CN106854660B - 携带人miR486基因的重组腺病毒、制备该病毒的载体及应用 - Google Patents
携带人miR486基因的重组腺病毒、制备该病毒的载体及应用 Download PDFInfo
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Abstract
本发明涉及生物医学领域，具体而言，涉及一种携带人miR486基因的腺病毒载体，并提供了所述载体表达的腺病毒的制备和纯化方法，该腺病毒可用于预防或治疗心肌缺血性相关疾病，对心肌梗塞等疾病有很好的疗效。
Description
技术领域
本发明涉及生物医学领域，具体而言，涉及一种携带人miR486基因的重组腺病毒、制备该病毒的载体及应用。
背景技术
缺血性心脏病已经成为全球范围的最主要死亡原因之一，是影响人类健康的重大疾病。随着医学科学技术不断发展，目前心脏外科手术及血管支架等治疗措施已经大大降低了缺血性心脏病病人的死亡率。另外，一些新型的治疗药物及治疗手段也在尝试应用于临床。如血管生长因子基因治疗、干细胞治疗等能够诱导治疗性血管新生从而达到改善心肌缺血目的。在研究缺血性心脏病病理生理学过程中发现，一些重要调控分子有可能成为防治的新靶点。
miRNAs是一类在转录后基因调控中发挥功能的非编码小RNA。miRNA由高等真核生物基因组编码，通过与其靶基因mRNA 3’非编码区碱基互补序列(UTRs)结合引导沉默复合体(RISC)降解mRNA或阻碍其翻译的方式调节特异基因的表达[FEBS J.2011；278(10):1610-8]。迄今为止已发现近千个特异miRNA，每一种miRNA可以调控数百种不同的蛋白编码基因，从而通过调控不同靶基因功能参与众多细胞生命活动的调节，包括调节细胞的分化、增殖、自噬以及凋亡等[Cell.2005；123:631–640]。在缺血心脏疾病中，愈来愈多的功能miRNA受到关注。研究显示在热休克小鼠的心脏中，miRNA-1，miRNA-2以及miRNA-24有显著的增加。许多miRNAs，包括miR-21、miR-29、miR-210、miR-373和miR-322/424在心肌细胞缺氧及纤维化时上调并涉及到血管化的调控[Cardiovasc Res.2009；82(1):21-29.CancerRes.2009；69:1221–1229.]，并且一些miRNA还可以调控重要的心脏保护蛋白包括HSP-70、eNOS、iNOS、HSP-20、Sirt-1和HIF-1α的合成[J Clin Invest.2010；120(11):4141–4154.Circulation.2010；122(13):1308-18]。这些功能microRNA在缺血性心脏病的发病及治疗中发挥重要作用，并有可能成为缺血性心脏疾病防治的重要靶点。
MicroRNA-486(miR-486)于2005年由军事医学科学院郑晓飞课题组从人胎肝组织中鉴定并注册[FEBS Lett.2005；579(17):3849-54.]。进一步分析表明miR-486基因(GeneID:619554)位于8号染色体Ankyrin-1(Ank-1)基因的最后一个个内含子(8p11.21位点)。Ank1在红细胞、肌肉细胞及脑组织中广泛存在。miR-486是心脏高丰度表达的重要miRNA之一，在miR-486的启动调控序列中，有两个重要心肌相关的转录因子(MRTF-A)和血清反应因子(SRF)的结合序列。研究显示在利用重组腺病毒技术转染了MRTF-A的乳鼠心肌细胞表达的一系列microRNAs中，miRNA-486升高的最明显[Proc Natl Acad Sci.2010；107(9),4218-4223]。而心肌缺血过程中，MRTF-A和SRF是参与心肌细胞适应及心脏纤维化基因的表达调控重要转录因子。在MRTF-A基因敲除的小鼠中，心肌缺血后胶原的基因表达及产生明显减低[Circ Res.2010；107(2):294-304.]。然而，miR-486在治疗心肌缺血性疾病方面未见报道。
有鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种携带人miR486基因的腺病毒载体，并提供了所述载体表达的腺病毒的制备和纯化方法，该腺病毒可用于预防或治疗心肌缺血性相关疾病。
为了实现本发明的上述目的，特采用以下技术方案：
一种携带人miR486基因的腺病毒载体，所述腺病毒载体由miR-486基因插入pHBAd-U6-GFP得到，由U6启动子调控miR-486基因表达。
本发明所构建的携带人miR486基因的腺病毒载体可以作为一种工具药，用于阻断/减少与调控重要的心脏保护蛋白相关的蛋白，从而具有治疗心肌缺血性疾病的作用。
优选的，如上所述的携带人miR486基因的腺病毒载体，miR-486基因插入pHBAd-U6-GFP的位点为EcoR I和BamH I位点。
携带人miR486基因的重组腺病毒的制备方法，包括：将如上所述的腺病毒载体及腺病毒辅助骨架质粒共转染宿主细胞，培养所述宿主细胞并收集整合包装得到的重组腺病毒。
腺病毒载体是目前运用最广的病毒载体之一，它被广泛用于基因治疗、基因功能性研究、反义治疗、疫苗开发等领域。
优选的，如上所述的制备方法，所述腺病毒辅助骨架质粒为pHBAd-BHG。
优选的，如上所述的制备方法，所述宿主细胞为HEK293细胞。
优选的，如上所述的制备方法，还包括：
将收集得到的重组腺病毒作为毒种进行扩大培养；
更优选的，所述扩大培养直至获得第三代重组腺病毒为止。
优选的，如上所述的制备方法，在收集得到所述重组腺病毒之后，还包括纯化步骤。
优选的，如上所述的制备方法，所述纯化步骤采用CsCl密度梯度超离心法。
更优选的，所述CsCl密度梯度超离心法经过两次超速离心，离心条件为90000×g～110000×g离心80～100分钟。
更优选的，在离心后CsCl密度梯度超离心之后还包括透析操作。
具体为每次用200x体积的透析buffer，透析三次，间隔一小时换一次液。
如上所述的腺病毒载体及如上所述的制备方法制备得到的重组腺病毒在制备用于治疗心肌缺血性疾病的药物中的应用。
如上所述的腺病毒载体及如上所述的制备方法制备得到的重组腺病毒在制备用于预防心肌缺血性疾病的药物中的应用。
优选的，所述心肌缺血性疾病即冠心病，包括心绞痛、心肌梗塞。
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为腺病毒载体结构示意图；
图2为腺病毒扩增时HEK293细胞形态图；
图3为4W后不同组大鼠梗死周围区域MVD(微血管密度)比较结果；上图：微血管密度检测结果代表图；A：Sham组；B：MI组；C：Ad组；D：miR-486组；下图为实验结果的统计图；
图4为TTC染色结果代表图(上图)及统计图(下图)；A：Sham组；B：MI组；C：Ad组；D：miR-486组；
图5HE染色结果代表图；A：Sham组；B：MI组；C：Ad组；D：miR-486组；
图6为Masson染色结果代表图(上图)及统计图(下图)；A：Sham组；B：MI组；C：Ad组；D：miR-486组；
图7为Tunel染色结果代表图(上图)及统计图(下图)；A：Sham组；B：MI组；C：Ad组；D：miR-486组。
具体实施方式
在描述本发明的化合物、组合物、蛋白质、肽等以及方法之前，应当理解，这些实施方式不限于所描述的特定方法、方案以及试剂，这是因为它们可以变化。还应当理解，本文所使用的术语仅用于描述特定实施方式目的，并且不旨在限制本实施方式或权利要求的范围。
除非另有限定，本发明使用的所有技术和科学术语具有与所公开的实施方案所属领域的普通技术人员通常理解的相同的含义。虽然与本发明所述的方法和材料类似或等同的方法和材料可用于本实施方式的实践或测试中，但下文仍然描述了合适的方法和材料。本发明提及的所有出版物、专利申请、专利以及其他参考文献通过引用全部内容结合于本文中。在冲突的情况下，本说明书(包括定义)将起支配作用。此外，材料、方法以及实施例仅仅是说明性的，而不旨在限制。实施方式的其他特征和优点将从以下详细的说明书和权利要求中变得明显。
为了促进理解本文描述的实施方式这一目的，将参考某些实施方式，并且将使用特定语言来描述这些实施方式。本文所使用的术语仅用于描述具体实施方式目的，而不旨在限制本公开的范围。
本发明的内容主要为一种治疗心肌缺血的方法，即利用携带人miR-486基因的腺病毒治疗心肌梗塞疾病。具体内容如下：
第一方面：腺病毒载体Ad-miR486的构建，足够量的病毒制备及纯化。
第二方面：建立大鼠心肌梗塞模型和合适的基因转移方法，观察Ad-miR486在心肌的表达，证明miR486可以改善、治疗心肌缺血。
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。所用试剂或仪器未注明生产厂商者，均为可以通过市购买获得的常规产品。
实施例1腺病毒载体Ad-miR486的构建及病毒制备和纯化
(1)腺病毒载体构建
使用pHBAd-U6-GFP过表达载体，EcoR I和BamH I为插入位点。目的片断miR-486基因插入EcoR I和BamH I位点，由U6启动子调控表达，该载体中含有CMV启动子调控的GFP基因表达(图1)。
(2)病毒制备和纯化
转染前一天，将293细胞接种于60mm培养皿，培养基为DMEM+10％Hyclon胎牛血清,置37℃含5％CO2的培养箱中培养过夜。待细胞生长至底面积的长满到70～80％时，取重组腺病毒载体质粒miR-486过表达及骨架质粒pHBAd-BHG，用LipofiterTM转染试剂进行转染。转染6小时后，更换新鲜的细胞培养液。每天观察细胞出毒迹象。出毒现象为细胞变大变圆，呈葡萄状，并开始出现明显噬斑。待细胞大部分病变并从底部脱落进行收毒。将60mm培养皿中所有细胞及培养液收于15ml离心管中。打开恒温水浴锅至37℃，将15ml离心管在液氮及37℃水浴反复冻融三次。3000rpm离心5分钟，收集含病毒的上清液，弃沉淀。该上清即为Ad-mir486过表达第一代毒种(P1)，将作为随后大量病毒扩增的毒种。从P1代病毒上清扩增，直至获得第三代病毒(P3)(图2)。
第一次超速离心。将离心机预冷到4℃。慢慢将8ml CsCl1.4加入离心管中，继续缓慢加入10ml CsCl1.2，在其上部缓慢加入约20ml的病毒溶液(不够20ml用10mMTris补齐)(体积共约37ml)。配平之，100000×g(24000rpm in SW31)90分钟，4℃，减速度为0。离心结束后，吸弃离心管上部的废液并用75％酒精擦拭管壁，用胶布贴住将要穿刺的部位(防止穿刺时管裂)。用5ml针管配1.22(18G)的针头在蓝白色条带下方穿刺吸出病毒溶液(针头开口向上)。用至少一倍体积的TE稀释所收集的病毒溶液。第二次超速离心：慢慢将12mlCsCl1.4加入离心管中，继续缓慢加入14ml CsCl1.2，在其上部非常缓慢的加入8～10ml稀释好的病毒溶液。之后同上述，用5ml针管配0.8(20G)的针头吸出蓝白条带(方法同上)或在离心管下部扎孔使液体自然流下来，收集蓝白色条带即可(可以减少cscl混入)。透析：每次用200x体积的透析buffer，透析三次，间隔一小时换一次液。透析结束后测滴度并分装-80℃保存病毒。感染性滴度检测，测定结果为2×1011PFU/ml。
实施例2 Ad-miR486治疗大鼠心肌梗塞的实验研究
(1)动物模型及基因转移
成年雄性SD大鼠120只，体重230～250g，所有大鼠均予以普通饲料，自由饮水，饲养环境均一。按照随机数表法将大鼠分为四组：Sham组(30只)，MI组(30只)，Ad组(30只)，miR-486组(30只)。大鼠称重后，采用10％的水合氯醛溶液(3mL/100g)腹腔注射麻醉大鼠。备皮后气管插管连接小动物呼吸机，胸骨左缘第3～4肋间开胸，与左心耳下缘与肺动脉圆锥连线终点下2～3mm处用6-0无创缝合针结扎左冠状动脉前降支。结扎后结扎点靠心尖部左室前壁呈灰白色，Ⅱ和AVL导联心电图S-T段抬高则证明造模成功。Sham组不予任何干预。MI组、Ad组和miR-486组用微量注射器分别吸取75μl生理盐水、腺病毒载体溶液、携带miR-486腺病毒溶液，于心肌梗死周围区域选取三点，斜行进针，直接注射入心肌内。闭合肋间伤口，通过软管抽取胸腔负压，然后依次缝合手术切口，待大鼠恢复自主呼吸后拔出气管插管。
(2)qRT-PCR检测miR-486在心肌的表达含量
分别于治疗后1W、2W和4W时间点处死老鼠，获取大鼠心脏。以SD大鼠心肌梗死的边带为标志，剪取梗死周边区域，应用Trizol法提取各个组心肌组织的RNA，Takara反转录试剂盒，通过特异性引物反转录miR-486和U6，SYBR Green荧光染料法检测miR-486表达情况。将U6作为内参，通过2-ΔΔCT方法计算miR-486相对表达水平。结果显示：Sham组、MI组、Ad组和miR-486组心梗大鼠在1W、2W和4W时心肌组织miR-486的表达量随时间逐渐下降。1W时Sham组、MI组、Ad组和miR-486组四组相比较，miR-486的表达量具有统计学差异(P＜0.05)；进一步分析，MI组和Ad组miR-486的表达量低于miR-486组(P＜0.05)，但高于Sham组(P＜0.05)，且MI组和Ad组之间miR-486的表达量无统计学差异(P＞0.05)。2W时Sham组、MI组、Ad组和miR-486组四组相比较，Sham组、MI组和Ad组的表达量低于miR-486组(P＜0.05)，Sham组、MI组和Ad组三组之间miR-486的表达量无统计学差异(P＞0.05)。4W时Sham组、MI组、Ad组和miR-486组四组相比较，miR-486的表达量具有统计学差异(P＜0.05)，MI组和Ad组miR-486的表达量低于Sham组(P＜0.05)和miR-486组(P＜0.05)，且MI组和Ad组miR-486的表达量不具有统计学差异(P＞0.05)(表1)。
表1
注：#P＜0.05vs miR-486组；*P＜0.05vs MI组和Ad组
(3)基因治疗效果
小动物心脏超声心动图检测
心梗4W后，各组大鼠异氟烷吸入持续麻醉，取仰卧位。使用Vevo2100小动物超声成像系统，超声探头于乳头肌水平垂直于室间隔及左心室后壁而获得M型超声心动图，记录左室收缩期末径和左室舒张期末径，由成像系统自带软件计算出左室射血分数(leftventricular ejection fraction，LVEF，％)和左室短轴缩短率(left ventricularfractional shortening，LVFS，％)，取连续3个以上心动周期的平均值。结果显示：4W后大鼠心梗模型超声结果示(表2)：与Sham组相比，心梗大鼠的LVEF和LVFS明显减低(P＜0.01)；miR-486组与MI组和Ad组比较，LVEF和LVFS明显升高(P＜0.05)；而MI组和Ad组间，LVEF和LVFS无明显差异(P＞0.05)。
表2
注：#P＜0.05vs Sham组；*P＜0.05vs MI组+Ad组
免疫组化法测定微血管密度(MVD)
切片完成后用小鼠抗大鼠CD31多克隆抗体免疫组织化学染色，内皮细胞被染成棕褐色。参照Weidner法计数MVD，在400倍光学显微镜下，在MI周围区域随机选取5个高倍视野计数微血管数目，求得计数的平均值作为该份标本的MVD。结果显示如图3：MI大鼠心肌MVD明显高于Sham组[(58.8±6.60)个/视野](P＜0.05)。且miR-486组心肌MVD[(117.88±10.81)个/视野]高于MI组[(95.76±9.18)个/视野]和Ad组[(98.32±9.70)个/视野(P＜0.05)]，MI组与Ad组之间心肌MVD没有统计学差异(P＞0.05)。(#P＜0.05vs Sham组；＊＊P＜0.05vs MI组&Ad组)
组织染色
TTC染色：4W后脱颈处死大鼠后迅速取出心脏，用PBS溶液冲洗血污，纱布吸干后放入-20℃冰箱中冻15分钟，垂直于心脏长轴以2mm厚度连续切片，置入1％的TTC溶液中，于水浴箱中37℃避光孵育15分钟，染色完成后用数码照相机拍照。砖红色为正常区域,灰白色为梗死区域，用Image Pro Plus 6.0图像采集分析系统计算梗死面积，梗死面积＝梗死面积/心脏切面总面积的百分数来表示。结果显示如图4：Sham组未见心肌梗死，其余三组可见明显的乳白色梗死区域，miR-486组梗死面积较小。miR-486组心肌梗死面积(30.75±1.96)％明显小于MI组和Ad组(P＜0.05)，且MI组(35.81±1.51)与Ad组(35.05±1.75)之间心肌梗死面积无明显差异(P＞0.05)(#P＜0.05vs Sham组；＊＊P＜0.05vs MI组&Ad组。)
HE染色、Masson染色及Tunel染色：4W后处死大鼠，将心脏置于多聚甲醛溶液中固定，石蜡包埋，切片后脱蜡、脱水，应用相应的试剂染色制片，在光学显微镜观察。大鼠心肌HE染色显示如图5：Sham组左室前壁心肌细胞排列整齐、边界清晰、形态正常，细胞间无纤维细胞增生现象；MI组和Ad组左室前壁心肌细胞排列紊乱，心肌细胞肿胀，细胞核固缩或碎裂，周围有大量炎症细胞浸润，存活心肌细胞呈岛样分布，周围纤维肉芽组织增生，坏死心肌为纤维组织取代：miR-486组左室前壁梗死病变程度轻于MI组和Ad组，心肌纤维排列轻度紊乱，细胞形态较为均一，有少量炎症细胞和纤维组织浸润。Masson染色后，通过Image ProPlus 6.0图像分析系统采图、分析，每张病例切片随机选取5个心梗周围区域视野，胶原组织面积占所测视野面积的百分比的平均值即为胶原容积分数(CVF)。结果显示如图6所示：胶原纤维呈蓝色，心肌细胞呈红色。Sham组大鼠心肌组织形态正常，胶原纤维含量较少；心梗后大鼠心肌组织排列紊乱，胶原纤维含量明显增多，纤维化程度明显。通过Image ProPlus 6.0图像分析系统量化分析，与Sham组(7.45±1.37)％相比MI组、Ad组和miR-486组CVF含量明显增加(P＜0.05)，且miR-486组CVF含量(53.72±2.89)较MI组(62.87±3.30)％和Ad组(64.38±5.25)％降低(P＜0.05)，MI组和Ad组之间CVF无差异(P＜0.05)(#P＜0.05vs Sham组；＊＊P＜0.05vs MI组&Ad组)。Tunel染色后，细胞系呈棕褐色或棕黄色颗粒且具备凋亡细胞形态学特征判定为凋亡细胞，在400倍光镜下，每张切片拍摄5个阳性视野，以平均阳性细胞数所占观察心肌细胞的比例作为凋亡指数(apoptotoc index，AI)，即心肌细胞凋亡指数(％)＝(凋亡心肌细胞系数/正常心肌细胞系数)x 100％。结果显示如图7：与Sham组相比，MI大鼠心肌凋亡细胞数显著增多、AI值显著增高(P＜0.05)。经过miR-486治疗，4W后miR-486组AI值(36.78±2.64)％明显低于MI组(45.96±3.49)％和Ad组(44.41±4.80)％(P＜0.05)，且MI组与Ad组之间差异无统计学意义(P＞0.05)。(#P＜0.05vs Sham组；＊＊P＜0.05vs MI组&Ad组)
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。
Claims (9)
1.腺病毒载体在制备用于治疗和/或预防心肌缺血性疾病的药物中的应用；
所述腺病毒载体由人miR-486基因插入pHBAd-U6-GFP得到，由U6启动子调控人miR-486基因表达。
2.根据权利要求1所述的应用，其特征在于，人miR-486基因插入pHBAd-U6-GFP的位点为EcoR I和BamH I位点。
3.携带人miR486基因的重组腺病毒在制备用于治疗和/或预防心肌缺血性疾病的药物中的应用；
所述腺病毒由权利要求1或2中所提及的腺病毒载体及腺病毒辅助骨架质粒共转染宿主细胞，培养所述宿主细胞并收集整合包装得到。
4.根据权利要求3所述的应用，其特征在于，所述腺病毒辅助骨架质粒为pHBAd-BHG。
5.根据权利要求3所述的应用，其特征在于，所述宿主细胞为HEK293细胞。
6.根据权利要求3所述的应用，其特征在于，还包括：
将收集得到的重组腺病毒作为毒种进行扩大培养。
7.根据权利要求6所述的应用，其特征在于，所述扩大培养直至获得第三代重组腺病毒为止。
8.根据权利要求3～7任一项所述的应用，其特征在于，在收集得到所述重组腺病毒之后，还包括纯化步骤。
9.根据权利要求8所述的应用，其特征在于，所述纯化步骤采用CsCl密度梯度超离心法。
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