CN115029351A - 一种shRNA或BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用 - Google Patents
一种shRNA或BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种shRNA或BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用,涉及生物医药技术领域。本发明提供了三种靶向沉默BACH1基因的shRNA。本发明还提供上述的shRNA或BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用。本发明通过利用shRNA沉默BACH1基因或利用BACH1缺失巨噬细胞来源的EVs,可以抑制BACH1关键靶点,可改善高血压异常的VSMCs增殖,进而抑制血管重构,延缓或阻滞高血压的发生发展和并发症形成。
Description
技术领域
本发明涉及生物医药技术领域,特别是涉及一种shRNA或BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用。
背景技术
高血压是心脑血管病的主要危险因素,血管重构和血管炎症在高血压的发生发展和并发症形成中起重要作用。目前对高血压的防治手段主要是药物,然而高剂量药物治疗出现了各种副作用并表现出耐药症状,致残率和致死率高,患者生存质量差,迫切需要探明高血压发病机制,寻找有效的防治方法。
巨噬细胞是免疫炎症的中心细胞,可促进炎症反应,导致高血压血管重构,在高血压的病理进程中发挥关键作用。血管平滑肌细胞(VSMCs)增殖是血管重构的关键因素,与高血压、动脉粥样硬化和血管成形术后再狭窄等心血管疾病的进展密切相关。巨噬细胞和VSMCs是血管重构的主要细胞成分,但巨噬细胞对VSMCs的调控作用及其在高血压血管重构中的作用与机制仍不清楚。
血管紧张素转化酶抑制剂(ACEI)等药物有降血压外的抗炎效应,慢性炎症在高血压的病理进程中起重要作用,提示可以从抗炎角度治疗高血压。在动脉粥样硬化早期和晚期病变中巨噬细胞产生丰富的血管紧张素转化酶(ACE),人单核细胞系THP1分化为巨噬细胞时,ACE增加。单核/巨噬细胞产生的效应细胞因子可调控氧化应激、内皮功能及血管炎症反应,导致高血压患者的靶器官损伤。巨噬细胞产生的VEGF-α可改善缺氧诱导的血管损伤,说明巨噬细胞浸润与缺氧诱导的血管重构密切相关。巨噬细胞在高血压的病理进程中发挥关键作用,但其具体作用机制尚不清楚。
BTB和CNC同源体1(BACH1)是一种应激反应性转录因子,具有增强氧化应激、调节细胞周期和免疫功能的作用。研究发现BACH1参与机体炎症反应和氧化应激,与心血管的氧化损伤相关。BACH1促进糖酵解依赖性的肺癌转移,靶向BACH1可使糖酵解正常,能够预防抗氧化剂诱导的转移。肺气肿患者肺组织和肺泡巨噬细胞中BACH1水平显著升高,氧化负荷增加。另外,BACH1缺失可激活心肌表达HO-1,减少缺血/再灌注损伤小鼠的心肌细胞死亡,改善横主动脉收缩(TAC)诱导的左心室肥大和重构,减少心血管的氧化损伤,提示BACH1与心血管的氧化损伤相关。然而,BACH1在高血压中的作用尚未报道。
发明内容
本发明的目的是提供一种shRNA或BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用,以解决上述现有技术存在的问题,通过沉默BACH1基因或利用BACH1缺失巨噬细胞来源的细胞外囊泡(EVs),可以抑制BACH1关键靶点,可改善高血压异常的VSMCs增殖,进而抑制血管重构,延缓或阻滞高血压的发生发展和并发症形成。
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沉默BACH1基因的shRNA,其特征在于,所述shRNA的正义链如SEQ ID NO.7所示,所述shRNA的反义链义链如SEQ ID NO.8所示。
本发明还提供一种靶向沉默BACH1基因的shRNA,所述shRNA的正义链如SEQ IDNO.9所示,所述shRNA的反义链如SEQ ID NO.10所示。
本发明还提供一种靶向沉默BACH1基因的shRNA,所述shRNA的正义链如SEQ IDNO.11所示,所述shRNA的反义链如SEQ ID NO.12所示。
本发明还提供一种重组shRNA干扰载体,包含上述的三个shRNA的其中之一。
本发明还提供一种慢病毒,包含上述的三个shRNA的其中之一或上述的重组shRNA干扰载体。
本发明还提供上述的shRNA、重组shRNA干扰载体或慢病毒在制备治疗高血压的药物中的应用。
本发明还提供上述的shRNA、重组shRNA干扰载体或慢病毒在制备抑制BACH1基因的表达的药物中的应用。
本发明还提供一种上述的重组shRNA干扰载体的构建方法,包括:首先合成shRNA的引物单链,然后退火配对产生双链,再将所述双链连入RNA干扰载体上,鉴定得到所述重组shRNA干扰载体。
进一步地,所述RNA干扰载体为pGmLV-SC5 RNAi载体。
本发明还提供BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用。
EVs在高血压、动脉粥样硬化和血管内皮细胞损伤等心血管领域均有研究,可调节炎症、氧化应激和钙化并可能影响血管疾病的发病机制,在高血压等心血管病中起重要作用。我们前期研究发现,血管外膜成纤维细胞(VAFs)来源的EVs调控VSMCs增殖、迁移和高血压血管重构。在此基础上,我们发现巨噬细胞来源的EVs在VSMCs增殖中起重要作用。
炎症细胞除了通过直接释放病毒蛋白、生长因子和细胞因子来促进肺血管重构外,还可能通过释放EVs参与细胞间通信。巨噬细胞来源的EVs在高血压条件下上调内皮细胞炎症因子的表达。巨噬细胞来源的EVs携带抗炎细胞因子IL-10,可改善肾小管损伤和缺血/再灌注损伤引起的炎症。动脉粥样硬化巨噬细胞释放的EVs促进动脉粥样硬化的发展,另外,巨噬细胞来源的EVs促进VSMCs迁移和泡沫细胞粘附。这些研究结果提示巨噬细胞来源的EVs在高血压血管重构中起重要作用。
III型纤连蛋白组件包含蛋白5(FNDC5)可以减轻胰岛素抵抗和肝脏脂肪变性,改善糖脂代谢,抑制高脂肪饮食诱导的肥胖小鼠脂肪组织炎症,减轻Ang II诱导的VSMCs氧化应激和NLRP3炎性小体激活。我们前期研究发现SHR的VAFs中增加的miR-135a-5p通过抑制FNDC5促进VSMCs增殖,这可能与FNDC5抑制炎症和氧化应激相关。另外,FNDC5预处理骨髓源性间充质干细胞分泌的EVs(FNDC5-BMMSCs-EVs)通过NF-κB信号通路和Nrf2/HO-1发挥抗炎作用,促进M2巨噬细胞极化,参与心肌梗死的治疗。BACH1/Nrf2可竞争性调控HO-1,调控机体炎症和氧化应激。我们推测FNDC5可能是BACH1的下游靶点,为此我们进行了一系列研究。
本发明公开了以下技术效果:
本发明从动物、细胞和分子水平多层次进行研究,并将细胞和分子水平的研究结果运用于整体动物,通过探讨巨噬细胞的EVs通过转运BACH1调控VSMCs增殖和高血压血管重构的作用与机制,为防治高血压和血管重构提供新思路和理论依。本发明发现:
(1)巨噬细胞可以分泌EV,巨噬细胞的EVs中外泌体相关标记蛋白CD9、CD63、TSG101表达阳性。这些结果符合外泌体的特征,确定本发明中分离的EVs是外泌体。
(2)巨噬细胞来源的EVs参与调控VSMCs增殖,其中京都种的正常Wistar大鼠(WKY)的巨噬细胞的EVs对WKY的VSMCs的增殖无明显影响,但抑制自发性高血压大鼠(SHR)的VSMCs增殖。SHR的VSMCs的增殖能力强于WKY的VSMCs,SHR的巨噬细胞的EVs促进WKY和SHR的VSMCs增殖。
(3)与WKY比较,SHR胸主动脉中BACH1水平显著升高,SHR的巨噬细胞中BACH1含量升高,但VSMCs中BACH1升高不明显,与WKY的EVs相比,SHR的EVs中BACH1升高更显著。
(4)SHR的BACH1缺失巨噬细胞来源的EVs对VSMCs增殖的促进作用减弱。
根据上述作用机制,本发明还设计了三种shRNA,可以成功沉默BACH1,进而为防治高血压提供新的治疗方法,对于防治高血压、改善患者预后具有重要意义。通过沉默BACH1基因或利用BACH1缺失巨噬细胞来源的EVs,可以抑制BACH1关键靶点,可改善高血压异常的VSMCs增殖,进而抑制血管重构,延缓或阻滞高血压的发生发展和并发症形成。
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pGMLV-SC5 RNAi载体示意图;
图2为shRNA载体的酶切片段电泳图,其中1:DL15000 DNA Ladder,2:酶切产物;
图3为慢病毒BACH1 siRNA1(PGMLV-SC5)感染细胞后96小时1号孔(A)和6号孔(B)的荧光照片;
图4为慢病毒BACH1 siRNA2(PGMLV-SC5)感染细胞后96小时1号孔(A)和6号孔(B)的荧光照片;
图5为慢病毒BACH1 siRNA3(PGMLV-SC5)感染细胞后96小时1号孔(A)和6号孔(B)的荧光照片;
图6为慢病毒BACH1 siRNA1-3对VSMCs中BACH1表达的影响;其中A-C为分别将慢病毒BACH1 siRNA1、慢病毒BACH1 siRNA2和慢病毒BACH1 siRNA3(MOI=80)加入WKY的VSMCs培养基中,48h后进行检测的结果,A为BACH1的mRNA水平,B为BACH1的抗体检测,C为BACH1抗体检测的代表性图像;D-F为对WKY和SHR来源的VSMCs给予慢病毒BACH1 siRNA1(MOI=80),48h后进行检测的结果,D为BACH1的mRNA水平,E为BACH1的抗体检测,F为BACH1抗体检测的代表性图像;*P<0.05vs Ctrl;vs WKY;
图7为巨噬细胞来源的EVs的鉴定,其中,A:TEM显示巨噬细胞(MФ)分泌的EVs呈杯形或球形;B:NTA检测发现巨噬细胞(MФ)来源的EVs直径主要为30-150nm;C:巨噬细胞(MФ)来源的EVs外泌体标志蛋白CD9、CD63、TSG101表达阳性;
图8为巨噬细胞来源的EVs对VSMCs增殖的作用,其中,A:EdU阳性细胞代表性图像;B:EdU阳性细胞百分比;C:CCK-8检测增殖;*P<0.05vs PBS;vs EVs of WKY;vs WKY-VSMCs;
图9为BACH1的水平,其中,A:与正常大鼠(WKY)比较,自发性高血压大鼠(SHR)胸主动脉中BACH1水平显著升高;B:与WKY相比,SHR的巨噬细胞(MФ)中BACH1含量升高但VSMCs中BACH1升高不明显;C:与WKY来源的EVs相比,SHR的巨噬细胞的EVs中BACH1升高更显著;*P<0.05vs WKY;vs MФ;
图10为用慢病毒BACH1 siRNA1构建的BACH1缺失巨噬细胞来源的EVs对VSMCs增殖的作用,其中A:CCK-8检测增殖;B:EdU阳性细胞百分比;C:EdU阳性细胞代表性图像;*P<0.05vs WKY-VSMCs;vs PBS;vs EVs of WKY;#P<0.05vs Ctrl;
图11为BACH1下游靶基因预测与FNDC5表达,其中,A:BACH1与FNDC5结合位点的预测;B:巨噬细胞(MФ)中FNDC5水平;*P<0.05vs WKY。
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干扰载体的构建
首先合成含干扰序列的单链DNAoligo,然后退火配对产生双链DNAoligo,再通过其两端所含酶切位点将其直接连入酶切后的RNA干扰载体上;将连接产物转入制备好的细菌感受态细胞,对长出的单克隆菌落送测序公司进行测序鉴定,比对正确的克隆即为构建成功的目的基因RNA干扰载体。
一、实验材料
1.载体信息
pGmLV-SC5 RNAi载体示意图见图1。
2.试剂(表1)
表1
3.仪器
表2
二、实验方法
1.shRNA靶点的选择
针对目的基因靶基因序列,利用公用网站中提供的RNA干扰序列设计原则,设计多个针对BACH1基因的RNA干扰靶点序列,根据我们的设计经验和设计软件进行评估测定,选择最佳的动力学参数靶点进入后续实验流程。
设计靶点如表3:
表3
注:Ctrl为阴性对照。
2.shRNA引物的设计
根据基因序列分别设计并合成shRNA寡聚单链DNA,oligo序列见下表4:
表4
3.RNAi重组质粒的构建
将寡聚单链DNA退火成双链,将双链的shRNAoligo分别插入到pGmLV-SC5 RNAi载体中,构建shRNA重组质粒BACH1 siRNA1(PGmLV-SC5)、BACH1 siRNA2(PGmLV-SC5)和BACH1siRNA3(PGmLV-SC5),并转化至感受态细胞Stbl3。
1)shRNA的退火
用无菌的TE buffer稀释引物使终浓度为100μM。分别吸取10μL的上下游引物混合并吹打均匀放入PCR管内进行退火,程序如下:
结束后置于冰上放置几分钟可直接连接或于-20℃冷冻保存。
2)shRNA载体的酶切及回收(载体为同一批次提前切好)
酶切体系如下:
于37℃酶切约30min。在此期间,可配置0.8%的琼脂糖凝胶,待酶切结束后进行核酸电泳。电泳结束后切下包含目的片段的胶条(电泳结果见图2)。用天平称量总重量并减去空管的重量算出凝胶的重量,按100mg约为100μL来计算凝胶的体积,并加入1倍凝胶体积的Binging Solution置于65℃水浴锅内将凝胶彻底融化。期间适当摇晃EP管,加快凝胶的溶解。将上述液体全部转移到滤柱内,13000转离心30s(可重复一次)。然后弃掉管内液体,向柱内加入500μL的WA Solution,13000转离心30s。弃掉管内液体,再向柱内加入500μL的Wash Solution,13000转离心30s(可重复一次)。然后空离3min。将滤柱放置在一个新的1.5mL EP管中,室温晾干。最后在柱子内加入35μL的ddH2O,静置5min,13000转离心1.5min。为了提高回收率,可将溶解的DNA再次加入柱子内离心一分钟。弃掉柱子,即为回收的载体片段,并测定浓度。
3)shRNA载体与引物的连接
连接体系如下:
于25℃水浴中孵育30min。
4)转化
将感受态细胞置于冰上待其自然解冻后,把连接产物全部加入感受态细胞中,于冰上放置20min,后于42℃水浴中热击90s。然后迅速置于冰上放置3min。加入1000μL不含抗生素的LB培养基于37℃,150rpm振荡培养45min。3000rpm离心2min,弃掉约850μL的上清液,将管底的菌液吹打散开,加入到含有相应抗性的培养皿中,用灭菌的涂布器涂匀,倒置于37℃恒温培养箱内过夜培养。
5)重组质粒的制备
挑取若干个单菌落,进行小量摇菌培养。
6)测序鉴定阳性克隆。
三、结果
3.1shRNA1干扰载体的测序结果
测序引物:
Hu6-F:GAGGGCCTATTTCCCATGATT(SEQ ID NO.13);CMV-R:GGGAACATACGTCATTATTG(SEQ ID NO.14)。
测序结果:
BACH1 siRNA1(PGmLV-SC5):
AATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAGgatccggaaccgacaagatccgaactctcgagagttcggatcttgtcggttccttttttAATTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT(SEQ ID NO.15)。其中,小写字母部分为shRNA1引物,下划线部分为shRNA1靶点。
3.2shRNA2干扰载体的测序结果
测序引物:
Hu6-F:GAGGGCCTATTTCCCATGATT;CMV-R:GGGAACATACGTCATTATTG。
BACH1 siRNA2(PGmLV-SC5):
ACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAGgatccggagacaaagcagaaccttacctcgaggtaaggttctgctttgtctccttttttAATTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCA(SEQ ID NO.16)。其中,小写字母部分为shRNA2引物,下划线部分为shRNA2靶点。
3.3shRNA3干扰载体的测序结果
测序引物:
Hu6-F:GAGGGCCTATTTCCCATGATT;CMV-R:GGGAACATACGTCATTATTG。
BACH1 siRNA3(PGmLV-SC5):
GACGGGGGAATAGGTAGTATGTACGCGGACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAATAACTAGaattaaaaaagcctcaatgaccagcggaagactcgagtcttccgctggtcattgaggcgGATCCTCGTCCTTTCCACAAGATATATAAAGCCAAGAAATCGAAATACTTTCAAGTTACGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAATTATTACTTTCTACGTCACGTATTTTGTACTAATATCTTTGTGTTTACAGTCAAATTAATTCTAATTATCTCTCTAACAGCCTTGTATCGTATATGCAAATATGAAGGAATCATGGGAAATAGGCCCTCTTCCTGCCCAGATCGATAAACTGGATCTCTGCTGTCCCTGTAATAAACCCGAAAATTTTGAATTTTTGTAATTTGTTTTTGTAATTCTTTAGTTTGTATGTCTGTTGCTATTATGTCTACTATTCTTTCCCCTGCACTGTACCCCCCAATCCCCCCTTTTCTTTTAAAGGCGATACCGTCGAGATCCGTTCACTAATCGAATGGATCTGTCTCTGTCTCTCTCTCCACCTTCTTCTTCTATTCCTTCGGGCCTGTCGGGTCCCCTCGGGGTTGGGAGGTGGGTCTGAAACGATAATGGTGAATATCCCTGCCTAACTCTATTCACTATAGAAAGTACAGCAAAAACTATTCTTAAACCTACCAAGCCTCCTACTATCATTATGAATAATTTTATATACCACAGCCAATTTGTTATGTTAAACCAATTCCACAAACTTGCCCATTTATCTAATTCCAATAATTCTTGTTCATTCTTTTCTTGCTGGTTTTGCGATTCTTCAATTAAGGAGTGTATTAAGCTTGTGTAATTGTTAATTTCTCTGTCCCACTCCATCCAGGTCGTGTGATTCCAAATCTGTTCCAGAGATTTATTACTCCAACTAGCATTCCAAGGCACAGCAGTGGTGCAAATGAGTTTTCCAGAGCAACCCCAAATCCCCAGGAGCTGTTGATTCTTTTAAGGTATC(SEQ ID NO.17)。其中,小写字母部分为shRNA3引物,下划线部分为shRNA3靶点。
经过比对,重组克隆中插入片段序列与设计的oligo序列完全一致,因此载体构建成功。
实施例2慢病毒的包装和病毒滴度的测定
抽提高纯度、无内毒素的慢病毒载体及其辅助包装原件载体质粒,使用HGtransgene reagent将构建好的慢病毒载体及其辅助包装原件载体质粒共转染进293T细胞,转染10~12h后加Enhancing buffer,接着8h后更换新鲜培养基,继续培养48h后,收集富含慢病毒颗粒的细胞上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。在一定滴度范围内的慢病毒颗粒可以满足大部分体内、体外实验需求。
一、实验材料
1.细胞株
293T(购自中国科学院典藏培养物保藏委员会细胞库的293T细胞或中科院公司,上海,中国),慢病毒的包装细胞,为贴壁依赖型成上皮样细胞,生长培养基为DMEM(含10%FBS)。贴壁细胞经培养生长增殖形成单层细胞。
2.大肠杆菌菌株stbl3(购自赛默飞,上海,中国)。用于扩增慢病毒载体和辅助包装载体质粒。
3.慢病毒包装系统
将构建成功的慢病毒重组质粒和包装质粒采用TIAGEN公司的质粒抽提试剂盒提取。所得的质粒DNA溶于无菌的TE中,以紫外光吸收法测定其浓度及纯度,保证所提质粒DNA的A260/A280在1.8~2.0之间。
4.试剂和耗材(见表5)
表5
5.仪器(见表6)
表6
二、实验方法
2.1慢病毒包装
2.1.1293T细胞分盘
转染前一天,将已经长好的细胞以合适的比例传代到10cm培养皿中,当细胞长到70%~80%时准备转染。
2.1.2转染
取无菌的1.5mL EP管或15mL离心管,转染体系按下列:
混匀后,室温放置15-20min后,均匀滴加到准备转染的培养皿中,后置于CO2培养箱中培养。
2.1.3加Enhancing buffer
转染10~12h后,均匀滴加100×Enhancing buffer促进转染,120μL/皿。
2.1.4换液
转染18~20h后,小心吸掉细胞培养液弃于盛有消毒液的废液杯中(注意:此时培养基中已含有少量的病毒,必须经处理后才能丢弃,所用的移液枪头等必须经消毒液浸泡至少15min后才能丢弃),然后加15mL新鲜的细胞培养基(或是无血清的DMEM或是含血清的DMEM)继续培养。
2.1.5病毒收集
换液48h后,吸取细胞上清液于50mL离心管,4℃,4500g离心5min,上清液用0.22μm滤器过滤后转移到新的离心管中,最后将滤液分批转移到浓缩装置中,4℃,4500g,离心10min,弃下层的液体于盛有消毒液的废液杯中,最后一次4℃,4500g,离心20min,此时可见滤器上层中的液体即为病毒浓缩液。
2.1.6病毒分装与保存
将病毒分装后保存于-80℃。
2.2Lentivirus滴度测定
1)将293T细胞培养至对数生长期,病毒稀释用培养液为含10%FBS的细胞培养基。
2)第一天,细胞胰酶消化计数后,按照每孔8000细胞接种96孔板,37℃培养过夜,感染时细胞长至30~50%的融合密度;
3)第二天,当天转染时,将保存于-80℃冰箱中病毒液冰浴融化,用含有10%FBS的细胞培养液(培养基:polybrene=1000:1)进行梯度稀释:
1号稀释液:10μL病毒液+90μL病毒稀释用培养基
2号稀释液:10μL 1号稀释液+90μL病毒稀释用培养基
3号稀释液:10μL 2号稀释液+90μL病毒稀释用培养基
4号稀释液:10μL 3号稀释液+90μL病毒稀释用培养基
5号稀释液:10μL 4号稀释液+90μL病毒稀释用培养基
……
4)选取所需的细胞孔,吸去90μL培养基,丢弃,轻轻混匀各管慢病毒稀释液,取90μL加入每孔细胞中,放入37℃的细胞培养箱中过夜培养;
5)第三天,去除含慢病毒的培养基,加入100μL的完全培养基;
6)第五天,在荧光显微镜下观察各孔中荧光细胞数量,病毒滴度为表达荧光的细胞数乘以相应稀释倍数。
2.2BACH1干涉病毒转染有效性验证
1)分别将慢病毒BACH1 siRNA1、BACH1 siRNA2和BACH1 siRNA3(MOI=80)干涉病毒,转染到WKY来源的VSMCs中,孵育48h后,收集样品进行下一步实验。
2)通过RT-PCR(Real-time PCR)检测待测样品中BACH1 mRNA水平。根据产品说明书,使用Trizol试剂提取总RNA。在260和280nm下,使用紫外分光光度计测定RNA的浓度和纯度。用RT试剂盒和ABI PRISM 7500序列检测PCR系统进行逆转录酶反应。使用SYBR扩增试剂盒,对样本中的mRNA水平进行相对定量,每个样品设置3个复孔,用GAPDH基因作为内参,以2-△△Ct相对定量分析法对结果进行分析。
3)采用蛋白质免疫印迹分析技术(Westernblot)检测待测样品BACH1和GAPDH的蛋白含量。将样品置于含有PMSF的RIPA裂解液裂中匀浆,提取上清液,用BCA蛋白浓度测定试剂盒测定样品总蛋白浓度。通过SDS-PAGE分离等量的总蛋白,并在三甘氨酸甲醇缓冲液中将其转移到PVDF膜上。用增强型化学发光试剂(ECL)对条带进行可视化成像。
三、结果
3.1病毒滴度
慢病毒原液滴度测定结果:
1号孔:1号稀释液,含有10*10-3mL慢病毒液。
2号孔:2号稀释液,含有10*10-4mL慢病毒液。
3号孔:3号稀释液,含有10*10-5mL慢病毒液。
4号孔:4号稀释液,含有10*10-6mL慢病毒液。
5号孔:5号稀释液,含有10*10-7mL慢病毒液。
6号孔:6号稀释液,含有10*10-8mL慢病毒液。
7号孔:7号稀释液,含有10*10-9mL慢病毒液。
8号孔:8号稀释液,含有10*10-10mL慢病毒液。
慢病毒BACH1 siRNA1(PGmLV-SC5)、BACH1 siRNA2(PGmLV-SC5)和BACH1 siRNA3(PGmLV-SC5)感染细胞后96小时部分孔(某一视野)的荧光照片如图3-5所示;如图3所示,6号孔中观察到表达绿色荧光的细胞至少为50个,病毒的滴度:50TU/(10*10-8)mL=5*108TU/mL;如图4所示,6号孔中观察到表达绿色荧光的细胞至少为50个病毒的滴度:50TU/(10*10-8)mL=5*108TU/mL;如图5所示,6号孔中观察到表达绿色荧光的细胞至少为50个,病毒的滴度:50TU/(10*10-8)mL=5*108TU/mL。
综上各病毒的滴度如下表7:
表7
3.2BACH1干涉病毒转染有效性验证结果
将慢病毒BACH1 siRNA1转染到WKY-VSMCs中,发现WKY-VSMCs中BACH1含量降低。与对照组相比,WKY的VSMCs中的BACH1水平和含量降低,其中加入慢病毒BACH1 siRNA1的BACH1水平和含量最低,而加入慢病毒BACH1 siRNA1的BACH1水平和含量变化不明显,说明慢病毒BACH1 siRNA1的敲低效果更明显(图6A-C)。
分别将慢病毒BACH1 siRNA1转染到WKY和SHR的VSMCs中,发现WKY和SHR的VSMCs中BACH1水平和含量均降低,进一步验证慢病毒BACH1 siRNA1基因敲低的有效性。
实施例3
一、实验材料和方法
(1)实验动物和动物模型
自发性高血压大鼠(SHR):SHR是人类原发性高血压的最理性动物模型。采用SHR作为高血压动物模型,以京都种的正常Wistar大鼠(WKY)作为对照。SHR大鼠和WKY大鼠购自(北京维通利华实验技术有限公司,北京,中国)。
(2)巨噬细胞的分离与培养
选取8-10周龄正常大鼠和自发性高血压模型大鼠,取外周血,对肝素化的血液进行梯度离心,纯化单核细胞,将得到的单核细胞置于加入20%FBS、5U/mLM-CSF和100U/mLGM-CSF的RPMI 1640培养基中培养,7天后,经单核细胞分化的贴壁粘附细胞即为单核细胞来源的巨噬细胞。
(3)VSMCs的分离与培养
选取8-10周龄正常大鼠和自发性高血压模型大鼠,分离胸主动脉,剥除血管周围脂肪组织,刮除内膜,分离外膜与中膜,将中膜置于含有0.4%胶原酶的PBS中孵育30min,待消化完全后,加入细胞培养液终止消化。悬浮液在200×g下离心10min。将分离的细胞重悬于细胞培养液中,放在细胞孵箱中培养,温度设置在37℃,CO2浓度为5%。
(4)巨噬细胞的EVs的分离与鉴定
巨噬细胞密度达到80%-90%后,用无血清培养基培养48h,收集培养液,分别使用外泌体分离试剂盒法和超速离心法将EVs从巨噬细胞培养液中分离出来;采用透射电子显微镜(TEM)鉴定EVs的形态;采用纳米粒子示踪技术(NTA)对EVs的浓度和粒径大小进行分析;用Pierce BCA蛋白测定试剂盒测定EVs中的蛋白质含量,作为EVs数量的指标,利用蛋白免疫印迹分析技术(Western blot)检测EVs中外泌体标志蛋白如CD9、CD63和TSG101的表达水平,Calnexin蛋白在外泌体中不表达,被作为阴性对照。
BACH1缺失巨噬细胞的EVs的提取:在巨噬细胞中加入BACH1-siRNA,5天后提取得到BACH1稳定敲低巨噬细胞来源的EVs(BACH1缺失巨噬细胞的EVs),将其与VSMCs共培养24h,收集样品进行下一步实验。
(5)荧光素酶报告基因实验
6孔板中VSMCs细胞汇合度达到85%-90%时,将6μL的DNAfectin TM Plus转染试剂加入200μL无血清无抗生素的DMEM培养基中稀释溶解,并将2μg的pLenti-UTR-GFP质粒分别和2μg的pre-BACH1或阴性对照加入其中制备成混合液,混匀后,室温孵育20min。将混合液加入VSMCs中,细胞培养箱中孵育6h后,更换为常规培养基继续培养12h,最后检测双荧光素酶报告基因活性。
(6)免疫荧光
细胞被接种在提前放入6孔板中的盖玻片上,4%多聚甲醛固定15min;PBS清洗5min,加0.2%的Tritonx-100孵育5min,PBS清洗5min,加1%BSA室温孵育1h,加BACH1一抗抗体(1:100;Abcam,Cambridge,MA,USA),4℃孵育过夜;PBS清洗3次,每次5min,加IgG二抗抗体(1:1000;Santa Cruz,CA,USA),室温孵育1h,最后用DAPI染色细胞核并封片,荧光显微镜下拍照。
(7)免疫组化
将分离的大鼠主动脉和肠系膜动脉,固定在4%多聚甲醛中,石蜡包埋,使用冰冻切片机横切成5μm切片。去石蜡后用0.1M PBS冲洗3次,用阻断缓冲剂阻断5min,加入BACH1一抗抗体(1:100;Abcam,Cambridge,MA,USA),4℃孵育24h,然后加入poly-HRP anti-Rabbit IgG,37℃孵育20min。利用3,3-二氨基联苯胺培养阳性细胞。切片用苏木精染色。
(8)Masson’s染色
分离大鼠胸主动脉和肠系膜动脉,4%多聚甲醛固定24h,石蜡包埋后切成5μm切片。切片复水后,染色。最后,用无水乙醇冲洗,脱水,滴加透明剂,封片,显微镜下拍照。
(9)血压和血管功能实验
血压:将正常状态下的大鼠放入动物固定盒内置于操作台,大鼠尾巴放在加压套内,用胶带固定鼠尾尖部,使得大鼠躯干和尾巴竖直,待脉搏稳定后测量收缩压(SBP)、舒张压(DBP)和心率(HR)。
血管功能:取动脉三级血管,制备血管环,采用多通道血管张力测定仪(620M,DMT,Denmark)测定血管张力,观察血管舒缩功能。
(10)基因过表达、RNA干涉
过表达研究:利用慢病毒作为载体,构建FNDC5过表达慢病毒,将其转染到VSMCs或大鼠,使之过表达。
RNA干涉:利用慢病毒作为载体,构建BACH1-siRNA慢病毒,将其转染到VSMCs或大鼠,阻断BACH1基因的表达。前期筛选发现慢病毒BACH1 siRNA1的敲低效果更明显,因此本实验均用BACH1 siRNA1作为干涉病毒。
二、结果
(1)我们成功分离了巨噬细胞来源的EVs。透射电子显微镜(TEM)显示巨噬细胞来源的EVs呈球形或杯形(图7A),根据纳米粒子示踪技术(NTA)分析巨噬细胞来源的EVs粒径约为30-150nm(图7B),说明巨噬细胞可以分泌EVs。
(2)我们发现巨噬细胞来源的EVs外泌体相关标记蛋白表达阳性。巨噬细胞EVs中外泌体相关标志蛋白CD9、CD63、TSG101表达阳性,而Calnexin表达阴性(图7C)。这些结果符合外泌体的特征,确定本实施例中分离的EVs是外泌体。
(3)巨噬细胞来源的EVs参与调控VSMCs增殖。WKY的巨噬细胞的EVs对WKY的VSMCs的增殖无明显影响,但抑制SHR的VSMCs增殖。SHR的VSMCs的增殖能力强于WKY的VSMCs,SHR的巨噬细胞的EVs促进WKY和SHR的VSMCs增殖(图8)。
(4)SHR中BACH1升高。与WKY比较,SHR胸主动脉中BACH1水平显著升高(图9A),SHR的巨噬细胞中BACH1含量升高但VSMCs中BACH1升高不明显(图9B),与WKY的EVs相比,SHR的EVs中BACH1升高更显著(图9C)。
(5)敲低巨噬细胞(MФ)中BACH1,使得BACH1蛋白表达降低,表明BACH1基因敲低的有效性(图6);在巨噬细胞中加入慢病毒BACH1 siRNA1(BACH1小干扰RNA),5天后提取得到BACH1稳定敲低巨噬细胞来源的EVs,将其与VSMCs共培养24h,采用CCK-8法和EdU掺入法检测VSMCs的增殖,干预巨噬细胞的EVs中BACH1,BACH1缺失巨噬细胞的EVs对高血压VSMCs增殖的促进作用减弱,说明巨噬细胞的EVs通过转运BACH1调控VSMCs增殖(图10)。
(6)根据Jaspar(http://jasper.genereg.net),推测FNDC5很可能是BACH1的靶基因,预测BACH1与FNDC53′-UTR的结合位点为899-912(图11A)。
(7)与WKY比较,FNDC5在SHR巨噬细胞中低表达(图11B)。
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。
序列表
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Claims (10)
1.一种靶向沉默BACH1基因的shRNA,其特征在于,所述shRNA的正义链如SEQ ID NO.7所示,所述shRNA的反义链如SEQ ID NO.8所示。
2.一种靶向沉默BACH1基因的shRNA,其特征在于,所述shRNA的正义链如SEQ ID NO.9所示,所述shRNA的反义链如SEQ ID NO.10所示。
3.一种靶向沉默BACH1基因的shRNA,其特征在于,所述shRNA的正义链如SEQ ID NO.11所示,所述shRNA的反义链如SEQ ID NO.12所示。
4.一种重组shRNA干扰载体,其特征在于,包含如权利要求1-3任一项所述的shRNA。
5.一种慢病毒,其特征在于,包含如权利要求1-3任一项所述的shRNA或权利要求4所述的重组shRNA干扰载体。
6.一种如权利要求1-3任一项所述的shRNA、如权利要求4所述的重组shRNA干扰载体或如权利要求5所述的慢病毒在制备治疗高血压的药物中的应用。
7.一种如权利要求1-3任一项所述的shRNA、如权利要求4所述的重组shRNA干扰载体或如权利要求5所述的慢病毒在制备抑制BACH1基因的表达的药物中的应用。
8.一种如权利要求4所述的重组shRNA干扰载体的构建方法,其特征在于,包括:首先合成shRNA的引物单链,然后退火配对产生双链,再将所述双链连入RNA干扰载体上,鉴定得到所述重组shRNA干扰载体。
9.根据权利要求8所述的构建方法,其特征在于,所述RNA干扰载体为pGmLV-SC5 RNAi载体。
10.BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用。
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