CN117530953A - circRcor3在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 - Google Patents
circRcor3在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 Download PDFInfo
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Abstract
本发明公开了circRcor3在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物,属于生物医学技术领域。本发明证明了circRcor3制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对主动脉弓缩窄引起的心力衰竭有很好治疗效果;具体包括增加circRcor3具有改善主动脉弓缩窄4周所致的心脏收缩功能不全和心脏纤维化的作用;过表达circRcor3能够抑制苯肾上腺素所诱导的心肌细胞病理性肥大。
Description
技术领域
本发明属于生物医学技术领域,具体涉及circRcor3在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物。
背景技术
心力衰竭是慢性高血压、机械性压力过载等各种刺激引起的病理性重构,常伴有心肌细胞大小异常、代谢紊乱等,心力衰竭已成为影响人口寿命的全球性负担。目前,临床上对心力衰竭的治疗尚处于对症治疗阶段,常见的药物主要有β受体阻滞剂、钙拮抗剂和血管紧张素转换酶抑制剂,患者预后较差。运动是一种众所周知的非药物作用干预方式,能够改善心血管健康,从运动心脏中发现的靶点可以促进心肌细胞健康肥大,促进心肌细胞增殖,抵抗心肌细胞凋亡,保护心脏免受病理性心脏重塑损害。研究运动引起的心脏保护生物学机制对生物学基础研究和潜在的药物开发都具有重要意义。
随着新一代测序技术的发展,在人类、小鼠等动物和植物中发现了大量的环状RNA(circularRNA,circRNAs),circRNAs具有封闭的共价环结构,具有RNA外切酶抵抗性。放线菌素D检测表明circRNAs的半衰期高达18h以上,明显长于其同源的线性mRNA。目前已通过RNA-seq和微阵列高通量测序方法鉴定出在心血管疾病中具有潜在作用的circRNAs,circRNAs与高血压、心力衰竭、心脏肥厚和其他心血管疾病之间存在密切的相关性,被认为是心血管疾病的生物标志物和潜在的治疗靶点。
发明内容
本发明的目的在于提供circRcor3在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物,利用circRcor3制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对主动脉弓缩窄引起的心力衰竭有很好治疗效果。
本发明提供了circRcor3或表达circRcor3的基因工具在制备治疗心力衰竭药物中的应用。
优选的,所述circRcor3的核苷酸序列如SEQ ID No.1所示。
优选的,所述表达circRcor3的基因工具包括表达或过表达circRcor3的成环载体,和经腺相关病毒包装的表达或过表达circRcor3的成环载体。
优选的,所述成环载体包括AAV过表达载体或慢病毒过表达载体。
本发明提供了一种表达circRcor3的成环载体,以腺病毒过表达载体为基础载体。
优选的,所述腺病毒过表达载体包括pK5ssAAVciR。
本发明提供了利用腺相关病毒包装上述表达circRcor3的成环载体得到的重组病毒。
优选的,所述腺相关病毒包括AAV9。
本发明提供了一种过表达circRcor3的成环载体,以慢病毒过表达载体为基础载体。
本发明提供了一种治疗心力衰竭的药物,活性成分包括上述重组病毒或上述过表达circRcor3的成环载体,还包括药学上可接受的辅料。
有益效果:本发明提供了circRcor3或表达circRcor3的基因工具在制备治疗心力衰竭药物中的应用。本发明通过实验证明了circRcor3制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对主动脉弓缩窄引起的心力衰竭有很好治疗效果;具体包括增加circRcor3具有改善主动脉弓缩窄4周所致的心脏收缩功能不全;过表达circRcor3能够抑制苯肾上腺素所诱导的心肌细胞病理性肥大。本发明提供了一种治疗心力衰竭的药物,并将以AAV过表达载体为基础载体的表达载体利用腺相关病毒包装,腺相关病毒不参与任何疾病的发生,免疫原性低,基因可持续表达半年以上,其中AAV9病毒对心脏组织存在强的亲和性,可以作为载体将目的基因序列在心脏组织中稳定高效的表达。
附图说明
图1为尾静脉注射AAV9-circRcor3 OE后小鼠心脏样本circRcor3的表达结果图,n=8:8:10:10;图中Sham:假手术;TAC:主动脉弓缩窄手术,**,P<0.01;
图2为过表达circRcor3改善主动脉弓缩窄4周导致的心功能下降结果图,n=8:8:10:10;图中Sham:假手术;TAC:主动脉弓缩窄手术,**,P<0.01;
图3为过表达circRocr3对苯肾上腺素(PE)刺激导致的病理性心肌细胞肥大的保护效应结果图,n=6;图中PBS:磷酸盐缓冲液,PE:苯肾上腺素,比例尺:100μm,**,P<0.01;
图4为分析病毒滴度的标准曲线。
具体实施方式
本发明提供了circRcor3或表达circRcor3的基因工具在制备治疗心力衰竭药物中的应用。
本发明所述circRcor3的核苷酸序列如SEQ ID No.1所示:5’-GGTAACGCTGACCAGCCGGTCCAAACCAGCAAGATCGGCCTGGGCCGG AGAGAGTACCAGAGTCTGCAGCACCGCCACCACTCTCAGCGCTCCAAGTGCCGCCCTCCTAAGGGCATGTATCTAACCCAGGAGGACGTGGTGGCTGTCTCCTGCAGCCCCAACGCAGCCAACACCATCCTGAGGCAACTGGACATGGAGCTGATCTCTCTCAAGCGCCAGGTTCAGAATGCTAAGCAAGTAAACAGTGCACTTAAGCAGAAAATGGAAGGTGGAATTGAAGAGTTCAAACCGCCTGAG-3’。在本发明中,所述circRcor3的circBase ID为mmu_circ_0008568。
本发明实施例证明了circRcor3制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对主动脉弓缩窄引起的心力衰竭有很好治疗效果;具体包括circRcor3具有改善主动脉弓缩窄4周所致的心脏收缩功能不全;过表达circRcor3能够抑制苯肾上腺素所诱导的心肌细胞病理性肥大。
本发明所述心力衰竭优选包括急性心力衰竭或慢性心力衰竭,更优选包括主动脉弓缩窄引起的心力衰竭。
本发明所述表达circRcor3的基因工具优选包括表达或过表达circRcor3的成环载体,和经腺相关病毒包装的表达或过表达circRcor3的成环载体。本发明所述成环载体包括AAV过表达载体或慢病毒过表达载体,如利用AAV过表达载体构建circRcor3的表达载体,利用慢病毒过表达载体构建circRcor3的过表达载体。
本发明提供了一种表达circRcor3的成环载体,以腺病毒过表达载体为基础载体。
本发明所述腺病毒过表达载体优选包括pK5ssAAVciR,在构建所述表达circRcor3的成环载体时,优选将所述circRcor3的序列(SEQ ID No.1)插入pK5ssAAVciR的EcoRI-HF和BamHI酶切位点之间。
本发明提供了利用腺相关病毒包装上述表达circRcor3的成环载体得到的重组病毒。
本发明所述腺相关病毒优选包括AAV9,并对所述腺相关病毒包装的方法并没有特殊限定。
本发明提供了一种过表达circRcor3的成环载体,以慢病毒过表达载体为基础载体。
本发明所述慢病毒过表达载体优选包括pLO-ciR(货号:GS0103),并将所述circRcor3的序列(SEQ ID No.1)插入pLO-ciR的EcoRI-HF和BamHI酶切位点之间。
本发明提供了一种治疗心力衰竭的药物,活性成分包括上述重组病毒或上述过表达circRcor3的成环载体,还包括药学上可接受的辅料。
本发明实施例中证实增加circRcor3具有改善主动脉弓缩窄4周所致的心脏收缩功能不全;过表达circRcor3能够抑制苯肾上腺素诱导的病理性心肌肥大。
为了进一步说明本发明,下面结合实施例对本发明提供的circRcor3在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
1、circRcor3过表达病毒(AAV9-circRcor3 OE)的构建和AAV9包装
1)将小鼠心脏组织总RNA逆转录后依次进行PCR扩增,双酶切,回收获得circRcor3的cDNA序列(如SEQ ID No.1所示);
其中PCR扩增的引物:
上游引物(SEQ ID NO.2):5’-CGGAATTCTAATACTTTCTAATACTTTCAGGGTAACGCTGACCAGCCGGT-3’,
下游引物(SEQ ID NO.3):5’-CGGGATCCAGTTGTTCTTACCTCAGGCGGTTTGAACTCTTCAA-3’;
50μLPCR扩增反应体系:上游引物(10μM)1.5μL(终浓度为0.3μM)、下游引物(10μM)1.5μL(终浓度为0.3μM)、10×KOD plus buffer 5μL,2mM dNTP 5μL(终浓度为0.2mM),25mMMgSO42μL(终浓度为1.0mM)KOD酶5μL(KOD plus,货号KOD-201)、cDNA模板(500ng/μL)1μL和无酶水33μL。
PCR反应程序:94℃预变性2min;94℃变性15s,60℃退火30s,68℃延伸60s,35个循环;4℃,保温。
所述双酶切的体系为:EcoRI-HF(星选酶)1μL、BamHI(星选酶)1μL、10x CutSmart缓冲液5μL、PCR产物/或者载体1μg和无酶水(体积定容到50μL)。
双酶切反应:37℃,4h;
双酶切后的PCR产物采用天根生物超薄DNA产物纯化试剂盒(货号DP203)清洁回收。
2)将成环载体(腺病毒过表达载体pK5ssAAVciR,购自广州吉赛生物科技股份有限公司,货号为GS0109)进行双酶切后,采用天根生物增强型琼脂糖凝胶DNA回收试剂盒(货号DP219-03)回收大片段载体。
3)将步骤1)回收的片段插入步骤2)回收的成环载体上,得到连接产物。具体反应体系如下:T4 DNA连接酶1μL、10×T4 DNA连接酶缓冲液1μL、双酶切的载体(100ng)2μL、双酶切的基因(100ng)3μL和无酶水2μL,总体积为10μL。
反应条件为:16℃,8h。
4)50μL感受态细胞(感受态细胞TransStbl3擎科生物,货号TSC-C06)冰上化开,加入5μL步骤3)制备的连接产物,轻弹混匀,在冰上放置25min;42℃热激45s,然后放置冰上2min;后加入500μL培养基(无抗LB培养基),混匀后37℃,180转/min培养1h,复苏细菌。涂布在氨苄抗性的培养平板上过夜培养。过夜培养后挑取单克隆摇菌,并对菌液进行保种,送Sanger测序。比对序列成功后,摇菌并大提质粒(TIANGEN,DP117),得mc-circRocr3-pK5ssAAVciR。
5)将10μg mc-circRocr3-pK5ssAAVciR或pK5ssAAVciR对照质粒、10μgpAAV 2/9质粒(Addgene#112865)、10μg pAd Delta F6(Addgene#112867)质粒及90μg PEI(Kingmorn,#KE1098)混匀并静置15分钟,加入10ml含1%青链霉素混合液的DMEM培养基,37℃恒温培养人肾上皮细胞系(HEK293T)。12h后更换为完全培养基,48h后收取细胞及其上清,4000rpm离心30min。取上清,加入1/5体积的PEG800,充分搅拌后静置过夜并再次离心,在沉淀中加入1MMgCl2和核酸酶(EMD Millipore Core,USA)。用碘二醇密度梯度10000g离心70min纯化AAV9病毒,然后用浓缩管(Amicon-Ultra-15,Millipore,Ireland)浓缩纯化的AAV9病毒,将该病毒命名为AAV9-circRcor3 OE。
提取病毒gDNA(TIANGEN,China)后采用qRT-PCR方法,用标准曲线(图4)分析病毒滴度。引物序列为
CMV-F(SEQ ID No.9):AAGTACGCCCCCTATTGACG;
CMV-R(SEQ ID No.10):CACGCCCATTGATGTACTGC;标准曲线是将浓度为0.1ng/μL的MCS质粒依次稀释2、4、6、8、10倍。通过标准曲线计算得到的病毒滴度为1.43*1013。通过尾静脉注射AAV9,每只老鼠的剂量是3*1012。
2.主动脉弓缩窄(TAC)模型建立:腹腔注射4%水合氯醛(0.1ml/10g),待小鼠不能自行翻身后将其固定于恒温手术台。沿颈部正中线剪开皮肤,钝性分离筋膜层和肌肉层,暴露气管,在气管上剪一小口插入气管插管,设置潮气量为0.2ml并观察胸廓起伏。沿小鼠胸骨正中纵向剪开0.8cm的开口,将胸骨剪到第二肋骨,拉开胸骨,分离胸腺及脂肪组织,暴露出主动脉,将垫扎针平行放置于主动脉弓旁边,使用显微镊夹取7-0缝合线穿过主动脉弓,与缝合线一起打结,再次打结固定后抽离垫扎针。逐层缝合胸骨、肌肉和皮肤并对伤口进行消毒。本实施例中所述假手术与上述TAC手术相似,唯一区别在于,假手术组只穿线不结扎。
实验分组分为4个组,具体分组信息:
第1组假手术组(Sham)+对照病毒(AAV9-Ctrl,指AAV9-pk5ssAAVciR病毒,即仅转染过表达环状载体的质粒而包装的病毒),对小鼠心脏进行假手术,样本数为8只;
第2组假手术组(Sham)+circRcor3过表达病毒(AAV9-circRcor3 OE),对小鼠进行假手术,样本数为8只;
第3组TAC 4w手术组(TAC 4w,使小鼠的主动脉弓缩窄持续4周,即主动脉弓缩窄手术4周后取样)+对照病毒(AAV9-Ctrl,所转染的质粒为pk5ssAAVciR,其余步骤与腺病毒AAV9-circRcor3 OE的构建过程一致),对小鼠心脏TAC手术,样本数为10只;
第4组TAC 4w+circRcor3过表达病毒(AAV9-circRcor3 OE),对小鼠心脏TAC手术,样本数为10只。
TAC或者Sham手术4周之后,对小鼠进行心脏彩色多普勒超声检测,检测完毕后处死小鼠,解剖得到小鼠心脏,使用荧光定量PCR验证AAV9-circRcor3OE是否成功在动物水平过表达心脏中circRcor3的表达。
2.qPCR检测circRcor3表达变化
TAC或者Sham手术4周之后,对小鼠进行心脏彩色多普勒超声检测,检测完毕后处死小鼠,解剖得到小鼠心脏,使用Trizol裂解液提取心脏组织中的总RNA,使用逆转录试剂盒获得cDNA。采用qPCR法检测circRcor3表达,并用2-ΔCT法计算分析circRcor3的表达变化。
qPCR反应体系(10μL):SYBR Green 5μL、上游引物F和下游引物R(10μM)各0.5μL、ddH2O 3μL、cDNA稀释液1μL;
qPCR反应用上游引物F和下游引物R的序列如下:
上游引物F(SEQ ID NO.4):5’-TGAAGAGTTCAAACCGCCTGA-3’;
下游引物R(SEQ ID NO.5):5’-ACCACGTCCTCCTGGGTTAG-3’。
内参基因为18s,qPCR反应用上游引物F和下游引物R的序列如下:
上游引物F(SEQ ID No.6):5’-TCAAGAACGAAAGTCGGAGG-3’;
下游引物R(SEQ ID No.7):5’-GGACATCTAAGGGCATCAC-3’;
qPCR反应程序如下:95℃预变性30sec;95℃变性15sec,60℃退火及延伸30sec,40次。
结果如图1所示,共有36只小鼠心脏被检测了circRcor3的表达情况,表达量数据以2-△△Ct计算得到。具体数据为:
Sham+AAV9-ctrl组circRcor3的表达值为:1.002168、0.981544、0.938302、1.030343、1.03392、0.998701、1.030343、0.988371;
Sham+AAV9-circRcor3 OE组circRcor3的表达为18.1026、20.29602、18.87134、18.67615、17.48596、18.67615、19.13477、19.26787;
TAC+AAV9-ctrl组circRcor3的表达值为:0.480672、0.546436、0.658897、0.593831、0.511613、0.492476、0.351873、0.485696、0.402797、0.518755;
TAC+AAV9-circRcor3 OE组circRcor3的表达为6.290283、7.126161、9.114257、6.268521、5.381935、5.808336、7.175727、5.271176、5.091621、5.127036。以上数据说明了TAC手术后,circRcor3的表达水平明显下降,而尾静脉注射AAV9-circRcor3 OE病毒后,心脏中circRcor3的表达明显上调。
实施例2
采用应用例1相同的分组及处理方式,选取C57BL/6J成年公鼠施行TAC手术,在TAC手术4周后,进行心脏超声测量上述四个组心功能相关指标(射血分数(EF)。
小动物心脏超声测定小鼠心功能:将小鼠胸部脱毛后,用异氟烷吸入麻醉法将小鼠麻醉,在超声平板上固定好小鼠四肢,并将适量螯合剂涂抹于小鼠腹部心脏处,用探头找到小鼠心脏,待心率稳定在400-500次/分钟,取胸骨旁左心室长轴及左心室短轴进行检查,测量并计算心功能,获得心功能相关指标:左室射血分数EF。结果见图2。
具体数据为:
第1组8只小鼠EF(%)值为64.718102、74.305677、67.77323、71.11175、67.531043、68.260482、65.458806、70.338886;
第2组8只小鼠EF(%)值为66.132828、63.15439、63.83195、65.644465、76.107628、65.208057、65.420813、68.876583;
第3组10只小鼠EF(%)值为38.706991、37.270527、32.075608、49.54786、48.431533、48.349237、46.438936、42.555319、42.174586、30.934425;
第4组10只小鼠EF(%)值为65.829352、61.959754、66.992898、57.602715、59.953252、57.964156、57.936344、59.513297、55.005428、68.525794。
统计结果如图2所示,**,P<0.01。
由图2可知,circRcor3治疗能够改善主动脉弓缩窄4周所致的心脏收缩功能不全。
实施例3
1.circRcor3过表达质粒的构建
1)将大鼠心脏组织总RNA逆转录后依次进行PCR扩增,双酶切,回收获得circRcor3的cDNA序列(如SEQ ID NO.1所示);
PCR扩增的上游引物(SEQ ID No.8):5’-CGGAATTCTAATACTTTCTAATACTTTCAGGGTAACTCTGAACAGCCAGTCC A-3’,
PCR扩增的下游引物(SEQ ID No.3):5’-CGGGATCCAGTTGTTCTTACCTCAGGCGGTTTGAACTCTTCAA-3’;
2)将成环载体(慢病毒过表达载体pLO-ciR,购自广州吉赛生物科技股份有限公司,货号为GS0103)进行双酶切后,采用天根生物增强型琼脂糖凝胶DNA回收试剂盒(货号DP219-03)回收大片段载体。
3)剩余步骤与实施例1中步骤3)4)一致。
2.苯肾上腺素(PE)刺激的心肌细胞病理性肥大模型建立及质粒转染:NRCM。
在新生大鼠心肌细胞中,转染circRcor3过表达的重组载体(0.02mg/L),处理48h后收集细胞,在实验终点前24小时构建PE模型完成功能获得性实验。
具体分组为:
1)对照组(对照空载体pLO5-ciR),
2)对照组(对照空载体pLO5-ciR)+PE处理组,
3)circRcor3过表达组(circRcor3 OE),
4)circRcor3过表达(circRcor3 OE)+PE处理组。
实验终点以α-actinin/Hoechst免疫荧光共染检测circRcor3对心肌细胞病理性肥大的保护作用。由图3可知,PE模型处理后,NRCM病理性肥大与对照组处理相比显著上升;而在PE模型下,过表达circRcor3能够保护NRCM PE实验引起的病理性心肌肥大。
具体的,
第1)组心肌细胞面积相对大小为1.008858、0.975985、0.949824、0.946376、1.04753、1.071426;
第2)组心肌细胞面积相对大小为1.152542、1.143512、1.233806、1.301356、1.211621、1.288814;
第3)组心肌细胞面积相对大小为1.989003、1.862821、1.898827、1.739483、1.765885、1.894278;
第4)组心肌细胞面积相对大小为1.760666、1.74601、1.604119、1.578863、1.458166、1.426038。统计结果如图3所示,**,P<0.01。
综上所述,增加circRcor3具有改善主动脉弓缩窄4周所致的心脏收缩功能不全;过表达circRcor3能够抑制苯肾上腺素诱导的病理性心肌肥大。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (10)
1.CircRcor3或表达circRcor3的基因工具在制备治疗心力衰竭药物中的应用。
2.根据权利要求1所述应用,其特征在于,所述circRcor3的核苷酸序列如SEQ ID No.1所示。
3.根据权利要求1所述应用,其特征在于,所述表达circRcor3的基因工具包括表达或过表达circRcor3的成环载体,和经腺相关病毒包装的表达或过表达circRcor3的成环载体。
4.根据权利要求3所述应用,其特征在于,所述成环载体包括AAV过表达载体或慢病毒过表达载体。
5.一种表达circRcor3的成环载体,其特征在于,以腺病毒过表达载体为基础载体。
6.根据权利要求5所述表达circRcor3的成环载体,其特征在于,所述腺病毒过表达载体包括pK5ssAAVciR。
7.利用腺相关病毒包装权利要求5或6所述表达circRcor3的成环载体得到的重组病毒。
8.根据权利要求7所述重组病毒,其特征在于,所述腺相关病毒包括AAV9。
9.一种过表达circRcor3的成环载体,其特征在于,以慢病毒过表达载体为基础载体。
10.一种治疗心力衰竭的药物,其特征在于,活性成分包括权利要求7或8所述重组病毒或权利要求9所述过表达circRcor3的成环载体,还包括药学上可接受的辅料。
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