CN117007816A - 靶点Nesfatin-1在预防或治疗血管钙化中的应用 - Google Patents
靶点Nesfatin-1在预防或治疗血管钙化中的应用 Download PDFInfo
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- CN117007816A CN117007816A CN202310907855.1A CN202310907855A CN117007816A CN 117007816 A CN117007816 A CN 117007816A CN 202310907855 A CN202310907855 A CN 202310907855A CN 117007816 A CN117007816 A CN 117007816A
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- nesfatin
- vascular calcification
- acetate
- gene
- smooth muscle
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Abstract
本发明公开了靶点Nesfatin‑1在预防或治疗血管钙化中应用,属于分子生物医疗技术领域。本发明确定了靶点Nesfatin‑1在血管钙化中的作用,提供了一个特异性敲降血管平滑肌Nesfatin‑1基因的腺相关病毒(adeno‑associated virus,AAV)shRNA干扰载体并筛选出能够靶向抑制Nesfatin‑1的天然产物Cedryl acetate等。本发明所述的干扰载体与天然产物能够治疗血管钙化,为用于预防和治疗血管钙化的药物制备提供扎实的研究基础。
Description
技术领域
本发明涉及靶点Nesfatin-1在预防或治疗血管钙化中的应用,属于分子生物医疗技术领域。
背景技术
血管钙化是动脉粥样硬化过程中的一个重要特征,指的是在动脉壁中沉积过多的钙盐。大量的研究表明,血管钙化与心血管事件(如心脏病发作和中风)之间存在密切关联。动脉壁中的钙盐沉积加剧了血管狭窄和血液不正常流动,使血管更容易发生血栓形成和血管阻塞,从而导致心血管事件的发生。针对血管钙化的有效预防和治疗方法一直是医学领域的一个重要挑战,目前研究主要关注于血管钙化的发生机制和影响因素,例如炎症、氧化应激、细胞凋亡等。
Nesfatin-1是一种由脑和胃部分泌的多肽激素,最初于2006年由日本研究人员在大鼠体内发现。Nesfatin-1的前体蛋白(nesfatin precursor protein)经酶切生成Nesfatin-1,Nesfatin-1被认为在多个生理过程中发挥重要作用,包括食欲调节、能量代谢、自主神经调节和体重控制等。已有研究发现,血清Nesfatin-1水平与冠状动脉疾病的发病率和严重程度负相关,但是不清楚具体机制。虽然动脉粥样硬化是冠状动脉疾病的主要病理基础,血管钙化又与动脉粥样硬化密切相关,但没有明确的证据表明Nesfatin-1与血管钙化之间存在直接的因果关系。
目前临床关于血管钙化的治疗存在诸多局限性,其中主要的治疗方法包括药物治疗、改变生活方式(如饮食和运动)以及手术干预(如血管成形术和搭桥手术)。然而这些方法并不适用于所有患者,并存在安全风险、疗效不确切、复发率高、副作用大等问题。因此,寻找有效的血管钙化治疗方法与药物具有积极意义,为血管钙化治疗与药物研发提供新思路。
发明内容
针对以上问题,本发明首次将Nesfatin-1作为一个可用于预防或治疗血管钙化的制药靶点,并基于Nesfatin-1设计具有预防或治疗血管钙化功能的靶向shRNA,并筛选了天然产物乙酸柏木酯(Cedryl acetate)等用于预防或治疗血管钙化。本发明具有突破性意义,为寻找有效的血管钙化治疗方法及药物制备提供了新思路。
本发明的第一个目的是提供靶点Nesfatin-1在制备预防或治疗血管钙化药物中的应用。
本发明的第二个目的是提供一种研究与血管钙化相关的基因的功能、或者筛选用于预防或治疗血管钙化的药物、或者构建血管钙化疾病模型、或者分析基因治疗血管钙化药物的方法,所述方法包括利用含有特异性敲降血管平滑肌Nesfatin-1基因的腺相关病毒(adeno-associated virus,AAV)shRNA干扰载体来降低Nesfatin-1的表达。
在一种实施方式中,所述应用能够在体外细胞中,或者在体内动物模型中,或者在筛选药物中,预防或治疗血管平滑肌血管钙化。
在一种实施方式中,所述干扰载体的构建和筛选方法包括如下步骤:
(1)通过基因测序技术确定Nesfatin-1基因的序列;
(2)设计候选AAV-shRNA序列,shRNA是双链RNA分子,由两个互补的单链RNA序列组成,所设计的AAV-shRNA序列与Nesfatin-1基因的特定区域具有互补性;
(3)最终选择对Nesfatin-1基因表达水平或钙化具有最显著抑制效果的AAV-shRNA作为最佳候选。
在一种实施方式中,所述AAV-shRNA的核苷酸序列是SEQ ID NO.1-6所示的序列,其中AAV-shRNA1的核苷酸序列为SEQ ID NO.1-2;AAV-shRNA2的核苷酸序列SEQ ID NO.3-4;AAV-shRNA3的核苷酸序列SEQ ID NO.5-6。
本发明的第三个目的是提供一种能够预防或治疗血管钙化的药物,所述药物能够抑制Nesfatin-1基因或蛋白的表达。
在一种实施方式中,所述药物是一种基因疗法的载体,包括基因编辑、基因表达调控或基因传递的载体;或者所述药物是细胞疗法的载体,包括干细胞、免疫细胞或修复细胞。
在一种实施方式中,所述药物通过调节Nesfatin-1信号通路或下游效应分子来预防或治疗血管钙化。
在一种实施方式中,所述药物包括如下一种或多种:香柏乙酸酯(Cedrylacetate)、酮异樟酮(Ketoisophorone)、尼古丁(Cotinine)、松油乙酸酯(Bornylacetate)、丁香酚(Eugenol)、青蒿素(Artesunate)、雌激素苯乙酸酯(Estropipate)、黄酮酮(Flavanone)、雌炔诺酮(Ethisterone)。优先地,所述药物中含有香柏乙酸酯(Cedrylacetate)。
在一种实施方式中,所述药物的剂型包括口服液、注射剂、片剂、丸剂、分散剂、胶囊剂、滴丸、颗粒剂、悬浮剂、乳剂。
本发明的第四个目的是提供天然产物在制备预防或治疗血管钙化的药物中的应用,所述天然产物包括如下一种或多种:香柏乙酸酯(Cedryl acetate)、酮异樟酮(Ketoisophorone)、尼古丁(Cotinine)、松油乙酸酯(Bornyl acetate)、丁香酚(Eugenol)、青蒿素(Artesunate)、雌激素苯乙酸酯(Estropipate)、黄酮酮(Flavanone)、雌炔诺酮(Ethisterone)。优先地,所述药物中含有香柏乙酸酯(Cedryl acetate)。
本发明的第五个目的是提供一种药物组合物,所述药物组合物是用于预防和治疗血管钙化的药物组合物或抑制Nesfatin-1表达的药物,所述药物组合物包括药学可接受的辅料和如下的一种或多种:香柏乙酸酯(Cedryl acetate)、酮异樟酮(Ketoisophorone)、尼古丁(Cotinine)、松油乙酸酯(Bornyl acetate)、丁香酚(Eugenol)、青蒿素(Artesunate)、雌激素苯乙酸酯(Estropipate)、黄酮酮(Flavanone)、雌炔诺酮(Ethisterone)。
本发明构建了血管平滑肌细胞特异性的短发夹核糖核酸(short hairpin RNA,shRNA),筛选得到了能够有效地降低血管平滑肌细胞中nesfatin-1基因的表达水平的shRNA,并验证了其在血管钙化中的作用。此外,本发明进行大规模的化合物筛选,寻找到了能够靶向抑制nesfatin-1的天然产物,并验证了其对血管钙化的预防及治疗效果。
本发明的优点和效果:
(1)本发明发现Nesfatin-1表达水平在高磷处理的血管平滑肌细胞和钙化的血管中均中显著增高,且血管平滑肌细胞的钙化程度越重,Nesfatin-1表达水平越高。
(2)本发明成功构建并筛选血管平滑肌特异性敲降Nesfatin-1基因的AAV-shRNA干扰载体,并证实其在预防和治疗血管钙化中的作用。
(3)本发明筛选出可以靶向抑制Nesfatin-1基因的天然产物,其中Cedrylacetate对血管钙化的抑制最好,达到53.6%;其次为Ethisterone、Eugenol、Cotinine、Flavanone、Ketoisophorone、Artesunate、Bornyl acetate和Ethisterone,对血管钙化的抑制分别达到45.5%、43.8%、41.5%、41%、39.5%、34.9%、32.3%、30.8%。为预防和治疗血管钙化药物的制备提供扎实的研究基础。
附图说明
图1为Nesfatin-1在钙化的血管平滑肌细胞及血管中的表达。(A)Western blot检测血管平滑肌细胞中Nesfatin-1的蛋白水平的代表图;(B)A图的定量统计分析;(C)钙化血管平滑肌细胞中Nesfatin-1的mRNA水平;(D)Western blot检测钙化血管中Nesfatin-1的蛋白水平的代表图;(E)D图的定量统计分析;(F)免疫荧光检测钙化血管中Nesfatin-1的蛋白表达,蓝色DAPI为细胞核,红色为Nesfatin-1蛋白,绿色α-SMA为血管平滑肌细胞的标记物,标尺=100μm。n=4-6。**p<0.01与day 0相比;***p<0.001与day 0/control相比。
图2为构建并筛选血管平滑肌特异性敲降Nesfatin-1基因的AAV-shRNA干扰载体图。(A)Western bolt检测构建的三条AAV-shRNA干扰载体对血管平滑肌细胞Nesfatin-1蛋白表达的干扰作用代表图;(B)A图的定量统计分析;(C)经三条AAV-shRNA干扰后血管平滑肌细胞中Nesfatin-1mRNA水平。n=4-6。*p<0.05与AAV-Con相比;**p<0.01与AAV-Con相比;***p<0.001与AAV-Con相比。
图3为AAV-shRNA3对于血管平滑肌细胞钙化和血管钙化的预防和治疗作用图。(A)AAV-shRNA3干预和治疗血管平滑肌细胞钙化的作用。上图为细胞培养皿,下图为茜素红S染色图;(B)血管平滑肌细胞ALP酶活性测定结果图;(C)血管平滑肌细胞钙含量测定结果图;(D)AAV-shRNA3干预和治疗血钙化茜素红S染色代表图;(E)血管ALP酶活性测定结果图;(F)血管钙含量测定结果图。n=4-6。*p<0.05与AAV-Con相比;**p<0.01与AAV-Con相比;***p<0.001与AAV-Con相比;#p<0.05与AAV-shRNA3相比。
图4为筛选靶向抑制Nesfatin-1的天然产物及验证实验图。(A)荧光素酶报告基因检测流程图;(B)可有效抑制Nesfatin-1的天然产物的RLU值;(C)天然产物对Nesfatin-1抑制率的排序,选择对Nesfatin-1抑制率最高的天然产物Cedryl acetate;(D)Western bolt不同浓度天然产物Cedryl acetate钙化血管平滑肌细胞Nesfatin-1蛋白水平影响的代表图;(E)D图定量统计分析;(F)三种不同浓度的天然产物处理钙化的血管平滑肌细胞Nesfatin-1的mRNA水平;(G)钙化血管平滑肌细胞及三种不同浓度的天然产物Cedrylacetate处理后的茜素红S染色图;(H)血管ALP酶活性测定结果图;(I)血管钙含量测定结果图;n=4-6。*p<0.05与Con相比;#p<0.05与High Pi组相比。
具体实施方式
一、本发明的技术方案
1.血管平滑肌细胞特异性敲低nesfatin-1基因的技术方案:
(1)设计和合成nesfatin-1基因特异性的shRNA序列;
(2)构建shRNA载体,包括一个适合血管平滑肌细胞的启动子和一个用于筛选的标记基因;
(3)将合成的shRNA序列克隆到载体中,验证克隆的准确性;
(4)制备nesfatin-1基因敲低的AAV病毒载体,并用于感染血管平滑肌细胞;
(5)通过Western blotting、PCR等分子生物学方法验证nesfatin-1基因的表达水平是否被成功敲降,并评估血管钙化的程度。
2.筛选可以靶向抑制nesfatin-1基因的天然产物Cedryl acetate的技术方案:
(1)设计和搭建化合物筛选平台,包括基于细胞模型和体外酶反应的筛选系统;
(2)准备天然产物样本库,包括来自植物、微生物或其他来源的天然产物样本;
(3)将天然产物样本与筛选平台相结合,筛选出对nesfatin-1表达具有抑制作用的化合物;
(4)验证筛选得到的天然产物Cedryl acetate的抑制效果,并通过细胞实验和动物模型实验来评估其对血管钙化的预防及治疗效果,从而推进下一步的药物制备。
二、本发明涉及的具体实验方法
1.高磷诱导的血管平滑肌细胞钙化模型
(1)细胞模型选择:选择大鼠血管平滑肌细胞系A7R5,在DMEM培养基中培养;
(2)高磷处理:血清饥饿24h后,更换含10%胎牛血清的DMEM培养液,并在其中加入10mmol/L的β-磷酸甘油诱导血管平滑肌细胞钙化,高磷培养液每三天更换1次。
(3)样本采集:收集高磷处理0、3、6、9、12天的细胞样本。
2.免疫印迹(Western blot):
(1)样本制备:a.收集钙化的血管平滑肌细胞;b.PBS缓冲液洗涤细胞样本以去除培养基或其他杂质;c.加入含有蛋白酶抑制剂细胞裂解缓冲液,裂解细胞获得总蛋白;
(2)蛋白质提取:a.离心:将裂解的样本离心以去除细胞碎片和细胞核等固体残渣,收集上清液;b.蛋白质浓度测定:使用BCA蛋白质定量方法测定蛋白质的浓度;
(3)SDS-PAGE凝胶电泳:a.样品制备:将提取的蛋白质样本与SDS-PAGE样品缓冲液混合,并加热变性;b.凝胶制备:制备聚丙烯酰胺凝胶;c.样品加载:将变性的蛋白质样本加载到凝胶孔中,同时加载分子量标记物作为参照;d.电泳:将凝胶置于电泳槽中,加入电泳缓冲液,并施加电场进行电泳;
(4)转膜:a.准备膜:将蛋白质从凝胶上转移到蛋白印迹膜上。准备合适大小的膜,并在转移前预处理膜;b.转膜装置:将凝胶和膜依次堆叠,放入蛋白转移装置中,加入适当的转移缓冲液。确保凝胶与膜之间没有气泡,并使其完全接触;c.转膜:将蛋白质从凝胶上转移到膜上;
(5)免疫印迹:a.阻断:将蛋白印迹膜置于阻断缓冲液中,阻断非特异性结合位点,减少后续抗体的非特异性结合;b.抗体结合:将特异性Nesfatin-1一抗加入抗体结合缓冲液中,将膜与Nesfatin-1一抗一起孵育,使Nesfatin-1一抗与膜上的Nesfatin-1蛋白结合;c.洗膜:对膜进行多次重复洗涤,去除未结合的Nesfatin-1一抗和其他非特异性结合物质;d.二抗结合:加入酶标记的二抗,将膜与二抗一起孵育,使二抗与Nesfatin-1一抗结合;e.洗涤:再次对膜进行多次洗涤,去除未结合的二抗和其他非特异性结合物质;f.信号检测:使用化学发光法检测蛋白质在膜上的信号;
(6)数据分析:通过image J图像分析软件、graphpad等工具,定量分析蛋白印迹图像,获得Nesfatin-1蛋白的相对表达水平,并比较各组Nesfatin-1蛋白水平。
3.逆转录聚合酶链反应(RT-PCR)
(1)样本制备:a.收集钙化的血管平滑肌细胞;b.使用PBS缓冲液冲洗以去除培养基或其他杂质;c.加入含有蛋白酶K的细胞裂解缓冲液,裂解细胞并释放DNA;
(2)PCR反应:a.准备PCR反应体系,包括DNA模板、引物、dNTPs、聚合酶、缓冲液和MgCl2;b.在PCR反应管中混合反应体系的组分,设置反应体系;c.进行PCR反应,包括初始变性步骤,循环变性步骤,退火步骤,延伸步骤,以及最终延伸步骤;
(3)凝胶电泳分析:a.准备琼脂糖凝胶和缓冲液;b.将PCR反应产物与DNA标记物混合,并加载到琼脂糖凝胶槽中;c.进行凝胶电泳,以分离和可视化PCR扩增产物;d.使用蛋白质成像系统观察凝胶,记录目标片段的大小和强度;
(4)数据分析:a.使用ImageJ图像分析软件,测量PCR产物的带位置和带强度;b.使用相对表达量计算方法,即△△Ct方法,根据Ct值计算Nesfatin-1基因相对于内参基因的表达量,并使用graphpad等工具比较各组Nesfatin-1基因水平。
4.构建血管钙化小鼠模型
(1)选择C57BL/6小鼠,正常环境饲养;
(2)高磷饮食造模:a.将小鼠分为两组,一组为高磷饮食组,另一组为对照组;b.高磷饮食组的小鼠给予富含添加无机磷盐的饲料6周;c.对照组的小鼠继续接受标准饮食6周;d.高磷饮食期间,对小鼠进行定期观察和记录,包括体重、食物摄入量和一般健康状况;
(3)评估钙化病变:造模完成后将小鼠处死,取胸主动脉进行进一步分析,如钙沉积染色(Alizarin Red S染色),测定钙含量等,以确定建模是否成功。
5.小鼠胸主动脉免疫荧光
(1)收集小鼠胸主动脉组织:a.选择血管钙化小鼠模型;b.安乐死小鼠并进行解剖,将胸主动脉迅速取出;
(2)动脉环状切片制备:a.将取得的胸主动脉组织置于PBS中冲洗以去除血液和其他污物;b.将胸主动脉组织用刀片或剪刀小心地剪开,取得动脉环状切片;c.将切片置于含有PBS的离心管中;
(3)固定和脱水:a.用4%多聚甲醛溶液固定切片,在室温下固定30分钟;b.将固定的切片进行脱水处理,将切片依次浸泡在不同浓度的乙醇溶液中(70%、80%、90%、95%和100%),每个浓度的乙醇溶液中浸泡5分钟;
(4)免疫荧光染色:a.在免疫荧光染色缓冲液中预处理切片以阻断非特异性结合;b.在Nesfatin-1和α-SMA抗体中孵育切片;c.洗涤切片以去除未结合的抗体;
(5)显微镜观察和成像:a.将染色后的切片放置在玻片上,并使用含DAPI的封片剂封盖;b.使用荧光显微镜观察切片,并在蓝色通道(DAPI)、红色通道(Nesfatin-1)和绿色通道(α-SMA)下进行图像获取;c.分析图像,观察共定位情况。
6.茜素红S(Alizarin Red S)染色
(1)样本准备:a.包括血管平滑肌细胞和小鼠胸主动脉血管;b.使用4%甲醛溶液对组织进行固定,在室温下固定约30分钟;c.用PBS进行冲洗,去除固定液;
(2)Alizarin Red S染色:a.准备Alizarin Red S染色溶液;b.PBS缓冲液配置0.1% Alizarin Red S溶液;c.将固定的组织样本置于Alizarin Red S溶液中,室温下孵育约30分钟至1小时,使其与钙离子发生络合反应;d.使用PBS缓冲液洗涤样本,以去除未结合的染色剂;
(3)观察和图像获取:将染色后的样本放置在显微镜载玻片上,显微镜下观察染色样本。Alizarin Red S与钙络合物呈现橙红色至红色沉积物,可用肉眼或显微镜进行观察。
7.钙含量测定
(1)样本收集:收集血管平滑肌细胞和血管样本;
(2)钙离子的提取:a.使用Tris-HCl缓冲液洗涤细胞样本,去除培养基和其他污染物;b.加入RIPA缓冲液细胞裂解缓冲液;c.将样本置于冰上或低温环境中,以避免钙离子的进一步流失;d.通过离心将细胞裂解液离心下来,以获得上清液;
(3)钙含量测定:a.使用钙检测试剂盒(Calcium Assay Kit)进行钙含量的测定;b.测定方法是比色法,根据钙离子与染色剂的反应产生的颜色进行定量测量;c.按照试剂盒说明书中的步骤,将上清液与钙检测试剂进行反应,并测量产生的信号;d.使用分光光度计进行吸光度检测;
(4)数据分析:通过graphpad等工具,分析获得钙含量的数据,并比较各组差异。
8.ALP酶活性的测定
(1)样本收集:收集血管平滑肌细胞和血管样本;
(2)细胞裂解:使用的细胞裂解缓冲液将细胞裂解,以释放细胞内的酶;
(3)蛋白质含量测定:通过BCA法测定细胞裂解液中的总蛋白质含量,以用于标准化ALP活性;
(4)ALP底物反应:将裂解液与ALP底物反应,并催化底物的水解反应;
(5)反应终止:通过加入终止液来终止ALP底物的反应;
(6)产物检测和数据分析:使用光度法测量反应后产物的吸光度,根据标准曲线或内部对照物质,将测量结果量化,并进行数据分析和解释。
9.荧光素酶报告基因检测
(1)构建荧光素酶报告基因载体:以大鼠血管平滑肌细胞基因组DNA为模板,将Nesfatin-1第一个外显子往前2000bp的范围视为启动子区域。应用Primer Premier 5.0软件设计扩增引物,同时加入正向酶切位点KpnⅠ和反向酶切位点HindⅢ以及相应的保护碱基,采用PCR方法扩增Nesfatin-1基因的启动子序列,采用可以用于快速PCR扩增的高保真DNA聚合酶TransStart FastPfu DNAPolymerase,PCR扩增Nesfatin-1启动子区的目标DNA片段。利用限制性内切酶KpnI和HindIII对上述PCR产物及载体质粒pGL3-Enhancer进行双酶切。酶切后的产物进行琼脂糖凝胶电泳,并切胶回收目的片段,经过菌液扩增、酶切、测序等方法获得目的质粒。
(2)转染目标细胞:将构建好的荧光素酶报告基因载体转染到血管平滑肌细胞中,确保其能够稳定地表达荧光素酶;
(3)处理天然产物样品:将待筛选的4160个天然产物样品(10μM)加入钙化诱导的含有nesfatin-1荧光素酶报告基因转染的血管平滑肌细胞,使其与细胞共同培养48h,然后采用荧光素酶报告基因检测试剂盒检测nesfatin-1的荧光素酶报告基因活性;
(4)荧光素酶活性测定:使用荧光素底物对处理后的细胞进行荧光素酶活性测定。添加荧光素底物后,荧光素酶会催化底物的反应,产生可测量的荧光信号;
(5)荧光测量和数据分析:a.使用荧光酶标仪测量处理后细胞的荧光素酶活性,RLU是指每秒钟产生的荧光信号强度,反映了荧光素酶活性的水平;b.比较和分析:比较和分析RLU值,进行统计学分析,筛选对Nesfatin-1活性的抑制效果的天然产物。
实施例1:Nesfatin-1在血管钙化中的作用
实施例1所涉及的实验方法包括:构建高磷诱导的血管平滑肌细胞钙化模型、构建血管钙化小鼠模型、Western blot、PCR和免疫荧光。
1、Nesfatin-1在钙化血管平滑肌细胞中显著升高
结果如图1所示,图1A检测血管平滑肌细胞中Nesfatin-1的蛋白表达量;图1B为A图的定量统计分析;图1C检测了检测血管平滑肌细胞中Nesfatin-1的基因表达量。通过Western blot检测和RT-PCR检测均发现,Nesfatin-1表达水平在高磷处理的血管平滑肌细胞中中显著增高,且血管平滑肌细胞的钙化程度越重,Nesfatin-1表达水平越高。
2、Nesfatin-1在钙化血管中显著升高
结果如图1所示,图1D检测血管中Nesfatin-1的蛋白表达量;图1E为D图的定量统计分析。图1F为免疫荧光检测钙化血管中Nesfatin-1的蛋白表达。通过Western blot检测和免疫荧光均发现,Nesfatin-1表达水平在钙化血管中中显著增高,且血管钙化程度越重,Nesfatin-1表达水平越高。
实施例2:血管平滑肌特异性敲降Nesfatin-1基因的AAV-shRNA干扰载体的构建与筛选。
实施例2所涉及的实验方法包括:Western blot、PCR、茜素红S染色、ALP酶活性测定、钙含量测定。
1、成功构建血管平滑肌特异性敲降Nesfatin-1基因的AAV-shRNA干扰载体。
成功构建了特异性敲降Nesfatin-1基因的三条AAV-shRNA干扰载体,其中AAV-shRNA1的核酸序列为SEQ ID NO.1-2;AAV-shRNA2的核酸序列为SEQ ID NO.3-4;AAV-shRNA3的核酸序列为SEQ ID NO.5-6。
结果如图2所示,图2A为示血管平滑肌细胞经三条AAV-shRNA干扰后Nesfatin-1的蛋白表达水平;图2B为图2A图的定量统计分析;图2C为AAV-shRNA3干扰后血管平滑肌细胞Nesfatin-1的基因表达水平。结果显示构建的三条AAV-shRNA干扰载体均可显著降低血管平滑肌细胞中Nesfatin-1蛋白和mRNA水平,其中AAV-shRNA3干扰载体效果最佳。
2、AAV-shRNA3在预防和治疗血管钙化中的作用。
结果如图3所示,图3A为AAV-shRNA3干预钙化血管平滑肌细胞,可以看出经AAV-shRNA3干预,血管平滑肌细胞钙化得到缓解;图3B为血管平滑肌细胞ALP酶活性测定结果图,可见AAV-shRNA3可显著逆转钙化血管平滑肌细胞的ALP酶活性,而对正常的血管平滑肌细胞无明显影响;图3C为血管平滑肌细胞钙含量测定结果图,可见AAV-shRNA3可显著逆转钙化血管平滑肌细胞中的钙含量,而对正常的血管平滑肌细胞无明显影响;图3D为AAV-shRNA3干预和治疗血钙化茜素红S染色代表图,可见AAV-shRNA3干预后能够显著缓解血管钙化;图3E为血管ALP酶活性测定结果图,可见AAV-shRNA3可显著逆转钙化血管异常升高的ALP酶活性,而对正常的血管无明显影响;图3F为血管钙含量测定结果图,可见AAV-shRNA3可显著逆转钙化血管异常升高的钙含量,而对正常的血管平滑肌细胞无明显影响。结果显示AAV-shRNA3可显著逆转钙化血管平滑肌细胞和钙化血管中异常升高的ALP酶活性以及钙含量,而对正常的血管平滑肌细胞和血管无明显影响。
实施例3:靶向抑制Nesfatin-1的天然产物Cedryl acetate的筛选及其在血管钙化的预防和治疗的应用
实施例3所涉及的实验方法包括:荧光素酶报告基因检测、Western bolt、PCR、茜素红S染色、ALP酶活性测定、钙含量测定。
结果如图4所示。
图4A为荧光素酶报告基因检测流程图,在4160个天然产物中进行筛选;
图4B为可有效抑制Nesfatin-1的天然产物的RLU值,可见高磷处理会显著增加RLU值,天然产物Cedryl acetate、Ethisterone、Eugenol、Cotinine、Flavanone、Ketoisophorone、Artesunate、Bornyl acetate和Ethisterone能够有效抑制RLU值,其中Cedryl acetate对RLU值的抑制效果最好。
图4C为部分筛选得到的天然产物对Nesfatin-1抑制效果,结果显示,Cedrylacetate对Nesfatin-1的抑制最好,达到53.6%;其次为Ethisterone、Eugenol、Cotinine、Flavanone、Ketoisophorone、Artesunate、Bornyl acetate和Ethisterone,对Nesfatin-1的抑制分别达到45.5%、43.8%、41.5%、41%、39.5%、34.9%、32.3%、30.8%;
图4D为Western bolt不同浓度天然产物Cedryl acetate钙化血管平滑肌细胞Nesfatin-1蛋白水平影响的代表图;
图4E为D图定量统计分析,可见三种不同浓度的天然产物Cedryl acetate均可显著抑制钙化血管平滑肌细胞Nesfatin-1的蛋白蛋白表达;
图4F为PCR检测Nesfatin-1基因表达量图,可见三种不同浓度的天然产物Cedrylacetate均可显著降低钙化的血管平滑肌细胞中Nesfatin-1的mRNA水平;
图4G为钙化血管平滑肌细胞及三种不同浓度(0μM、3μM、10μM)的天然产物Cedrylacetate处理后的茜素红S染色图。结果显示,经三种不同浓度的天然产物Cedryl acetate处理后,钙化血管平滑肌细胞得到了显著的缓解。
图4H为血管ALP酶活性测定结果图,可见三种不同浓度(0μM、3μM、10μM)的天然产物Cedryl acetate均可显著逆转钙化血管异常升高的ALP酶活性;
图4I为血管钙含量测定结果图,可见三种不同浓度(0μM、3μM、10μM)的天然产物Cedryl acetate均可显著逆转钙化血管异常升高的钙含量。
本实施例从4160种天然产物中筛选出对Nesfatin-1基因抑制程度最高的Cedrylacetate,其对血管钙化的抑制率达到53.6%。Cedryl acetate可显著逆转钙化血管平滑肌细胞中Nesfatin-1蛋白和mRNA水平,并逆转钙化血管中异常升高的ALP酶活性以及钙含量。
本发明涉及的序列:
SEQ ID NO.1Sense:5’-GTTTGTTTGTGTAATTCTA-3’;
SEQ ID NO.2Antisense:5’-TAGAATTACACAAACAAAC-3’
SEQ ID NO.3Sense:5’-GGTTGGTGCTCCTGTTGAA-3’;
SEQ ID NO.4Antisense:5’-TTCAACAGGAGCACCAACC-3’;
SEQ ID NO.5Sense:5’-GGCTTGCTGTTGAGTTCTA-3’;
SEQ ID NO.6Antisense:5’-TAGAACTCAACAGCAAGCC-3’
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.靶点Nesfatin-1在制备预防或治疗血管钙化药物中的应用。
2.一种研究与血管钙化相关的基因的功能、或者筛选用于预防或治疗血管钙化的药物、或者构建血管钙化疾病模型、或者分析基因治疗血管钙化药物的方法,所述方法包括利用含有特异性敲降血管平滑肌Nesfatin-1基因的腺相关病毒(adeno-associated virus,AAV)shRNA干扰载体来降低Nesfatin-1的表达。
3.根据权利要求2所述的方法,其特征在于,所述方法能够在体外细胞中,或者在体内动物模型中,或者在筛选药物中,预防或治疗血管平滑肌血管钙化。
4.一种能够预防或治疗血管钙化的药物,其特征在于,所述药物能够抑制Nesfatin-1基因或蛋白的表达。
5.根据权利要求4所述的药物,其特征在于,所述药物是一种基因疗法的载体,包括基因编辑、基因表达调控或基因传递的载体;或者所述药物是细胞疗法的载体,包括干细胞、免疫细胞或修复细胞。
6.根据权利要求4所述的药物,其特征在于,所述药物通过调节Nesfatin-1信号通路或下游效应分子来预防或治疗血管钙化。
7.根据权利要求4所述的药物,其特征在于,所述药物包括如下一种或多种:香柏乙酸酯(Cedryl acetate)、酮异樟酮(Ketoisophorone)、尼古丁(Cotinine)、松油乙酸酯(Bornyl acetate)、丁香酚(Eugenol)、青蒿素(Artesunate)、雌激素苯乙酸酯(Estropipate)、黄酮酮(Flavanone)、雌炔诺酮(Ethisterone)。
8.根据权利要求4所述的药物,其特征在于,所述药物的剂型包括口服液、注射剂、片剂、丸剂、分散剂、胶囊剂、滴丸、颗粒剂、悬浮剂、乳剂。
9.天然产物在制备预防或治疗血管钙化的药物中的应用,其特征在于,所述天然产物包括如下一种或多种:香柏乙酸酯(Cedryl acetate)、酮异樟酮(Ketoisophorone)、尼古丁(Cotinine)、松油乙酸酯(Bornyl acetate)、丁香酚(Eugenol)、青蒿素(Artesunate)、雌激素苯乙酸酯(Estropipate)、黄酮酮(Flavanone)、雌炔诺酮(Ethisterone)。
10.一种用于预防和治疗血管钙化的药物组合物或抑制Nesfatin-1表达的药物组合物,其特征在于,所述药物组合物包括药学可接受的辅料与如下的一种或多种:香柏乙酸酯(Cedryl acetate)、酮异樟酮(Ketoisophorone)、尼古丁(Cotinine)、松油乙酸酯(Bornylacetate)、丁香酚(Eugenol)、青蒿素(Artesunate)、雌激素苯乙酸酯(Estropipate)、黄酮酮(Flavanone)、雌炔诺酮(Ethisterone)。
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