CN108379555A - Fgf21在制备治疗由血管紧张素ⅱ引发的高血压和/或血管病损伤的药物中的应用 - Google Patents
Fgf21在制备治疗由血管紧张素ⅱ引发的高血压和/或血管病损伤的药物中的应用 Download PDFInfo
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Abstract
本发明涉及具有药理作用的蛋白,具体而言,提供FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管病损伤的药物中的应用。FGF21是一个由约210个氨基酸组成的分泌蛋白,本发明发现FGF21对由血管紧张素Ⅱ引发的高血压和/或血管损伤具有良好的缓解和治疗作用,这为由血管紧张素Ⅱ引发的高血压和/或血管损伤的治疗药物的开发提供了依据。由FGF21作为活性物质制备的由血管紧张素Ⅱ引发的高血压和/或血管损伤的治疗药物具有安全性好,药效持续时间长,降血压效果明显同时保护器官无损伤,无明显副作用等优点。
Description
技术领域
本发明涉及具有药理作用的蛋白,具体而言,涉及FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管病损伤的药物中的应用。
背景技术
高血压是指以体循环动脉血压(收缩压和/或舒张压)增高为主要特征 (收缩压≥140毫米汞柱,舒张压≥90毫米汞柱),可伴有心、脑、肾等器官的功能或器质性损害的临床综合征。高血压作为心脑血管疾病发病的主要危险因素,可诱发心脑肾和血管功能与结构的改变,最终引起脑卒中、心肌梗死、心力衰竭及慢性肾病等严重的并发症。
2012年中国心血管病报告指出,心血管疾病死亡率为44.1%,占死亡疾病谱中第一位,高于肿瘤、呼吸系统和其它疾病。我国每天因心血管疾病死亡5990人,每小时因心血管疾病死亡约400人,每10秒钟就有一人死于心血管疾病。估计我国每年约350万人死于心血管疾病,其中70%脑卒中和50%以上心肌梗死发生与高血压有关。流行病调查结果显示,我国因为高血压导致心血管疾病死亡率有逐年上升趋势,1990年心血管病死亡率约为160/10万,而2010年高达260/10万,在十年间使死亡率增加近一倍。因此,我国高血压发展趋势很不乐观,其防治任务十分繁重,认识我国高血压流行病学特点,对于制定相关对策有效防治高血压具有重要的现实意义。高血压分为原发性高血压和继发性高血压两种,是一种常见的中老年病,并呈现年轻化的趋势。高血压的诊断标准为舒张压≥90mmHg,收缩压≥140mmHg。2016年,美国心脏学会修订了高血压的诊断指标,即舒张压≥80mmHg,收缩压≥130mmHg就可认定为高血压。这意味着高血压人群的大量增加。根据近年的流行病学研究,我国18岁以上公民的高血压患病率不断上升,从2010年公布的25.2%的患病率上升到2014年的 27.2%。
目前在治疗高血压的药物方面,虽然种类繁多,但各类药物均出现不同的负面效应,如噻嗪类利尿剂降低收缩压的作用优于舒张压,适于老年单纯收缩期高血压的患者或有心衰表现的患者,但在应用中要注意避免血钾过低;β受体阻滞剂适用于高血压伴心绞痛、心肌梗死、心衰、快速心律失常、青光眼和怀孕的患者,但其直接影响糖脂代谢,可增加糖尿病发病风险;血管紧张素转换酶抑制剂、血管紧张素II受体阻滞剂虽然适于有胰岛素抵抗、糖尿病、左心功能不全、心力衰竭、心肌梗死的患者,但存在引发血管神经性水肿、高钾血症、白细胞减少、低血糖等不良反应,同时也不可用于孕妇。因此,开发一种安全的、同时具有降压和器官保护效应的、无相关明显副作用的、安全的治疗由血管紧张素II引起的高血压或血管损伤的药物是目前研发的理想目标。
有鉴于此,特提出本发明。
发明内容
本发明的第一目的在于提供FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管病损伤的药物中的应用,以缓解现有技术中治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物缺乏安全性高,效果明显同时没有副作用的特性的现状。
本发明的第二目的在于提供一种治疗由血管紧张素Ⅱ引发的高血压和/ 或血管损伤的药物,以缓解现有技术中治疗高血压和/或血管损伤的药物治疗效果较差并且伴随有副作用的问题。
本发明的第三目的在于提供一种FGF21的制备方法。
为了实现本发明的上述目的,特采用以下技术方案:
本发明提供FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物中的应用。
进一步地,所述FGF21具有如SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列。
进一步地,所述FGF21作用于脂肪细胞,并促进血管紧张素转化酶2 的表达,将血管紧张素II转变为血管紧张素-(1-7),抑制高血压和/或逆转血管损伤。
本发明又提供一种治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物,所述药物的活性成分包括FGF21。
进一步地,所述FGF21具有如SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列。
进一步地,所述FGF21作用于脂肪细胞,并促进血管紧张素转化酶2 的表达,将血管紧张素II转变为血管紧张素-(1-7),抑制高血压和/或逆转血管损伤。
进一步地,所述药物还包括药学上可接受的载剂或辅料。
进一步地,所述药物的给药方式为腹腔给药。
本发明最后提供一种FGF21的制备方法,构建表达FGF21的重组病毒进行表达并纯化收集所述FGF21。
进一步地,所述构建表达FGF21的重组病毒进行表达的方法包括:将小鼠FGF21的编码基因插入到质粒载体pAAV-IRES-ZsGreen后导入 HEK293AAV细胞产生高滴度含目的基因的AAV病毒并进行小鼠FGF21蛋白的过表达。
与现有技术相比,本发明的有益效果为:
本发明提供FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物中的应用。FGF21是一个由约210个氨基酸组成的分泌蛋白,本发明发现FGF21对由血管紧张素Ⅱ引发的高血压和/或血管损伤具有良好的缓解和治疗作用,这为由血管紧张素Ⅱ引发的高血压和/或血管损伤的治疗药物的开发提供了依据。由FGF21作为活性物质制备的治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物具有安全性好,药效持续时间长,降血压效果明显同时保护器官无损伤,无明显副作用等优点。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,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为本发明实施例1中pAAV-mFGF21-IRES-ZsGreen质粒用EcoR I 和BamH I双酶切鉴定结果图;
图2为本发明实施例2中1μL的rAAV9-mFGF21-IRES-ZsGreen病毒感染48孔板HEK293细胞荧光图片结果图;
图3为本发明实施例3的10)试验结果(a)中正常生理和血管紧张素 II诱导的高血压状态下,FGF21KO小鼠与野生型小鼠的收缩压结果图;
图4为本发明实施例3的10)试验结果(a)中AAV-FGF21给予后FGF21 KO高血压小鼠血压变化结果图;
图5a为本发明实施例3的10)试验结果(b)中正常小鼠血管的HE 染色结果;
图5b为本发明实施例3的10)试验结果(b)中给予小鼠血管紧张素 II和对照病毒AAV-GFP后的血管HE染色结果;
图5c为本发明实施例3的10)试验结果(b)中给予小鼠血管紧张素 II和AAV-FGF21给药后的血管HE染色结果;
图5d为本发明实施例3的10)试验结果(b)中对不同处理组小鼠血管厚度测定的结果;
图6a为本发明实施例3的10)试验结果(c)中正常小鼠血管的Masson 染色结果;
图6b为本发明实施例3的10)试验结果(c)中给予小鼠血管紧张素 II和对照病毒AAV-GFP后的血管Masson染色结果;
图6c为本发明实施例3的10)试验结果(c)中给予小鼠血管紧张素 II和AAV-FGF21给药后的血管Masson染色结果;
图6d为本发明实施例3的10)试验结果(c)中对不同处理组小鼠血管纤维化面积的统计结果;
图7为本发明实施例3的10)试验结果(c)中AAV-FGF21治疗对小鼠血管纤维化因子表达的影响结果图;
图8为本发明实施例3的10)试验结果(d)中AAV-FGF21治疗对小鼠血管巨噬细胞浸润的影响结果图;
图9为本发明实施例3的10)试验结果(d)中AAV-FGF21治疗对小鼠血管炎症因子表达的影响结果图;
图10为本发明实施例3的10)试验结果(e)中AAV-FGF21治疗对小鼠血管氧化损伤的影响结果图;
图11为本发明实施例3的10)试验结果(f)中AAV-FGF21治疗对小鼠颈动脉内皮依赖的血管舒张作用的影响结果图;
图12为本发明实施例3的10)试验结果(f)中AAV-FGF21治疗对小鼠颈动脉内皮非依赖的血管舒张作用的影响结果图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。
本发明提供FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物中的应用。
血管紧张素Ⅱ是由血管紧张素Ⅰ在血管紧张素转化酶的作用下,将其 C-末端水解切去2个氨基酸残基,产生的一个八肽(l-8)。人体的血管平滑肌、肾上腺皮质球状带细胞以及脑的一些部位、心脏和肾脏器官的细胞上存在有血管紧张素受体。血管紧张素Ⅱ与血管紧张素受体结合,会引起相应的生理效应,包括使全身微动脉、静脉收缩,从而导致血压升高,回心血量增多。血管紧张素Ⅱ可以引起高血压,进而引发血管损伤,或者因为血管紧张素Ⅱ引起血管损伤,进而伴有高血压等症状。
FGF21(成纤维细胞生长因子21,Fibroblast Growth Factor 21)是一个约210个氨基酸序列组成的,主要由肝脏细胞表达的分泌蛋白。不同于传统的其它FGF家族成员,其主要以内分泌荷尔蒙因子的角色参与机体的物质代谢调控作用。本发明发现FGF21对由血管紧张素Ⅱ引发的高血压和/ 或血管损伤具有良好的缓解和治疗作用,这为治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物的开发提供了依据。FGF21可以降低血压,减少高血压病变过程中血管壁的增厚,减少血管的纤维化因子的表达和纤维化过程,减少血管巨噬细胞的浸润及炎症因子的表达,从而减少高血压引发的炎症反应,缓解血管损伤,或者减少高血压引发的血管氧化损伤和血管损伤引发的高血压,从而从多方面对高血压和/或血管损伤的病程起到治疗作用。
在本发明的一个优选地实施方式中,FGF21具有如SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列。
在本发明的一个优选地实施方式中,FGF21作用于脂肪细胞,并促进血管紧张素转化酶2的表达,将血管紧张素II转变为血管紧张素-(1-7),抑制高血压和/或逆转血管损伤。
本发明提供一种治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物,药物的活性成分包括FGF21。
由FGF21作为活性物质制备的治疗由血管紧张素Ⅱ引发的高血压和/ 或血管损伤的药物具有安全性好,药效持续时间长,降血压效果明显同时保护器官无损伤,无明显副作用等优点。
在本发明的一个优选地实施方式中,FGF21具有如SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列。
在本发明的一个优选地实施方式中,药物还包括药学上可接受的载剂或辅料。
在本发明的一个优选地实施方式中,药物的给药方式为腹腔给药。
本发明最后提供一种FGF21的制备方法,构建表达FGF21的重组病毒进行表达并纯化收集FGF21。
在本发明的一个优选地实施方式中,构建表达FGF21的重组病毒进行表达的方法包括:将小鼠FGF21的编码基因插入到质粒载体 pAAV-IRES-ZsGreen后导入HEK293AAV细胞产生高滴度含目的基因的 AAV病毒并进行小鼠FGF21蛋白的过表达。
为了有助于更清楚的理解本发明的内容,现结合具体的实施例详细介绍如下。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。
下述的实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中的实验材料,如无特殊说明,均为可自常规生化试剂商店购买得到的。
实施例1pAAV-mFGF21-IRES-ZsGreen质粒构建
1)获得目标基因
以质粒pMD18-T-mFGF21(武汉维诺赛生物技术有限公司提供)为模板,PCR扩增小鼠FGF21目的基因(SEQ ID NO.3),在引物两端分别引入EcoR I和BamH I的酶切位点。其中,引物的序列如SEQ ID NO.4和SEQ ID NO.5所示,详见下表;小鼠FGF21目的基因的序列大小为633bp。
引物名称 | 引物序列(5’-3’) | 引物序列号 |
FGF21-EcoR I-F | attgaattcgccaccatggaatggatgagatctagagttg | SEQ ID NO.4 |
FGF21-BamH I-R | agaggatcctcaggacgcatagctggggcttcgg | SEQ ID NO.5 |
PCR反应条件如下:
PCR反应的反应程序如下:
1%琼脂糖凝胶回收小鼠FGF21目的基因的DNA条带(633bp)。
2)质粒载体pAAV-IRES-ZsGreen的EcoR I和BamH I双酶切
将质粒载体pAAV-IRES-ZsGreen(武汉维诺赛生物技术有限公司提供) 用限制性内切酶EcoR I和BamH I进行双酶切,酶切反应在37℃水浴反应 3h,载体酶切体系如下:
1%琼脂糖凝胶电泳回收质粒pAAV-IRES-ZsGreen酶切的大片段。
3)小鼠FGF21目的基因的EcoR I和BamH I双酶切
酶切反应在37℃水浴反应3h,酶切体系如下:
1%琼脂糖凝胶电泳回收小鼠FGF21的EcoR I和BamH I酶切片段
4)重组质粒的连接
连接反应在22℃反应3h,连接反应体系如下:
5)连接产物转化
取10μl 4)中的连接产物与100μl JM109感受态细菌(武汉维诺赛生物技术有限公司)混匀后冰浴30min,42℃热激45s,立即置冰上放置2min, 加入预热至室温的400μl LB培养基,37℃恒温摇床培养1h,4000rpm离心 1min,弃去400μl培养上清,剩余100μl用移液器混匀后均匀涂布于含 100μg/ml Ampicillin抗性的LB平板上,37℃恒温培养箱倒置培养过夜。
6)鉴定
挑取3个单菌落接种于含5ml,100μg/ml Ampicillin抗性的LB培养液中, 250rpm,37℃恒温摇床培养过夜,用小量质粒抽提试剂盒抽提质粒(全式金生物),用EcoR I和BamHI做酶切鉴定,同时送菌液去测序。其中,测序引物为FGF21-EcoR I-F(SEQ ID NO.4)和IRES-R(SEQ ID NO.6),其引物序列如下表所示:
引物名称 | 引物序列(5’-3’) | 引物序列号 |
FGF21-EcoR I-F | attgaattcgccaccatggaatggatgagatctagagttg | SEQ ID NO.4 |
IRES-R | cctcacattgccaaaagacg | SEQ ID NO.6 |
实验结果如附图1所示,其中,M为Maker,1为鉴定泳道。经重组质粒双酶切鉴定结果表明,成功得到阳性克隆。此外测序结果表明插入的 FGF21序列完全正确。将得到的重组质粒命名为 pAAV-mFGF21-IRES-ZsGreen质粒。
实施例2rAAV9-mFGF21-IRES-ZsGreen病毒包装
将含目的基因的重组质粒pAAV-mFGF21-IRES-ZsGreen导入293AAV 细胞(武汉维诺赛生物技术有限公司),产生高滴度含目的基因的AAV病毒(以下简称病毒rAAV9-mFGF21-IRES-ZsGreen);同时用质粒 pAAV-IRES-ZsGreen做对照病毒。具体实验步骤如下:
1)按照每皿1.5×107个293AAV细胞接种到12个15cm细胞培养皿中, 37℃,5%CO2培养箱培养过夜。
2)培养30-48h后,每皿用15ml高糖DMEM+10%FBS+P/S的培养基换液。
3)质粒转染(每个15cm培养皿):
(a)取两个洁净的2ml EP管,分别标记为A管和B管。
(b)在A管中加入1ml CPT Buffer A。
(c)在B管中按下表依次加入以下质粒和试剂:
(d)用1ml移液器吸入B管的溶液一滴一滴加入到A管溶液中,用 1ml移液器反复吹气泡10次,以达到轻微混匀的目的。
(e)室温静置30min,然后将混和溶液均匀一滴一滴加入到15cm细胞培养皿中,37℃,5%CO2培养箱培养4-6h。
4)培养4-6h后,用20ml高糖DMEM+10%FBS+P/S的培养基换液, 37℃,5%CO2培养箱培养过夜。
5)继续培养60-72h后,用10ml移液管反复吹打细胞,使所有细胞从培养皿上完全脱落下来,收集细胞和培养上清到一个新的50ml离心管中。
6)-80℃和37℃反复冻融3次,3000rpm离心10min,收集上清,用 0.45μm PVDF滤器过滤去除细胞碎片。
7)过病毒纯化柱纯化,最后用2ml Buffer洗脱。
生物滴度测定:
1)在滴度测定的前一天取一块48孔板,按照每孔5×104个细胞的密度接种HEK293细胞(武汉维诺赛生物技术有限公司)到48孔板中。
2)用完全培养基10倍梯度稀释病毒,具体做法如下:取一个48孔板,每孔含有90μl培养基,第一个孔加入10μl待测病毒原液,混匀后吸取10μl 病毒混合液至第二个孔(混匀时尽量不要产生气泡),如此稀释至第六个孔 (即稀释105倍)。具体数据如下表所示:
3)各吸取10μl病毒稀释液到相对应的48孔板中,置于37℃,5%CO2培养箱培养过夜,16h-24h后用新鲜的完全培养基换液(DMEM+10% FBS+P/S)。
4)置于37℃,5%CO2培养箱继续培养48h-96h后,用荧光显微镜计数荧光细胞数量(如果观察不清晰可以先用PBS(pH7.4)换液后再进行观察)。一般情况下,在最高稀梯度10m的孔数出现N(N<10)个带荧光的细胞,则病毒滴度为N×10mTU/μl(10m为稀释倍数),即病毒滴度为N×10m+3TU/ml,若N>10,则需要继续稀释。
荧光显微镜下观测结果表明构建的rAAV9-mFGF21-IRES-ZsGreen病毒能够转染HEK293细胞,并呈现绿色荧光(见附图2)。此外,用上述病毒滴度检测的方法检测,结果表明所得的rAAV9-mFGF21-IRES-ZsGreen病毒滴度为1×107TU/mL,即1×1012vg/mL。
实施例3AAV9-mFGF21对血管紧张素II诱导的高血压的治疗作用
1)动物分组及高血压小鼠模型建立
取8-10周龄雄性FGF21KO(FGF21基因缺失)小鼠20只,分为A/B 两组,每组10只,取年龄匹配的雄性野生型WT小鼠20只,分为C/D两组,每组10只。利用渗透泵灌注血管紧张素II的方式制作小鼠高血压模型。选用ALZET渗透泵(Model 2004),先将渗透泵浸泡于无菌的生理盐水在37℃恒温细胞培养箱孵育48h以激活渗透泵。手术前根据小鼠(A组和C 组)体重配好Ang II溶液,并注入渗透泵,对照组C、D两组注入PBS缓冲液。以上操作均需在超净台中操作。用4%水合氯醛(0.1ml/10g)腹腔注射麻醉,并将小鼠固定在无菌手术操作台上,腹部向下,然后用脱毛膏将小鼠颈后背部的毛脱去,碘酒消毒,在小鼠颈后皮肤做垂直于尾巴的切口,长度为1cm。用灭菌的直止血钳在皮肤下制作一个皮肤囊袋,并将渗透泵置入小鼠皮下,用无菌缝合线缝合切口。将手术后的小鼠置于保温箱等待完全苏醒。之后每天观察小鼠状况,以防小鼠发生意外死亡。
另外取30只8-10周的雄性FGF21KO小鼠,10只一组,分为A、B 和C共三组。按上述高血压模型建立方式,A组渗透泵中给予生理盐水,B 和C两组给予血管紧张素。此外,在小鼠造模之前第5天,B组小鼠每只腹腔注射5-10×1010vgAAV-GFP,C组小鼠每只腹腔注射5-10×1010vg AAV-FGF21。
2)实验动物处理
在实验动物埋泵前4天到处死前,每个两天监测各组小鼠的尾动脉收缩压,灌泵4周后,进行葡萄糖耐受实验和胰岛素耐受实验。随后,称量小鼠体重,用4%水合氯醛(0.1ml/10g)腹腔内注射麻醉,将小鼠四肢固定后,酒精喷洒胸腹部,剪开胸部表皮,于第二至第三肋骨间插入1ml注射器,缓慢心脏取血,低温超速离心机4℃3000rpm×10min离心取上清,分装后-80℃保存;剪开右心耳,装有约20ml生理盐水的注射器进入小鼠左心室进行灌流。然后在体视显微镜下,用显微镊以及显微剪仔细分离,将小鼠的颈动脉和主动脉组织分离出来。颈动脉置于Krebs缓冲液,用于血管舒张功能的检测,取材是尽量避免损伤。主动脉中,取长约0.5cm胸主动脉置于OCT中,并放于-20℃冰箱缓慢冻存用于冰冻切片,其余主动脉组织于 -80℃保存。
3)小鼠尾动脉收缩压测定
采用BP2000仪器通过尾套法测定小鼠尾动脉收缩压,恒温37℃。术前一周开始适应测血压,即小鼠每天在同一时间点放入仪器进行测量,从而使其熟悉检测环境,减少环境因素的影响。然后分别在灌泵前,灌泵后2、 4、6、8、10、12、14天检测小鼠尾动脉收缩压并记录。每只小鼠测量5组数据以上。
4)组织切片H&E染色
冰冻切片切取5μm厚度的主动脉切片进行H&E染色。主要步骤如下所述:
(a)将冰冻片子从-20℃冰箱取出,于室温放置5min;
(b)蒸馏水浸洗10min;
(c)苏木素染色5min,用蒸馏水稍洗,置于蒸馏水中;
(d)1%盐酸酒精溶液分化2s,蒸馏水稍洗,弱氨水返蓝30s,蒸馏水稍洗,置于蒸馏水中;
(e)伊红染色1min,蒸馏水稍洗,置于蒸馏水中;
(f)酒精梯度脱水:85%酒精10s,95%酒精30s,100%乙醇I 2min, 100%乙醇II2min;
(g)透明:二甲苯I 5min;二甲苯II 15min;
(h)中性树胶封片,镜下观察。
5)Masson三色染色
(a)将冰冻片子从-20℃冰箱取出,于室温放置5min;
(b)蒸馏水浸洗10min;
(c)用Harris氏苏木素染液或Weigert苏木素液染核1-2min;
(d)充分水洗,如过染可盐酸酒精分化2-3s;
(e)温水返蓝;
(f)用Masson丽春红酸性复红液5-10min;
(g)1%磷钼酸水溶液分化3-5min;(镜下观察,着色程度)
(h)1%苯胺蓝或光绿液染5min;
(i)1%冰醋酸水溶液分化几秒;(镜下观察,着色程度)
(j)95%酒精、无水酒精、二甲苯透明、中性树胶封固。
6)流式细胞仪分析血管巨噬细胞浸润
主动脉弓至股分叉的降主动脉,用手术刀切碎后,37℃于含60U/ml DNase I,60U/ml hyalronidase,450U/ml Collagenase和125U/ml Collagenase XI的消化液中消化(所有酶都购自Sigma-Aldrich公司)。用过滤器过滤得到单细胞悬浮液,用于随后的流式细胞术染色。离体主动脉细胞用CD45 percp-cy5.5,CD3PE-CF594,CD11b APC-Cy7,和F4/80APC,以及它们同源的isotypematched阴性对照染色(BD)后,上流式细胞仪分析。
7)DHE染色
(a)将冰冻片子从-20℃冰箱取出,于室温放置5min;
(b)PBS浸洗5min;
(c)10μM的二氢乙锭溶液孵育30min,37℃,避光;
(d)DAPI盖片。
8)荧光定量PCR
(a)主动脉组织总RNA提取:
将存于-80℃冰箱中的四组小鼠主动脉组织取出置于1.5ml EP管中并加入1mltrizol,使用电动匀浆器高速匀浆组织,然后按照以下步骤进行总RNA 的提取:
(1)将匀浆后的裂解液低温超速离心4℃12000g×5min,上层澄清液置于新的1.5ml EP管中;
(2)各管中加入0.2ml氯仿,盖紧EP管盖,上下剧烈震荡混匀15s,室温静置5min;4℃离心12000g×15min;
(3)取出EP管,此时EP管分三层,取上层液相置于新的1.5ml EP 管中;
(4)每管加入0.5ml异丙醇,轻轻摇晃,室温静置10min;4℃离心 12000g×10min;
(5)弃上清,加1ml 75%乙醇清洗沉淀;4℃离心7500g×5min;
(6)弃上清,用移液器尽量吸去残余的乙醇溶液,干燥RNA;
(7)待干燥后加入适当DEPC水溶解,备用;
(8)用NanoDrop测定总RNA纯度及浓度。
(b)cDNA的合成:
根据RNA浓度,以20μl的反应体系,每次逆转0.5-5μg总RNA量。具体逆转录步骤如下:
(c)QPCR检测:
根据实验的设计,我们根据美国国立图书馆基因库中小鼠的基因序列,设计了小鼠下面相关基因表达扩增引物,序列如下所示:
取上述所获得的cDNA,采用SYBR-Green荧光定量PCR法,根据下面的反应体系和进行QPCR扩增:
SYBR-Green QPCR反应体系:
SYBR-Green QPCR扩增条件:
9)血管环功能的评价方法
(a)Krebs液的配制(mmol/L):NaCl 119,KCl 4.7,CaCl2 2.5,MgSO4 1.2,KH2PO41.2,NaHCO3 25,Glucose 11.1,pH 7.35-7.45;
(b)主动脉分离:主动脉放入装有Krebs液的盘中,在解剖显微镜下仔细分离;
(c)血管环的固定:在解剖显微镜下取3mm长的主动脉环用25μm 的钢丝固定在浴槽中,调节温度使水温保持37℃;
(d)平衡:最初调节张力时逐渐调至1mN,在浴槽中平衡30min;
(e)KCl预激实验:加入浓度为60mmol/L的KCl,待达到反应平台后,用新鲜的Krebs液抽洗3次;
(f)用1μmol/L phenylephrine收缩,然后在池子里加不同药物不同浓度的acetylcholine和sodium nitroprusside(SNP)刺激,测得各值与KCl刺激后所测值相比,分别测定内皮依赖和内皮非依赖血管舒张功能。
10)试验结果
(a)AAV-FGF21给药降低血管紧张素II的升血压作用
血管紧张素II能够诱导小鼠血压的升高。在实施例中,观察血管紧张素II对FGF21KO小鼠和野生型WT小鼠的升血压作用,结果如图3所示,血管紧张素II对野生型WT小鼠的升血压作用要显著低于FGF21KO小鼠。该结果提示FGF21缺失后,小鼠更容易接受血管紧张素II的刺激,血压升高的幅度更大。接下来,为明确FGF21确实能够抑制血管紧张素II的升血压作用,观察给予FGF21KO小鼠AAV-FGF21及血管紧张素II刺激后,小鼠血压的变化。结果如图4所示,FGF21的过表达能够显著的抑制血管紧张素II的升血压作用,FGF KO小鼠在实验前后的血压能够与对照小鼠的血压无显著性差异,其中,图4中AAV-CFP为空载体病毒对照。
(b)AAV-FGF21给药抑制血管紧张素II引发的血管增厚作用
在实施例中,观察给予AAV-FGF21对血管紧张素II引发的血管增厚的影响,结果如图5a、图5b、图5c和图5d所示:图5a为正常小鼠主动脉的形态图,图5b表明血管紧张素II能够显著地增加血管厚度,引发血管的损伤,图5c表明AAV-FGF21给药能够显著地抑制血管紧张素II引发的FGF21 KO小鼠的血管的增厚。对血管厚度的测定结果也表明AAV-FGF21给药能够抵抗血管紧张素II引发的血管壁的增厚(图5d)。
(c)AAV-FGF21给药抑制血管紧张素II引发的血管纤维化作用
在实施例中,观察给予AAV-FGF21对血管紧张素II引发的血管纤维化的影响,结果如图6a、图6b、图6c和图6d所示:Masson染色结果显示血管紧张素II能够显著地增加血管胶原的堆积,增加血管纤维化,并引发血管的损伤,AAV-FGF21给药能够显著地抑制血管紧张素II引发的FGF21KO 小鼠的血管胶原堆积纤维化。此外,定量荧光PCR的结果也显示(图7),AAV-FGF21给药能够显著地降低血管紧张素II引发的血管纤维化因子的表达,如CollagenI、Collagen III和Fibronectin等纤维化因子基因的表达,从而降低血管的纤维化。
(d)AAV-FGF21给药抑制血管紧张素II引发的血管巨噬细胞浸润和炎症作用
血管紧张素II能够显著地增加血管巨噬细胞浸润,引发血管的炎症反应并进一步诱发血管的损伤。在实施例中,观察给予AAV-FGF21对血管紧张素II引发的血管巨噬细胞浸润的影响。图8a-c及g-i表明AAV-FGF21给药能够减少血管紧张素II引发的血管CD45+巨噬细胞的浸润,图8d-f表明 AAV-FGF21给药能够减少血管紧张素II引发的血管CD11+巨噬细胞的浸润结果如图8所示:血管紧张素II能够增加FGF21KO小鼠血管CD45+和 CD11+等类型的巨噬细胞的浸润,而AAV-FGF21给药能够显著地抑制血管紧张素II引发的FGF21KO小鼠的血管巨噬细胞的浸润。此外,定量荧光 PCR的结果也显示(图9),AAV-FGF21给药能够显著地降低血管紧张素II 引发的血管炎症因子的表达,如IL-1β、IL-6、TNF-α、MCP-1、VCAM-1 等炎症因子基因的表达,从而降低血管的炎症。
(e)AAV-FGF21给药抑制血管紧张素II引发的血管氧化损伤
血管紧张素II能够显著地增加血管的氧化损伤,增加氧自由基的堆积,引发血管的损伤。在实施例中,观察给予AAV-FGF21对血管紧张素II引发的血管氧化损伤的影响。结果如图10所示:正常小鼠血管过氧化物含堆积很少(图10a),给予了血管紧张素II的小鼠血管在内膜(E)、中膜(M) 和外膜(A)都堆积了大量的活性氧(图10b);AAV-FGF21给药能够显著地抑制血管紧张素II引发的FGF21KO小鼠的血管活性氧的堆积(图10c)。定量结果也表明AAV-FGF21能够显著地减少血管紧张素II引起的血管活性氧的堆积(图10d)。
(f)AAV-FGF21给药保护血管紧张素II引发的血管功能的损伤
血管紧张素II能够引发血管功能损伤。在实施例中,观察给予 AAV-FGF21对血管紧张素II引发的血管功能的影响。结果如图11和图12所示:血管紧张素II能够引起内皮依赖性血管舒张功能的显著性改变,但是对内皮非依赖性血管舒张功能没有显著的改变;AAV-FGF21给药能够显著地抑制血管紧张素II引发的FGF21KO小鼠的内皮依赖性血管舒张功能的变化,但是对FGF KO小鼠的内皮非依赖性血管舒张功能没有显著的影响。
尽管已用具体实施例来说明和描述了本发明,然而应意识到,在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此,这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
SEQUENCE LISTING
<110> 暨南大学
<120> FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管病损伤的药物中的应用
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 208
<212> PRT
<213> 人属人
<400> 1
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1 5 10 15
Val Leu Ala Gly Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp
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Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu
35 40 45
Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu
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Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu
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Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys
85 90 95
Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser
100 105 110
Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu
115 120 125
Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His
130 135 140
Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro
145 150 155 160
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro
165 170 175
Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro
180 185 190
Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
195 200 205
<210> 2
<211> 210
<212> PRT
<213> 鼠属小家鼠
<400> 2
Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly Leu Trp Val Arg
1 5 10 15
Leu Leu Leu Ala Val Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile
20 25 30
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg
35 40 45
Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile
50 55 60
Arg Glu Asp Gly Thr Val Val Gly Ala Ala His Arg Ser Pro Glu Ser
65 70 75 80
Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly
85 90 95
Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr
100 105 110
Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu
115 120 125
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro
130 135 140
Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp
145 150 155 160
Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln
165 170 175
Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser
180 185 190
Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr
195 200 205
Ala Ser
210
<210> 3
<211> 633
<212> DNA
<213> 鼠属小家鼠
<400> 3
atggaatgga tgagatctag agttgggacc ctgggactgt gggtccgact gctgctggct 60
gtcttcctgc tgggggtcta ccaagcatac cccatccctg actccagccc cctcctccag 120
tttgggggtc aagtccggca gaggtacctc tacacagatg acgaccaaga cactgaagcc 180
cacctggaga tcagggagga tggaacagtg gtaggcgcag cacaccgcag tccagaaagt 240
ctcctggagc tcaaagcctt gaagccaggg gtcattcaaa tcctgggtgt caaagcctct 300
aggtttcttt gccaacagcc agatggagct ctctatggat cgcctcactt tgatcctgag 360
gcctgcagct tcagagaact gctgctggag gacggttaca atgtgtacca gtctgaagcc 420
catggcctgc ccctgcgtct gcctcagaag gactccccaa accaggatgc aacatcctgg 480
ggacctgtgc gcttcctgcc catgccaggc ctgctccacg agccccaaga ccaagcagga 540
ttcctgcccc cagagccccc agatgtgggc tcctctgacc ccctgagcat ggtagagcct 600
ttacagggcc gaagccccag ctatgcgtcc tga 633
<210> 4
<211> 40
<212> DNA
<213> 人工序列
<400> 4
attgaattcg ccaccatgga atggatgaga tctagagttg 40
<210> 5
<211> 34
<212> DNA
<213> 人工序列
<400> 5
agaggatcct caggacgcat agctggggct tcgg 34
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<400> 6
cctcacattg ccaaaagacg 20
<210> 7
<211> 21
<212> DNA
<213> 人工序列
<400> 7
aggtcggtgt gaacggattt g 21
<210> 8
<211> 23
<212> DNA
<213> 人工序列
<400> 8
tgtagaccat gtagttgagg tca 23
<210> 9
<211> 20
<212> DNA
<213> 人工序列
<400> 9
tgtgacaact gccgtagacc 20
<210> 10
<211> 21
<212> DNA
<213> 人工序列
<400> 10
gaccaactgt caccattgag g 21
<210> 11
<211> 22
<212> DNA
<213> 人工序列
<400> 11
gcaactgttc ctgaactcaa ct 22
<210> 12
<211> 21
<212> DNA
<213> 人工序列
<400> 12
atcttttggg gtccgtcaac t 21
<210> 13
<211> 23
<212> DNA
<213> 人工序列
<400> 13
tagtccttcc taccccaatt tcc 23
<210> 14
<211> 21
<212> DNA
<213> 人工序列
<400> 14
ttggtcctta gccactcctt c 21
<210> 15
<211> 19
<212> DNA
<213> 人工序列
<400> 15
gaggccaagc cctggtatg 19
<210> 16
<211> 19
<212> DNA
<213> 人工序列
<400> 16
cgggccgatt gatctcagc 19
<210> 17
<211> 23
<212> DNA
<213> 人工序列
<400> 17
ttaaaaacct ggatcggaac caa 23
<210> 18
<211> 23
<212> DNA
<213> 人工序列
<400> 18
gcattagctt cagatttacg ggt 23
<210> 19
<211> 19
<212> DNA
<213> 人工序列
<400> 19
gctcctctta ggggccact 19
<210> 20
<211> 20
<212> DNA
<213> 人工序列
<400> 20
attggggacc cttaggccat 20
<210> 21
<211> 23
<212> DNA
<213> 人工序列
<400> 21
ctgtaacatg gaaactgggg aaa 23
<210> 22
<211> 23
<212> DNA
<213> 人工序列
<400> 22
ccatagctga actgaaaacc acc 23
<210> 23
<211> 21
<212> DNA
<213> 人工序列
<400> 23
agttggggat tcggttgttc t 21
<210> 24
<211> 20
<212> DNA
<213> 人工序列
<400> 24
cccctcattc cttaccaccc 20
Claims (10)
1.FGF21在制备治疗由血管紧张素Ⅱ引发的高血压和/或血管病损伤的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述FGF21具有如SEQ ID NO.1或SEQ IDNO.2所示的氨基酸序列。
3.根据权利要求1或2所述的应用,其特征在于,所述FGF21作用于脂肪细胞,并促进血管紧张素转化酶2的表达,将血管紧张素II转变为血管紧张素-(1-7),抑制高血压和/或逆转血管损伤。
4.一种治疗由血管紧张素Ⅱ引发的高血压和/或血管损伤的药物,其特征在于,所述药物的活性成分包括FGF21。
5.根据权利要求4所述的药物,其特征在于,所述FGF21具有如SEQ ID NO.1或SEQ IDNO.2所示的氨基酸序列。
6.根据权利要求4或5所述的药物,其特征在于,所述FGF21作用于脂肪细胞,并促进血管紧张素转化酶2的表达,将血管紧张素II转变为血管紧张素-(1-7),抑制高血压和/或逆转血管损伤。
7.根据权利要求4或5所述的药物,其特征在于,所述药物还包括药学上可接受的载剂或辅料。
8.根据权利要求4或5所述的药物,其特征在于,所述药物的给药方式为腹腔给药。
9.一种FGF21的制备方法,其特征在于,构建表达FGF21的重组病毒进行表达并纯化收集所述FGF21。
10.根据权利要求9所述的制备方法,其特征在于,所述构建表达FGF21的重组病毒进行表达的方法包括:将小鼠FGF21的编码基因插入到质粒载体pAAV-IRES-ZsGreen后导入HEK293AAV细胞产生高滴度含目的基因的AAV病毒并进行小鼠FGF21蛋白的过表达。
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