℃加热5min使蛋白酶K失活,然后12000rpm,离心2min,取上清进行qPCR检测。
37℃孵育30min,95℃加热5min使DNA酶失活。
5.3qPCR
将5.2得到的上清,取5μL进行10倍梯度稀释,即病毒原液稀释了1000倍。分别取2μL待测样品及标准品作为模板进行qPCR检测。
qPCR反应体系:2×SYBR Green mix 10μL,Primers-F 0.8μL,Primers-R 7.2μL,DNA2μL,Total 20μL。Primers-F如SEQ ID NO.1所示;Primers-R如SEQ ID NO.2所示。
qPCR反应程序:95℃预变性3min,95℃5s,60℃15s,72℃15s,39个循环。
实施例2Agrin基因的腺相关病毒对动脉粥样硬化的作用
将apoE-/-小鼠(购北京维通利华实验动物技术有限公司)分为两组,每组6只,在10周龄时进行处理,分别尾静脉注射实施例1获得的腺相关病毒AAV-Agrin-shRNA及对照AAV空载体(AAV-GFP),两组的小鼠及处理情况如下:
实验组:ApoE-/-小鼠,尾静脉注射AAV-Agrin-shRNA;
对照组:ApoE-/-小鼠,尾静脉注射AAV-GFP。
两组小鼠给予连续12周高脂喂养后,安乐死小鼠,取小鼠主动脉根部切片,小鼠主动脉根部粥样斑块负荷情况如图2所示,对照组小鼠产生了严重的主动脉根部粥样硬化现象,试验组小鼠尾静脉注射腺相关病毒AAV-Agrin-shRNA后能够显著缓解主动脉根部粥样硬化。
取上述小鼠主动脉,分别剥去主动脉外膜,加蛋白裂解液研磨样品,各取40μg蛋白上样做Western blot验证,分别用Agrin、Notch-1和Hes-1抗体检测Agrin、Notch-1和Hes1蛋白的表达情况,结果如图3所示,发现小鼠尾静脉注射腺相关病毒AAV-Agrin-shRNA可以抑制Agrin蛋白表达,减少Notch-1和Hes-1蛋白的表达量,从而缓解主动脉根部粥样硬化。
以上结果证实了Agrin基因在调控Notch-1/Hes-1通路中的关键作用,对照组小鼠主动脉根部粥样斑块进展明显,Agrin基因表达量升高,而通过给ApoE-/-小鼠尾静脉注射Agrin-shRNA腺相关病毒的方法加以治疗,能够缓解小鼠主动脉根部粥样硬化程度。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (4)
1.一种靶向平滑肌细胞的Agrin-shRNA或包含所述Agrin-shRNA的载体在制备抗动脉粥样硬化的药物中的应用,其特征在于,所述Agrin-shRNA包括正义链和反义链;所述正义链的核苷酸序列如SEQ ID NO .1所示;所述反义链的核苷酸序列如SEQ ID NO .2所示。
2.根据权利要求1所述的应用,其特征在于,所述Agrin-shRNA通过抑制平滑肌细胞中Agrin基因的表达,减少Notch-1和Hes-1蛋白的表达量,进而缓解动脉粥样硬化。
3.根据权利要求2所述的应用,其特征在于,所述Agrin-shRNA的靶点序列如SEQ ID NO.3所示。
4.根据权利要求1所述的应用,其特征在于,所述动脉粥样硬化由高脂饮食引起。
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