CN113481237A - 一种预防和治疗新生血管眼部疾病的基因药物 - Google Patents
一种预防和治疗新生血管眼部疾病的基因药物 Download PDFInfo
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Abstract
一种预防和治疗新生血管眼部疾病的基因药物。本发明涉及基因药物技术领域,具体是一种重组载体,其包含PEDF或PEDF功能片段基因,和靶向人VEGFA的shRNA片段。本发明还提供所述的重组载体在制备预防或治疗血管新生相关的眼部疾病的药物中的应用。本发明涉及的PEDF过表达联合VEGFA干扰腺相关病毒能在血管内皮细胞中干扰VEGFA的表达水平,有效下调VEGFA的蛋白水平,进而抑制血管内皮细胞的增殖和迁移活力,抑制血管内皮细胞的血管生成能力,为湿性黄斑病变的治疗提供新的靶点和途径。
Description
技术领域
本发明涉及基因药物技术领域,具体地说,是一种预防或治疗血管新生相关的眼部疾病的基因药物。
背景技术
血管新生疾病主要由促血管生成与抗血管生成因子的失衡引起。如生物力,高氧,血栓,免疫和炎症反应及遗传突变等刺激因素都会引起血管生成的异常。眼内血管增生可发生在视网膜,脉络膜和角膜等位置,不受抑制的血管新生可以导致严重的视觉损伤。新生的血管具有分泌性,期分泌物积累最终导致视网膜功能损伤。此外,血管的异常生长还可以导致组织结构异常和角膜透明性受损。与新生血管相关的眼科疾病包括,湿性黄斑病变,糖尿病性视网膜症,早产儿视网膜病变和角膜血管新生,视网膜血管阻塞以及新生血管性青光眼。在各种相关疾病中,湿性黄斑病变和糖尿病性视网膜症是造成全球视觉疾病的主要因素。
在新生血管疾病中,公认最重要的功能蛋白为VEGF(血管内皮生长因子)。其功能包括调控细胞增殖,生存,运动和血管通透性。VEGF可以通过分泌到胞外或者结合细胞外基质的方式与血管内皮细胞上的VEGFR2结合,激活下游通路包括受体磷酸激酶。VEGFR2下游激活的通路包括PI3K,PLCg,FAK和p38MAPK。之后PI3K激活AKT and Rac导致凋亡抑制并减少细胞黏附,增加了血管通透性。PLCg激活MAPK级联效应导致细胞增值及细胞骨架重组。而FAK和p38MAPK公认与内皮细胞黏附和迁移有关。这些由VEGF引发的效应是血管生成中内皮细胞迁移,微管形成以及出芽等过程的主要功能组成部分。临床研究表明VEGF表达异常增高在湿性黄斑病变,糖尿病视网膜病等眼科疾病中都有存在,而且玻璃体腔注射抗VEGF药物可以有效治疗这些疾病。但是这些治疗方法存在需要多次注射,且容易产生耐受和副作用的问题。
基因治疗特别是以腺相关病毒为载体的基因治疗技术在新生血管疾病领域经过近二十年的发展取得了一定成果。腺相关病毒(Adeno-associated virus,AAV)是一种复制缺陷的细小病毒,在人群众存在多种天然血清型,并且无致病性报道。因此腺相关病毒是目前公认的安全的基因递送载体。2017年美国Spark公司的AAV-RPE65基因治疗获得临床治疗许可成为AAV基因治疗眼科疾病应用的里程碑。目前在新生血管领域,以AAV介导VEGF捕获蛋白如sFlt在眼内的表达以达到中和游离VEGF,并产生抑制新生血管的作用。相关疗法已经进入临床II期研究,并有望在未来2~3年内获得临床使用批准。
RNA干扰技术是一种沉默基因表达的技术,两名美国科学家Andrew Z.Fire和Craig C.Mello因对此项技术的贡献于2006年被授予诺贝尔生理学奖。RNA干扰技术的原理是较长双链RNA被特异核酸酶Dicer切割加工成21-23nt的由正义和反义链组成的小干扰RNA。小干扰RNA随后形成沉默复合体(RNA-induced silencing complex,RISC)解旋成单链。反义链引导该沉默复合体通过碱基配对特异性结合到目标mRNA上,使mRNA分解。小发夹RNA(short hairpin RNA,shRNA)是一种形成急转弯结构的RNA序列,可以经由RNA干扰使基因沉默。
色素内皮源因子(pigment epithelium-derived factor,PEDF),被认为是与VEGF共同调节血管增殖和通透性的分子。两者之间的动态平衡对血管保持正常的结构和功能具有重要作用。曾有基因治疗研究利用腺病毒载体递送PEDF进行玻璃体腔注射治疗湿性黄斑病变及糖尿病视网膜新生血管,并取得了一定效果。但由于其使用腺病毒载体,因此受试者容易出现感染和炎症等症状,因此难以获得进一步的应用(Mori K,Gehlbach P,YamamotoS,Duh E,Zack DJ,Li Q,Berns KI,Raisler BJ,Hauswirth WW,Campochiaro PA.AAV-mediated gene transfer of pigment epithelium-derived factor inhibitschoroidal neovascularization.Invest Ophthalmol Vis Sci.2002Jun;43(6):1994-2000;Haurigot V,Villacampa P,Ribera A,Bosch A,Ramos D,Ruberte J,Bosch F.Long-Term Retinal PEDF Overexpression Prevents Neovascularization in a MurineAdult Model of Retinopathy.PLoS One.2012;7(7):e41511.doi:10.1371/journal.pone.0041511.Epub 2012Jul 20.Askou AL,Alsing S,Benckendorff JNE,Holmgaard A,Mikkelsen JG,Aagaard L,Bek T,Corydon TJ.Suppression of ChoroidalNeovascularization by AAV-Based Dual-Acting Antiangiogenic Gene Therapy MolTher Nucleic Acids.2019Jun 7;16:38-50.doi:10.1016/j.omtn.2019.01.012.Epub2019Feb 2.)。
但是关于一种从VEGF和PEDF协同性出发,治疗新生血管性眼病的基因药物目前还未见报道。
发明内容
本发明的目的在于提供一种包含PEDF或PEDF功能片段基因,和靶向人VEGFA的shRNA片段的重组载体,本发明的另一目的在于提供所述的重组载体在制备预防或治疗血管新生相关的眼部疾病药物中的应用。本发明从VEGF和PEDF协同性出发,治疗新生血管性眼病,具有重要的临床意义。
为了实现上述目的,本发明采用以下技术方案:
1.PEDF过表达和VEGFA干扰质粒的制备;
2.腺相关病毒颗粒(AAV)的包装;
3.血管内皮细胞感染不同组AAV后VEGFA的表达检测;
4.血管内皮细胞感染不同组AAV后增殖活力的检测;
5.血管内皮细胞感染不同组AAV后迁移活力的检测;
6.血管内皮细胞感染不同组AAV后成管活力的检测;
7.小鼠注射不同组AAV后血管生成的检测。
本发明的第一方面,提供一种重组载体,其包含PEDF或PEDF功能片段基因,和靶向人VEGFA的shRNA片段。
进一步的,所述的重组载体可选自质粒、慢病毒载体、腺相关病毒载体。
更进一步的,所述的重组载体为包含PEDF或PEDF功能片段基因,靶向人VEGFA的shRNA片段,和腺相关病毒外壳的重组病毒载体。
在本发明的一个优选实施方式中,所述的重组载体为VEGFA干扰联合PEDF过表达的双表达框载体pAAVE2099-PEDF-shVEGFA-9(全序列如SEQ ID NO.32所示);其中PEDF序列如SEQ ID NO.1所示,shVEGFA-9序列的正义链和反义链分别如SEQ ID NO.2和SEQ ID NO.3所示。所述的shVEGFA-9的正义链和反义链转录DNA序列分别如SEQ ID NO.4和SEQ ID NO.5所示。
shVEGFA-9序列正义链:
5'-AAUUCGCCAGCACAUAGGAGAGAUGACUCGAGUCAUCUCUCCUAUGUGCUGGCUUUUUA-3'(SEQID NO.2)。
shVEGFA-9序列反义链:
5'-CGCGUAAAAAGCCAGCACAUAGGAGAGAUGACUCGAGUCAUCUCUCCUAUGUGCUGGCG-3'(SEQID NO.3)。
shVEGFA-9序列正义链转录DNA序列:
5'-AATTCGCCAGCACATAGGAGAGATGACTCGAGTCATCTCTCCTATGTGCTGGCTTTTTA-3'(SEQID NO.4)。
shVEGFA-9序列反义链转录DNA序列:
5'-CGCGTAAAAAGCCAGCACATAGGAGAGATGACTCGAGTCATCTCTCCTATGTGCTGGCG-3'(SEQID NO.5)。
在本发明的一个优选实施方式中,所述的重组载体为包含如SEQ ID NO.1所示的PEDF基因,如SEQ ID NO.2和SEQ ID NO.3所示的靶向人VEGFA的shRNA片段,和腺相关病毒外壳的重组病毒载体。
本发明的第二方面,提供一种如上所述的重组载体在制备预防或治疗血管新生相关的眼部疾病的药物中的应用。
进一步的,所述的血管新生相关的眼部疾病为角膜、脉络膜血管新生相关的眼部疾病。
更进一步的,所述的血管新生相关的眼部疾病为湿性黄斑病变、年龄相关黄斑变性、糖尿病视网膜病变等。
本发明的第三方面,提供一种靶向人VEGFA基因的shRNA,所述的shRNA为shVEGFA-9或shVEGFA-1,其核苷酸序列分别如下所示:
shVEGFA-9序列正义链:
5'-AAUUCGCCAGCACAUAGGAGAGAUGACUCGAGUCAUCUCUCCUAUGUGCUGGCUUUUUA-3'(SEQID NO.2);
shVEGFA-9序列反义链:
5'-CGCGUAAAAAGCCAGCACAUAGGAGAGAUGACUCGAGUCAUCUCUCCUAUGUGCUGGCG-3'(SEQID NO.3);
shVEGFA-1序列正义链:
5'-AAUUCGCAGAUUAUGCGGAUCAAACCCUCGAGGGUUUGAUCCGCAUAAUCUGCUUUUUA-3'(SEQID NO.6);
shVEGFA-1序列反义链:
5'-CGCGUAAAAAGCAGAUUAUGCGGAUCAAACCCUCGAGGGUUUGAUCCGCAUAAUCUGCG-3'(SEQID NO.7)。
本发明还提供了一种如上所述的shRNA的转录DNA序列,其核苷酸序列分别如下所示:
shVEGFA-9序列正义链转录DNA序列:
5'-AATTCGCCAGCACATAGGAGAGATGACTCGAGTCATCTCTCCTATGTGCTGGCTTTTTA-3'(SEQID NO.4);
shVEGFA-9序列反义链转录DNA序列:
5'-CGCGTAAAAAGCCAGCACATAGGAGAGATGACTCGAGTCATCTCTCCTATGTGCTGGCG-3'(SEQID NO.5);
shVEGFA-1序列正义链转录DNA序列:
5'-AATTCGCAGATTATGCGGATCAAACCCTCGAGGGTTTGATCCGCATAATCTGCTTTTTA-3'(SEQID NO.8);
shVEGFA-1序列反义链转录DNA序列:
5'-CGCGTAAAAAGCAGATTATGCGGATCAAACCCTCGAGGGTTTGATCCGCATAATCTGCG-3'(SEQID NO.9)。
本发明的第四方面,提供一种重组载体,含有如上所述的靶向人VEGFA基因的shRNA或所述shRNA的转录DNA序列。
本发明的第五方面,提供一种如上所述的shRNA或转录DNA序列在制备VEGFA基因表达抑制剂中的应用。
本发明的第六方面,提供一种如上所述的shRNA或转录DNA序列在制备预防或治疗血管新生相关的眼部疾病的药物中的应用。
进一步的,所述的血管新生相关的眼部疾病为湿性黄斑病变。
本发明的第七方面,提供一种基因治疗药物,其包含如上所述的重组载体,或靶向人VEGFA基因的shRNA,或转录DNA。
进一步的,所述的基因药物的给药方式为角膜、视网膜下或玻璃体分腔注射重组载体。
本发明优点在于:
本发明经实验证明在湿性黄斑病变进程中,VEGFA呈现较高的表达水平,调节VEGFA的表达能影响湿性黄斑病变进程中血管内皮细胞的增殖及眼底血管生成。
本发明设计靶向VEGFA基因的shRNA载体,特异性抑制VEGFA基因的表达,从而抑制湿性黄斑病变进程中血管内皮细胞的增殖和眼底血管增生。
本发明结合VEGFA分子在湿性黄斑病变进程中的功能,利用RNA干扰技术开发新的治疗药物。
本发明提供用于预防或治疗血管新生相关的眼部疾病的包含PEDF或其功能片段基因的重组载体表达单元和靶向人VEGFA的shRNA片段的合适的腺相关病毒外壳的重组病毒载体,其通过角膜、玻璃体腔或视网膜下注射可预防或治疗血管新生相关的眼部疾病,如角膜新生血管、年龄相关黄斑变性和糖尿病视网膜病变等。
本发明涉及的PEDF过表达联合VEGFA干扰AAV能在血管内皮细胞中干扰VEGFA的表达水平,有效下调VEGFA的蛋白水平,进而抑制血管内皮细胞的增殖和迁移活力,抑制血管内皮细胞的血管生成能力,为湿性黄斑病变的治疗提供新的靶点和途径。
附图说明
图1.QPCR实验检测HUVEC细胞感染各组AAV后VEGFA的mRNA水平;
图2.Western blot实验检测HUVEC细胞感染各组AAV后VEGFA蛋白水平;
图3.CCK8实验检测HUVEC细胞感染各组AAV后的增殖活力;
图4.Transwell实验检测HUVEC细胞感染各组AAV后的迁移活力;
图5.血管生成实验检测HUVEC细胞感染各组AAV后的成管活力;
图6.小鼠感染各组AAV后眼球血管分布;
图7.pAAVE2099-PEDF-shVEGFA-9载体图谱。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。以下实施方式只是较佳的实施方式中的一种,并非对本发明的限制。其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。未作特殊说明的实验试剂和方法,均指常规试剂和方法。
本发明所用试剂和原料均市售可得或可按文献方法制备。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人《分子克隆:实验室指南》(NewYork:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照常规条件,或按照制造厂商所建议的条件。
实施例1:VEGFA shRNA序列设计与PEDF过表达腺相关病毒质粒构建
(1)VEGFA干扰载体构建
首先在NCBI上搜索人源VEGFA(NM_001025366.3)序列信息,然后使用在线设计软件http://rnaidesigner.thermofisher.com/rnaiexpress/design.do预测VEGFA的shRNA干扰序列。在序列中间加入酶切位点XhoI对应的序列作为发卡结构,按照“EcoRI酶切位点粘性末端-正义链-XhoI-反义链-MluI酶切位点粘性末端进行组装。合成,并通过退火方式,将其构建到质粒pAAVE2099(经EcoRI和MluI酶切)中,通过酶切验证、测序成功,最终获得pAAVE2099-shVEGFA-1、2、3、4、5、6、7、8、9、10、11、12合计12个VEGFA干扰载体。
shVEGFA-1到12的转录DNA序列如下:
shVEGFA-9序列正义链转录DNA序列:
5'-AATTCGCCAGCACATAGGAGAGATGACTCGAGTCATCTCTCCTATGTGCTGGCTTTTTA-3'(SEQID NO.4)
shVEGFA-9序列反义链转录DNA序列:
5'-CGCGTAAAAAGCCAGCACATAGGAGAGATGACTCGAGTCATCTCTCCTATGTGCTGGCG-3'(SEQID NO.5)
shVEGFA-1序列正义链转录DNA序列:
5'-AATTCGCAGATTATGCGGATCAAACCCTCGAGGGTTTGATCCGCATAATCTGCTTTTTA-3'(SEQID NO.8)
shVEGFA-1序列反义链转录DNA序列:
5'-CGCGTAAAAAGCAGATTATGCGGATCAAACCCTCGAGGGTTTGATCCGCATAATCTGCG-3'(SEQID NO.9)
shVEGFA-2序列正义链转录DNA序列:
5'-AATTCGCAAGAAATCCCGGTATAAGTCTCGAGACTTATACCGGGATTTCTTGCTTTTTA-3'(SEQID NO.10)
shVEGFA-2序列反义链转录DNA序列:
5'-CGCGTAAAAAGCAAGAAATCCCGGTATAAGTCTCGAGACTTATACCGGGATTTCTTGCG-3'(SEQID NO.11)
shVEGFA-3序列正义链转录DNA序列:
5'-AATTCGCTCTCTCTTGCTCTCTTATTCTCGAGAATAAGAGAGCAAGAGAGAGCTTTTTA-3'(SEQID NO.12)
shVEGFA-3序列反义链转录DNA序列:
5'-CGCGTAAAAAGCTCTCTCTTGCTCTCTTATTCTCGAGAATAAGAGAGCAAGAGAGAGCG-3'(SEQID NO.13)
shVEGFA-4序列正义链转录DNA序列:
5'-AATTCAGGGCAGAATCATCACGAAGTCTCGAGACTTCGTGATGATTCTGCCCTTTTTTA-3'(SEQID NO.14)
shVEGFA-4序列反义链转录DNA序列:
5'-CGCGTAAAAAAGGGCAGAATCATCACGAAGTCTCGAGACTTCGTGATGATTCTGCCCTG-3'(SEQID NO.15)
shVEGFA-5序列正义链转录DNA序列:
5'-AATTCGACGTGTAAATGTTCCTGCAACTCGAGTTGCAGGAACATTTACACGTCTTTTTA-3'(SEQID NO.16)
shVEGFA-5序列反义链转录DNA序列:
5'-CGCGTAAAAAGACGTGTAAATGTTCCTGCAACTCGAGTTGCAGGAACATTTACACGTCG-3'(SEQID NO.17)
shVEGFA-6序列正义链转录DNA序列:
5'-AATTCATGCGGATCAAACCTCACCAACTCGAGTTGGTGAGGTTTGATCCGCATTTTTTA-3'(SEQID NO.18)
shVEGFA-6序列反义链转录DNA序列:
5'-CGCGTAAAAAATGCGGATCAAACCTCACCAACTCGAGTTGGTGAGGTTTGATCCGCATG-3'(SEQID NO.19)
shVEGFA-7序列正义链转录DNA序列:
5'-AATTCGGTGCTGGAATTTGATATTCACTCGAGTGAATATCAAATTCCAGCACCTTTTTA-3'(SEQID NO.20)
shVEGFA-7序列反义链转录DNA序列:
5'-CGCGTAAAAAGGTGCTGGAATTTGATATTCACTCGAGTGAATATCAAATTCCAGCACCG-3'(SEQID NO.21)
shVEGFA-8序列正义链转录DNA序列:
5'-AATTCGAGTACATCTTCAAGCCATCCCTCGAGGGATGGCTTGAAGATGTACTCTTTTTA-3'(SEQID NO.22)
shVEGFA-8序列反义链转录DNA序列:
5'-CGCGTAAAAAGAGTACATCTTCAAGCCATCCCTCGAGGGATGGCTTGAAGATGTACTCG-3'(SEQID NO.23)
shVEGFA-10序列正义链转录DNA序列:
5'-AATTCGCATTTGTTTGTACAAGATCCCTCGAGGGATCTTGTACAAACAAATGCTTTTTA-3'(SEQID NO.24)
shVEGFA-10序列反义链转录DNA序列:
5'-CGCGTAAAAAGCATTTGTTTGTACAAGATCCCTCGAGGGATCTTGTACAAACAAATGCG-3'(SEQID NO.25)
shVEGFA-11序列正义链转录DNA序列:
5'-AATTCGCGAGGCAGCTTGAGTTAAACCTCGAGGTTTAACTCAAGCTGCCTCGCTTTTTA-3'(SEQID NO.26)
shVEGFA-11序列反义链转录DNA序列:
5'-CGCGTAAAAAGCGAGGCAGCTTGAGTTAAACCTCGAGGTTTAACTCAAGCTGCCTCGCG-3'(SEQID NO.27)
shVEGFA-12序列正义链转录DNA序列:
5'-AATTCGAGTTAAACGAACGTACTTGCCTCGAGGCAAGTACGTTCGTTTAACTCTTTTTA-3'(SEQID NO.28)
shVEGFA-12序列反义链转录DNA序列:
5'-CGCGTAAAAAGAGTTAAACGAACGTACTTGCCTCGAGGCAAGTACGTTCGTTTAACTCG-3'(SEQID NO.29)。
(2)PEDF过表达载体构建
从NCBI中查找人源PEDF(NM_002615.7)序列信息,找到相应CDs区域,设计引物。
F:ttttggcaaagaattggatccATGCAGGCCC TGGTGCTAC(SEQ ID NO.30)
R:aggttgattatcgataagcttTTAGGGGCCCCTGGGGTC(SEQ ID NO.31)。
将其构建到pAAVE2099(经BamHI和HindIII酶切)中,经酶切验证、测序成功后,最终获得PEDF过表达载体pAAVE2099-PEDF。
PEDF的DNA序列如SEQ ID NO.1所示。
(3)VEGFA干扰联合PEDF过表达的双表达框载体构建
将干扰效果最佳的shVEGFA-9序列经退火方式,构建到pAAVE2099-PEDF(经EcoRI和MluI酶切)中,通过酶切验证、测序成功后,最终获得VEGFA干扰联合PEDF过表达的双表达框载体pAAVE2099-PEDF-shVEGFA-9。
pAAVE2099-PEDF-shVEGFA-9全序列如SEQ ID NO.32所示。
pAAVE2099-PEDF-shVEGFA-9载体图谱如图7所示。
PEDF的DNA序列如SEQ ID NO.1所示;shVEGFA-9DNA序列如SEQ ID NO.4和SEQ IDNO.5所示。将pAAVE2099-shVEGFA-1、2、3、4、5、6、7、8、9、10、11、12分别转染到HUVEC细胞,经QPCR筛选发现pAAVE2099-shVEGFA-1和pAAVE2099-shVEGFA-9可显著干扰VEGFA的表达,其中pAAVE2099-shVEGFA-9干扰效果最佳,所以选取shVEGFA-9与过表达PEDF联用。
实施例2:腺相关病毒颗粒的包装
在37℃5%CO2的细胞培养箱中培养293T细胞,培养基使用添加了10%胎牛血清的DMEM培养基。传代培养293T细胞于直径为10cm的培养皿中,24h后(细胞处于代数生长期),挑选细胞汇合度为50%左右的培养皿,换成含2%胎牛血清的DMEM培养基培养细胞。
准备好目的质粒(pAAVE2099、pAAVE2099-shVEGFA-9、pAAVE2099-PEDF、AAV-PEDF-shVEGFA-9)、包装质粒(pHelper、pAAV-RC2)与脂质体转染试剂的转染混合物。把混合物添加到已经换液的细胞中进行转染,12小时后更换新鲜含5%胎牛血清的DMEM培养基。转染72h后收集细胞。加入PBS重悬细胞沉淀,-80℃/37℃反复冻融3次,4000rpm/min离心20min,上清经0.22μm孔径的膜过滤后,加入50U/ml的benzonase,37℃孵育1小时,4000rpm/min离心20min收集上清,上清液经层析纯化后得到高纯度腺相关病毒(AAV-NC、AAV-shVEGFA-9、AAV-PEDF、AAV-PEDF-shVEGFA-9)。
腺相关病毒的滴度测定:取5ul纯化好的腺相关病毒进行梯度稀释,取稀释好的病毒用QPCR方法进行定量分析。
实施例3:VEGFA mRNA水平的检测
HUVEC细胞分为四组(AAV-NC组、AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组)。各组细胞分别感染相应腺相关病毒(感染复数为10),继续培养4d。采用Trizol法提取细胞总RNA,并使用随机引物将RNA反转录为cDNA。qPCR反应体系包括2×SYBRGreen Mix 5μL、上下游引物(5pmol/mL)各0.2μL、cDNA模板0.2μL、ddH2O补充至10μL。反应程序为[95℃2min;(95℃15s,60℃30s,72℃30s)×40循环;熔解曲线]。以GAPDH作为内参,使用2-△△CT法计算相对表达量。实验涉及的引物序列如下:
VEGF-F:5'-AGGGCAGAATCATCACGAAGT-3'(SEQ ID NO.33);
VEGF-R:5'-AGGGTCTCGATTGGATGGCA-3'(SEQ ID NO.34);
GAPDH-F:5'-ACAGCCTCAAGATCATCAGC-3'(SEQ ID NO.35);
GAPDH-R:5'-GGTCATGAGTCCTTCCACGAT-3'(SEQ ID NO.36)。
QPCR检测结果如图1所示:相比较于AAV-NC组,AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组细胞中VEGFA的mRNA水平显著降低,且AAV-PEDF-shVEGFA-9组细胞中VEGFA的mRNA水平最低,说明本发明中的shVEGFA-9与PEDF可协同降低VEGFA的mRNA水平。
上述的shRNA序列,包括正义链和反义链:
shVEGFA-9序列正义链:
5'-AAUUCGCCAGCACAUAGGAGAGAUGACUCGAGUCAUCUCUCCUAUGUGCUGGCUUUUUA-3'(SEQID NO.2);
shVEGFA-9序列反义链:
5'-CGCGUAAAAAGCCAGCACAUAGGAGAGAUGACUCGAGUCAUCUCUCCUAUGUGCUGGCG-3'(SEQID NO.3)。
实施例4:VEGFA蛋白水平的检测
HUVEC细胞分为四组(AAV-NC组、AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组)。各组细胞分别感染相应腺相关病毒(感染复数为10),继续培养4d。使用含有苯甲基磺酰氟(PMSF)和蛋白酶抑制剂cocktail的RIPA细胞裂解液收集细胞蛋白样品,二喹啉甲酸(CBA)法测定蛋白浓度。将蛋白样品和等体积的2×蛋白上样缓冲液混合煮沸5min,使蛋白质充分变性。配制十二烷基硫酸钠聚丙烯酰胺(SDS-PAGE)凝胶,每孔上样30μg蛋白样品,以100V恒压电泳约1h,直至溴酚蓝指示剂电泳至距离凝胶底部1-2cm处。随后以0.2A恒流转膜约30min,将蛋白转移至聚偏氟乙烯(PVDF)膜上。使用5%脱脂奶粉溶液室温封闭30min,随后使用稀释的一抗4℃过夜孵育。次日使用稀释的二抗室温孵育30min,使用电化学发光(Electrochemiluminescence,ECL)显影液显影,用化学发光仪呈像。使用Image J软件计算测量蛋白条带灰度值,以GAPDH为内参,计算蛋白相对表达量。
蛋白表达检测结果如图2所示:相比较于AAV-NC组,AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组细胞中VEGFA的蛋白水平显著降低,且AAV-PEDF-shVEGFA-9组细胞中VEGFA的蛋白水平最低,说明本发明中的shVEGFA-9与PEDF可协同降低VEGFA的蛋白水平。
实施例5:CCK8检测HUVEC细胞的增殖活力
HUVEC细胞分为四组(AAV-NC组、AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组)。各组细胞分别感染相应腺相关病毒(感染复数为10),继续培养4d。使用胰蛋白酶将细胞消化为单细胞悬液,重新接种于96孔培养板中,5×103细胞/孔,分别于24h和48h后检测。检测时每孔更换新鲜培养基,并加入10μL的CCK8试剂,混匀后继续培养3h。使用多功能酶标仪测定450nm波长下各孔OD值。
细胞增殖活力检测结果如图3所示:相比较于AAV-NC组,AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组HUVEC细胞增殖活力显著降低,且AAV-PEDF-shVEGFA-9组HUVEC细胞增殖活力最低,说明本发明中的shVEGFA-9与PEDF可协同降低HUVEC细胞增殖活力。
实施例6:血管内皮细胞感染不同组AAV后迁移活力的检测
HUVEC细胞分为四组(AAV-NC组、AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组)。各组细胞分别感染相应腺相关病毒(感染复数为10),继续培养4d。使用胰蛋白酶将细胞消化,用无血清培养基重悬为单细胞悬液,调整细胞密度为5×104细胞/mL。取Transwell小室置于24孔细胞培养板中。下室加入完全培养基,上室中接种200μL细胞悬液,继续培养24h。擦去上室中未迁移细胞,4%多聚甲醛固定膜上迁移细胞,0.1%龙胆紫溶液染色。在显微镜下观察并采集图像,利用Image J软件对迁移细胞计数。
细胞迁移检测结果如图4所示:相比较于AAV-NC组,AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组HUVEC细胞迁移活力显著降低,且AAV-PEDF-shVEGFA-9组HUVEC细胞迁移活力最低,说明本发明中的shVEGFA-9与PEDF可协同抑制HUVEC细胞迁移活力。
实施例7:血管内皮细胞感染不同组AAV后成管活力的检测
HUVEC细胞分为四组(AAV-NC组、AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组)。各组细胞分别感染相应腺相关病毒(感染复数为10),继续培养4d。使用胰蛋白酶将细胞消化为单细胞悬液,调整细胞密度为2×105细胞/mL。取50μL细胞悬液接种于基质胶包被的96孔培养板中,静置培养3h。在显微镜下观察并采集图像,评价各组细胞的成管能力。
细胞成管实验结果如图5所示:相比较于AAV-NC组,AAV-shVEGFA-9组、AAV-PEDF组、AAV-PEDF-shVEGFA-9组HUVEC细胞成管活力显著降低,且AAV-PEDF-shVEGFA-9组HUVEC细胞成管活力最低,说明本发明中的shVEGFA-9与PEDF可协同抑制HUVEC细胞成管活力。
实施例8:小鼠注射不同组AAV后血管生成的检测
通过碱烧伤来制造角膜新生血管模型。造模前,用裂隙灯检查C57BL/6小鼠眼睛无血管且无损伤。麻醉小鼠,将1.8ul 1M NaOH溶液滴在圆形滤纸片(直径2mm)上使其润湿,用镊子夹住滤纸片并轻轻地将其覆盖在小鼠眼睛的角膜正中央位置(整个处理过程中,要时刻在显微镜下观察滤纸片是否移位或脱落,若出现这种情况,要及时将滤纸片复位至角膜中央,否则将影响造模效果)。持续作用40s后,迅速移走滤纸片,立即用适量PBS冲洗小鼠眼睛。造模结束24h后,用裂隙灯检查小鼠角膜有新生血管出现,此时,对小鼠眼睛进行基因治疗。设置对照组(AAV-NC)和治疗组(AAV-shVEGFA-9、AAV-PEDF、AAV-PEDF-shVEGFA-9),麻醉小鼠后,分别在各组小鼠上、下眼睑一侧角膜缘处注射相同剂量的相应病毒颗粒。在治疗后的第15d进行裂隙灯检查,上眼睑一侧的新生血管如图6所示,各组角膜均无穿孔现象,对照组中新生血管由角膜缘向角膜中央生长侵袭,致密地布满整个角膜;AAV-shVEGFA-9和AAV-PEDF治疗组中新生血管分布较对照组更稀疏;而PEDF联合shVEGFA-9治疗后,与其他三组相比新生血管明显更少,注射点处几乎没有血管,注射点两边的血管长度更短,没有侵袭到角膜中央。表明过表达PEDF或抑制VEGFA-9表达在一定程度上能抑制角膜新生血管,而两者联合作用时,可产生明显的角膜新生血管抑制作用。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
序列表
<110> 厦门朔望医药科技有限公司
<120> 一种预防和治疗新生血管眼部疾病的基因药物
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acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 180
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tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 480
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 540
agatccttga gagttttcgc cccgaagaac gttttccatg atgagcactt ttaaagttct 600
gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 660
acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 720
tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 780
caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 840
gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 900
cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 960
tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 1020
agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 1080
tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 1140
ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 1200
acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1260
ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 1320
gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 1380
gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 1440
ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 1500
gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 1560
tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 1620
cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 1680
cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 1740
ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 1800
tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 1860
cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 1920
ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 1980
aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 2040
ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 2100
tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 2160
gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 2220
gccgattcat taatgcagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc 2280
gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga 2340
gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta 2400
tctacgtagc catgctctag gaagatcctt atcgatgcta gaaaaaattc tccgaacgtg 2460
tcacgtctcg agacgcgtaa gcttaaaaag ccagcacata ggagagatga ctcgagtcat 2520
ctctcctatg tgctggcgaa ttcacgtgac acgttcggag aaccggagtt tcgtcctttc 2580
cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag 2640
tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt 2700
cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattccaat tatctctcta 2760
acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcggtgaag 2820
gggatcgatt ttaccacatt tgtagaggtt ttacttgctt taaaaaacct cccacatctc 2880
cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct 2940
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 3000
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgctcgaag 3060
cggccgcccg ggtcgacggt atcgatgcgg ggaggcggcc caaagggaga tccgactcgt 3120
ctgagggcga aggcgaagac gcggaagagg ccgcagagcc ggcagcaggc cgcgggaagg 3180
aaggtccgct ggattgaggg ccgaagggac gtagcagaag gacgtcccgc gcagaatcca 3240
ggtggcaaca caggcgagca gccaaggaaa ggacgatgat ttccccgaca acaccacgga 3300
attgtcagtg cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg cggcgatgag 3360
ttccgccgtg gcaataggga gggggaaagc gaaagtcccg gaaaggagct gacaggtggt 3420
ggcaatgccc caaccagtgg gggttgcgtc agcaaacaca gtgcacacca cgccacgttg 3480
cctgacaacg ggccacaact cctcataaag agacagcaac caggatttat acaaggagga 3540
gaaaatgaaa gccatacggg aagcaatagc atgatacaaa ggcattaaag cagcgtatcc 3600
acatagcgta aaaggagcaa catagttaag aataccagtc aatctttcac aaattttgta 3660
atccagaggt tgattatcga taagctttta ggggcccctg gggtccagaa tcttgccaat 3720
gaagagaagg gcccctgtgt ctgtgtccct cagtacgaag atgaaaggct ggttaaggtg 3780
atagtccagc gggaaggtga ggtgggcagg ctgcagccct gggctggggg tggttcccgc 3840
cccatcctcg ttccactcaa agccagcccg gtgttccacc tgagtcagct tgatgggttt 3900
gcctgtgatc ttgctaaagt ctggtgaatc aaacaaggat tgcagcttca tctcctgcag 3960
ggacttggtg acttcgcctt cataactcag cttcagcttg gggacagtga ggaccgcctg 4020
cacggtcttc agttctcggt ctatgtcatg aatgaactcg gaggtgaggc tctcctctat 4080
caaggtcaaa ttctgggtca ctttcagggg caggaagaag atgatactca tgcttccggt 4140
caagggcagc tgggcaatct tgcagctgag atctgaatcc aagccatagc gtaaaacagc 4200
cttagggtcc gacatcatgg ggaccctcac ggtcctctct tcatccaagt agaaatcctc 4260
gagggaagtc tttctggagt caaactttgt tacccactgc cccttgaagt gcgccacacc 4320
gagaaggaga atgctgatct catcgggaat ttcctttgtg gacctggcga gcttcccttt 4380
catctgcgcc tgcacccagt tgttgatctc ttgcaggtcc aagcgagggt tgcccgtcag 4440
gactctgggc ctggtcccat atgacttttc cagaggtgcc acaaagctgg attttatgcg 4500
cagcttcttc tcaaagacga tccgggaggc actcttgagg ttcttctggg gggcagtgac 4560
cgtgtcaagg agctccttat aggtaccatg gatgtctggg ctgctgatca agtcatagta 4620
gagagcccgg tgaatgatgg attctgttcg ctgctccgct cccagcgaga gggccgagag 4680
ggccgtggcc acactgagag gagacaggag cacgttggtc gtggggctcg tgctggatcg 4740
cacccggtac aggtcatagc cgaagttgga gacagccgct gccagcttgt tcacggggac 4800
tttgaagaaa ggatcctcct cctccaccag cgcccctgtg ctgtcggggt ctggggagcc 4860
ctcctccggg gggctggcag ggttctggca gctgctgtgc ccgaggaggg ctccaatgca 4920
gaggagtagc accagggcct gcatggatcc aattctttgc caaaatgatg agacagcaca 4980
ataaccagca cgttgcccag gagctgtagg aaaaagaaga aggcatgaac atggttagca 5040
gaggctctag agccgccggt cacacgccag aagccgaacc ccgccctgcc ccgtcccccc 5100
cgaaggcagc cgtccccccg cggacagccc cgagccgctc cccccgcaca aagggccctc 5160
ccggagcccc tcaaggcttt cacgcagcca cagaaaagaa acaagccgtc attaaaccaa 5220
gcgctaatta cagcccggag gagaagggcc gtcccgcccg ctcacctgtg ggagtaacgc 5280
ggtcagtcag agccggggcg ggcggcgcga ggcggcggcg gagcggggca cggggcgaag 5340
gcagcgcgca gcgactcccg cccgccgcgc gcttcgcttt ttatagggcc gccgccgccg 5400
ccgcctcgcc ataaaaggaa actttcggag cgcgccgctc tgattggctg ccgccgcacc 5460
tctccgcctc gccccgcccc gcccctcgcc ccgccccgcc ccgcctggcg cgcgcccccc 5520
cccccccccc gcccccatcg ctgcacaaaa taattaaaaa ataaataaat acaaaattgg 5580
gggtggggag gggggggaga tggggagagt gaagcagaac gtggggctca cctcgaccat 5640
ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat 5700
gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact 5760
tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca 5820
ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt 5880
caatggtact cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta 5940
actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 6000
ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 6060
gcgagcgagc gcgcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 6120
agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg 6180
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 6240
tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 6300
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 6350
<210> 33
<211> 21
<212> DNA
<213> 人工序列(Artificial)
<400> 33
agggcagaat catcacgaag t 21
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial)
<400> 34
agggtctcga ttggatggca 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial)
<400> 35
acagcctcaa gatcatcagc 20
<210> 36
<211> 21
<212> DNA
<213> 人工序列(Artificial)
<400> 36
ggtcatgagt ccttccacga t 21
Claims (12)
1.一种重组载体,其特征在于,其包含PEDF或PEDF功能片段基因,和靶向人VEGFA的shRNA片段。
2.根据权利要求1所述的重组载体,其特征在于,所述的重组载体可选自质粒、慢病毒载体、腺相关病毒载体。
3.根据权利要求1所述的重组载体,其特征在于,所述的重组载体为包含PEDF或PEDF功能片段基因,靶向人VEGFA的shRNA片段,和腺相关病毒外壳的重组病毒载体。
4.一种如权利要求1-3任一所述的重组载体在制备预防或治疗血管新生相关的眼部疾病的药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的血管新生相关的眼部疾病为角膜、脉络膜血管新生相关的眼部疾病。
6.根据权利要求4所述的应用,其特征在于,所述的血管新生相关的眼部疾病为湿性黄斑病变、年龄相关黄斑变性、糖尿病视网膜病变。
7.一种靶向人VEGFA基因的shRNA,所述的shRNA为shVEGFA-9或shVEGFA-1,其中shVEGFA-9的正义链和反义链的核苷酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示,shVEGFA-1的正义链和反义链的核苷酸序列分别如SEQ ID NO.6和SEQ ID NO.7所示。
8.一种如权利要求7所述的shRNA的转录DNA序列,其正义链和反义链的核苷酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.8、SEQ ID NO.9所示。
9.一种重组载体,其含有如权利要求7所述的靶向人VEGFA基因的shRNA或所述shRNA的转录DNA序列。
10.一种如权利要求7或8所述的shRNA或转录DNA序列在制备VEGFA基因表达抑制剂中的应用。
11.一种如权利要求7或8所述的shRNA或转录DNA序列在制备预防或治疗血管新生相关的眼部疾病的药物中的应用。
12.一种基因治疗药物,其包含如权利要求1或3任一所述的重组载体,或如权利要求7所述的靶向人VEGFA基因的shRNA,或如权利要求8所述的转录DNA序列。
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