CN115137740B - miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用 - Google Patents
miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物医学技术领域,尤其涉及了miRNA497b或miRNA5106在制备治疗缺血心肌的药物中的应用,包括miRNA‑497b与miRNA‑5106核苷酸序列,如SEQ ID NO:1、SEQ ID NO:2所示,miRNA‑497b或miRNA‑5106作为活性物质能够促进缺血心肌的恢复,miRNA‑497b或miRNA‑5106作为一种治疗缺血性心脏病的靶向药物。该miRNA497b或miRNA‑5106在制备治疗缺血心肌的药物中的应用,miRNA‑497b或miRNA‑5106可以直接作为活性物质治疗缺血心肌,使机体miRNA的表达量增多,广泛用于临床括冠心病以及心肌梗死的疾病治疗。
Description
技术领域
本发明涉及生物医学技术领域,具体为miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用。
背景技术
缺血性心脏病(IHD),也称为冠心病,包括由冠状动脉狭窄或阻塞引起的一种威胁人类健康的重大疾病。随着社会的不断发展,人民的生活节奏也在逐渐加快,抽烟、喝酒、熬夜以及高脂高热量的摄入这些不健康的生活方式正在威胁着人们的心血管健康。目前,冠心病较为有效的临床治疗方法有冠状动脉内溶栓法、冠脉内支架法、切割球囊技术以及心脏移植。虽然以上方法均可以对病情有所改善,但是大部分手术治疗都有一定程度的风险且并不适用于所有患者,比如,很大一部分的患者由于现有的并发症而不能进行心脏移植手术。因此,心脏再生疗法的最新进展已成为一种有吸引力的替代治疗方案。
微小RNA(microRNA,miRNA)是一类非编码的小分子单链RNA,长度大约为22个核苷酸。研究表明,miRNA可以对基因的表达进行调节,在细胞的生长发育、增殖、凋亡以及分化中起到了重要的作用。越来越多的证据表明miRNA可以通过不同的机制来对心脏起到保护作用,可以为心肌缺血的预防与治疗提供新的靶点与思路。
发明内容
本发明首次揭示了miRNA-497b或miRNA-5106与缺血心肌修复之间的关系,证明了miRNA-497b或miRNA-5106调节剂能够促进受损心肌细胞恢复。
为实现上述目的,本发明提供如下技术方案:miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用,所述miRNA-497b与miRNA-5106核苷酸序列为
5’-CACCACAGUGUGGUUUGGACGUGG-3’(SEQ ID NO:1)、
5’-AGGUCUGUAGCUCAGUUGGCAGA-3’(SEQ ID NO:2)。
优选的,所述miRNA-497b或miRNA-5106作为活性物质,或者作为药物活性靶点设计miRNA-497b或miRNA-5106增效剂,miRNA-497b或miRNA-5106或它们的增效剂能够修复缺血的心肌细胞。
优选的,所述增效剂是miRNA-497b或miRNA-5106模拟核酸。
优选的,所述miRNA的模拟物是通过化学合成的方法模拟生物体内源的miRNA,通过转染的方法转染进细胞内,可以达到增强内源性miRNA的功能的目的。
优选的,一种miRNA-497b或miRNA-5106增效剂,具体为miRNA-497b或miRNA-5106模拟核酸。
优选的,所述miRNA-497b或miRNA-5106或它们的增效剂治疗缺血心肌药物包括治疗冠心病以及心肌梗死的药物。
与现有技术相比,本发明的有益效果是:
1.该miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用,miRNA-497b或miRNA-5106可以直接作为活性物质治疗缺血心肌或者作为药物的靶点设计miRNA-497b或miRNA-5106的增效剂,来使机体miRNA的表达量增多,从而调控机体内其他因子的表达来治疗缺血心肌。
2.该miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用,miRNA-497b或miRNA-5106的模拟物能够调节心肌细胞的功能,达到治疗缺血心肌的目的,可以广泛用于临床括冠心病以及心肌梗死的疾病治疗。
附图说明
图1为miRNA-497b或miRNA-5106对缺血再灌注诱导H9C2损伤后细胞形态观察及其存活率情况图;
图2显示miRNA-497b或miRNA-5106对IR诱导H9C2损伤后LDH、SOD的影响图;
图3显示miRNA-497b或miRNA-5106对IR诱导H9C2损伤后ROS的影响图;
图4显示miRNA-497b或miRNA-5106对IR诱导H9C2损伤后PI3K/AKT蛋白水平的影响图;
图5显示miRNA-497b或miRNA-5106对缺血再灌注诱导小鼠心肌的影响图;
图6显示miRNA-497b或miRNA-5106对缺血再灌注诱导小鼠心肌纤维化的影响图;
图7显示miRNA-497b或miRNA-5106对缺血再灌注诱导后血清中BNP、CK-MB和hs-TnT含量的影响图。
图8显示miRNA-497b或miRNA-5106对缺血再灌注诱导后小鼠心功能的影响图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1-8,本发明提供一种技术方案:miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用,
实施例一:H9C2细胞的培养以及体外缺血模型的建立。
1)用含有10%FBS和2%青霉素-链霉素双抗溶液的DMEM培养基培养H9C2细胞,培养条件为5%CO2和37℃,每隔2-3天更换一次新鲜培养液,3-4天传代一次,取对数生长期的细胞用于实验。
2)将H9C2细胞以1×106个/孔接种至6孔板上,将其分为空白对照组(Con)、缺血组(MI)、和两组缺血后转染组(miRNA-497b与miRNA-5106),用培养基培养24小时后,去除旧培养基,将细胞用PBS清洗3遍。
3)缺血组与转染组用缺血液处理20min后,缺血组直接加入新培养基,转染组转染miRNA-497b与miRNA-5106于细胞中,空白对照组不做缺血处理。
4)加入后24h移出观察细胞形态。
实施例二:细胞存活率的检测
将H9C2细胞以1×105个/孔接种至96孔板上,每孔体积为200μL,用实施例二的方法制造体外缺血模型并分组,每组做6个平行组。根据试剂盒说明书要求在避光条件下向每孔加入20μLCCK-8溶液后,在培养箱孵中育2小时,用酶标仪测定450nm处的吸光值。
如图1所示,对比MI组(45.13±2.52)和Control组可以发现,MI组不仅细胞数量很少,且在缺血后细胞形态扭曲、部分细胞膜破碎,证明该缺血模型造模成功。转染组比MI组细胞存活率显著升高,但与Control组比较仍未恢复至正常水平。其中,空白对照组(Con):心脏细胞在无任何处理的对照培养24h;缺血组(MI):心脏细胞在缺血损伤后无任何后处理培养24h;实验组(miRNA-497b与miRNA-5106):心脏细胞在缺血损伤后转染miRNA-497b与miRNA-5106培养24h。
将miRNA模拟物转染到H9C2细胞后细胞形态以及细胞存活率并没有发生明显的改变,证明用PEI试剂转染6小时对细胞的影响并不大。对细胞缺血处理后,可以发现,转染miRNA的H9C2细胞的形态扭曲程度较弱、凋亡减少,且转染miRNA-5106后的细胞存活率要高于转染miRNA-497b。
注:图中星号表示与空白对照组存在显著性差异:p<0.05。
实施例三:Hoechst33258染色法观察细胞核型变化
用实施例一的方法制造体外缺血模型并分组,向6孔板中加入预冷的PBS对每孔细胞进行清洗,在室温的条件下用4%多聚甲醛固定液对细胞进行固定。固定20min后使用PBS进行清洗3次,每次3分钟。在避光条件下,加入Hoechst33258染色液,室温下孵育15min,最后再使用PBS进行清洗3次,每次3分钟,于超净台中避光风干,在荧光显微镜下用400×的放大倍数下观察细胞核的形态。
Control组的细胞核呈现出弥散均匀的淡蓝色荧光,色泽均一,形态规则且表面光滑。MI组与Control组相比,可见较强的蓝色荧光,这是程序性凋亡的细胞急剧增多,细胞出现细胞膜出泡、细胞核缩聚的现象,甚至可以观察到一些细胞的细胞核破裂染色质逸出并在核边聚集的现象。转染miRNA-497b后细胞核聚缩、破裂这些程序性细胞凋亡的特征愈逐渐减弱,呈现与Control组相似的弥散均匀的淡蓝色荧光。
实施例三:乳酸脱氢酶(LDH)细胞毒性试验与总SOD活性检测(WST-8法)
将H9C2细胞接种于96孔板上,并按实施例1的方法处理细胞并分组,加药孵育24小时后吸取120μL上清液至新的96孔板上,按照试剂盒说明书配制LDH检测工作液,分别向每孔加入60μLLDH检测工作液,混匀后在室温且避光的条件下水平摇床缓慢摇动30min,用酶标仪测定在490nm处的吸光值。
用实施例一的方法制造体外缺血模型并分组,用预冷的PBS洗涤细胞一次,再加入PBS用细胞刮刀将细胞刮下转移到离心管中,在4℃、1500rpm的条件下离心10分钟,去除上清液,每管加入70μL的裂解液(将RIPA裂解液与苯甲基硫酰氟按100:1的比例混匀,现配现用,置于冰上保存)并适当吹打以充分裂解细胞,在4℃的条件下水平摇床快速摇动30分钟。在4℃、13200rpm的条件下离心10分钟,将上清液转移至新的离心管,用BCA试剂盒测定蛋白浓度。依照说明书要求配制WST-8/酶工作液和反应启动液后,并按照说明书在低温条件下依次加入待测样品和其它工作液。
如图2所示,转染组的LDH释放量(112.17±6.59、119.00±5.17)明显低于MI组(157.17±4.54),且具有显著性差异。同时我们发现SOD的活性在缺血处理后相较于空白对照组明显降低(47.11±3.87),转染后SOD(84.70±5.22、81.94±3.48)活性增加,且具有显著性差异。以上结果表明转染miRNA-497b或miRNA-5106后可以增强H9C2细胞的抗氧化应激作用。
实施例四:活性氧(ROS)检测
用实施例一的方法制造体外缺血模型并分组,用预冷的PBS洗涤细胞一次。用培养液按照1000:1的比例稀释DCFH-DA,每孔加入1mL稀释后的DCFH-DA,然后在细胞培养箱中孵育20分钟,用无血清细胞培养液洗涤细胞三次后在激光共聚焦显微镜下观察并拍照。
如图4所示,MI组与Control组比较,MI组产的细胞数量急剧减少并产生大量的活性氧,表明该缺血再灌注模型可以导致氧化应激损伤从而致使细胞逐渐凋亡。转染组的细胞能够有效的阻止缺血再灌注所引起的活性氧含量的增加,并且对比MI组细胞的数量也明显的上升趋势。这些结果表明转染miRNA-497b或miRNA-5106能够减弱氧化应激所带来的损伤,从而来减少缺血心肌细胞的凋亡。
实施例五:蛋白印迹法检测
用实施例一的方法制造体外缺血模型并分组,用预冷的PBS洗涤细胞一次,再加入PBS用细胞刮刀将细胞刮下转移到离心管中,在4℃、1500rpm的条件下离心10分钟,去除上清液,每管加入70μL的裂解液(将RIPA裂解液与苯甲基硫酰氟按100:1的比例混匀,现配现用,置于冰上保存)并适当吹打以充分裂解细胞,在4℃的条件下水平摇床快速摇动30分钟。在4℃、13200rpm的条件下离心10分钟,将上清液转移至新的离心管,取5μL用BCA试剂盒定量。向剩余蛋白样品中加入3×的SDSloadingbuffer(使loadingbuffer的终浓度为1×),充分混匀后在100℃的沸水中煮5分钟,可存放于-80℃长期保存。
将上述蛋白样品解冻后按每孔10μg的蛋白量加入制作好的凝胶电泳槽中,进行聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳。电泳结束后,将蛋白转移到硝酸纤维膜上,再用TBST清洗3次,每次5分钟。用5%脱脂牛奶在室温下封闭2小时后取出膜,继续用TBST清洗3次,每次5分钟。孵一抗(用含有5%BSA的TBST按照1000:1的比例稀释)过夜。第二天取出PVDF膜,用TBST清洗3次,每次10分钟。在室温下孵育相应的二抗(用含有5%BSA的TBST按照5000:1的比例稀释)1.5小时,取出PVDF膜,用TBST清洗3次,每次10分钟。加入ECL发光液并在分子成像系统中显影、分析。
如图4所示,与Control组相比,MI组PI3K、p-AKT蛋白水平显著降低(P<0.01)转染组相应的蛋白水平得到增加,暗示转染miRNA-497b或miRNA-5106可以上调心肌细胞的PI3K/AKT通路发挥心肌缺血保护效应的作用。
实施例六:小鼠心肌缺血再灌注模型
用戊巴比妥钠80mg/kg腹腔注射麻醉,剃除小鼠胸部及腋下毛发以充分暴露手术区域,用碘伏和75%乙醇对手术区进行消毒。打开呼吸机,设置呼吸频率为110次/分,洗呼比为1:1.5,潮气量为1.5mL,将插管沿声门插入小鼠气管,连接呼吸机,可观察到小鼠胸廓起伏与呼吸机频率保持一致。小鼠采用右侧卧位,用显微剪在左侧第3-4根肋骨之间剪开皮肤和肋间隙,用拉钩撑开肋骨充分暴露心脏。用显微直镊轻轻撕开左心耳处的心包膜,使左冠状动脉前降支(LAD)所在区域(左心耳中下1-2mm处)充分暴露。用7-0缝线垂直于LAD走向的方向打活结以结扎冠状动脉,假手术组以同样的手法暴露心脏但不结扎冠状动脉。缺血45分钟后,解开结扎线。转染组用微量进样器在梗死区域多点注射富含miRNA-497b的C-kit+心脏细胞外泌体25μg,缺血组用微量进样器在梗死区域多点注射等体积PBS。用4-0缝线由内向外逐层缝合各层肌肉和皮肤。术后每天关注小鼠状态,正常饲养。术后3天,取血后处死小鼠,用生理盐水对小鼠进行全身灌流后取出心脏,再用生理盐水冲洗掉表面血迹并用纱布擦干。用4%多聚甲醛进行固定。
实施例七:苏木素伊红(HE)染色
将实施例六的心脏,脱水包埋后切成厚度约为5μg的薄片。用二甲苯I、II、III、IV以及各个浓度的酒精对切片进行处理,再用流水冲洗。用苏木素染液将切片染色7分钟后用流水冲洗,再用盐酸酒精分化液分化,然后用稀氨水返蓝。用伊红染液将切片染色3分钟,再用流水冲洗。使用不同浓度的酒精和二甲苯I、III对切片进行脱水透明处理,待切片透明后封片。将染好的切片在显微镜下观察并进行分析。
结果如图5所示,取手术后3天的小鼠心脏进行HE染色,可以看出MI组与假手术组相比较比较,细胞周围间隙增宽且存在很多大小不等的空泡,细胞排列更为松散,细胞质染色程度不均匀,细胞核皱褶变形,具有明显的凋亡特征。外泌体组与MI组相比较,细胞周围间隙缩短、轮廓逐渐清晰、排列更为有序,细胞核恢复正常,心肌细胞数量增加,说明富含miRNA-497b或miRNA-5106的C-kit+心脏细胞外泌体对缺血心肌具有保护效应。
实施例八:Masson染色
用实施例六的方法对切片进行脱蜡水化,用Weigert铁苏木素染液将切片染色7分钟后用流水冲洗,再用盐酸酒精分化液分化,然后用稀氨水返蓝。用丽春红染液将切片染色5分钟,再用流水冲洗。用2%的冰醋酸水溶液清洗切片后,用1%磷钼酸水溶液处理切片并倾倒。苯胺蓝染液复染5分钟后将其倾去,2%的冰醋酸水溶液清洗切片至无蓝色脱出。使用不同浓度的酒精和二甲苯I、III对切片进行脱水透明处理,待切片透明后封片。将染好的切片在显微镜下观察并进行分析。
结果如图6所示,取手术后3天的小鼠心脏进行Masson染色,观察小鼠心肌纤维化情况,可以观察到MI组梗死区域周边发生大规模的纤维化,而外泌体给药组相较于MI组梗死区域周边纤维化明显减少,说明富含miRNA-497b或miRNA-5106的C-kit+心脏细胞外泌体可以通过减少缺血所导致的纤维化来达到保护缺血心肌的目的。
实施例九:酶联免疫吸附实验
用实施例6的方法制造小鼠心肌缺血再灌注模型,术后3天,用异氟烷麻醉小鼠,眼球取血,3000rpm的条件下离心5分钟,取上层血清于-80℃保存。使用ELISA试剂盒检测各组小鼠血清中BNP、CK-MB和hs-TnT的含量,并采用酶标仪进行定量分析,严格按照试剂盒说明书操作。
如图7所示,我们发现IR诱导后小鼠的血清中BNP、CK-MB和hs-TnT含量(分别为523.14±15.26pg/mL、83.11±2.66ng/mL、473.89±11.43ng/L)相较于空白对照组明显升高(分别为369.16±6.20pg/mL、50.37±1.40ng/mL、322.42±9.15ng/L),外泌体给药后小鼠的血清中BNP、CK-MB和hs-TnT含量(分别为448.87±13.58pg/mL、64.66±2.12ng/mL、382.02±10.01ng/L)显著降低,且具有显著性差异。以上结果表明富含miRNA-497b或miRNA-5106的C-kit+心脏细胞外泌体对缺血性心脏病具有一定的治疗作用。
实施例十:心脏超声检测心功能
分别将每组小鼠手术前以及手术后的第一天、第三天进行心超检测。用戊巴比妥钠麻醉小鼠后,在不扯开缝合处的情况下小心地剃除手术区域附近的毛发。将彩超仪的探头垂直于第3和第4根肋骨之间靠左放置,获取小鼠左心室(Leftventricle,LV)的胸骨旁长轴及短轴切面,得到M型超声心动图。分别测量左室收缩末期内径(Leftventricle end-systolic diameter,LVDs)、左室舒张末期内径(Leftventricle end-diastolicdiameter,LVDd)、左室收缩末期容积(Left ventricle end-systolic volume,LVVs)和左室舒张末期容积(Left ventricle end-diastolic volume,LVVd)。
如图8所示,在手术之前三组小鼠的心脏功能都保持在同一水平,但经过手术后,MI组和EXO组小鼠的左室短轴缩短率与左室射血分数均急剧降低,而sham组这两项指标无明显变化。EXO组小鼠的左室短轴缩短率与左室射血分数在手术后的第1天和第3天都要高于MI组,表明了向缺血心肌内注射C-kit+心脏细胞外泌体能够使心功能得到明显的恢复。手术后第三天,EXO组的左心室的舒张末期以及收缩末期内径要明显低于MI组,表明富含miRNA-497b或miRNA-5106C-kit+心脏细胞外泌体可以减轻缺血性心脏病所导致的心室形态的变化。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (3)
1.miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用,包括miRNA-497b或miRNA-5106核苷酸序列,其特征在于:所述miRNA-497b、miRNA-5106核苷酸序列分别如SEQID NO:1、SEQ ID NO:2所示。
2.根据权利要求1所述的miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用,其特征在于:所述miRNA-497b或miRNA-5106作为活性物质,miRNA-497b或miRNA-5106能够促进缺血心肌的恢复。
3.根据权利要求2所述的miRNA-497b或miRNA-5106在制备治疗缺血心肌的药物中的应用,其特征在于:所述miRNA-497b或miRNA-5106作为一种用于治疗缺血性心脏病的靶向药物。
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