CN116173062A - MiR-410-3p在制备预防或治疗肝损伤药物中的用途 - Google Patents
MiR-410-3p在制备预防或治疗肝损伤药物中的用途 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种MiR‑410‑3p在制备预防或治疗肝损伤药物中的用途。针对现有研究仅发现干细胞来源的外泌体在肝损伤修复中具有积极作用,但扩增时间长,不适宜产业化的问题,本发明提供了一种MiR‑410‑3p在制备预防或治疗肝损伤药物中的用途。本发明首次发现了人脐带血血浆来源的外泌体对肝脏损伤的修复作用,并进一步发现是其中的MiR‑410‑3p对肝脏损伤修复起关键作用,进而为肝脏损伤修复提供了一种新的药物。本发明的人脐带血血浆取样简单安全,还可进行产业化提取,使用方便,具有很好的应用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及一种MiR-410-3p在制备预防或治疗肝损伤药物中的用途。
背景技术
肝脏是人体五大重要器官之一,在人体新陈代谢中起着关键作用。病毒感染、药物中毒、脂质沉积和自身免疫反应各种肝毒性因可导致急性或慢性肝损伤。急性肝损伤(ALI)其临床特征是无肝病患者的肝细胞功能迅速丧失,以血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、γ-谷氨酰转肽酶(GGT)等肝胆酶活性来评价。严重急性肝损伤后,肝再生失败,肝衰竭随之发生,治疗难度大,恢复几率极低。持续的肝损伤会导致细胞外基质过度累积,导致肝实质逐渐被瘢痕组织所取代,从而失去正常结构,进展成肝纤维化,若不加以干预,病情可急剧恶化,失去正常的肝功能。目前,有研究表明外源性的肝细胞移植可能是治疗肝损伤一种有前景的新方法,但原代肝细胞获取困难,体外难以扩增、储存以及细胞移植后的免疫排斥反应等制约了肝细胞移植的临床转化应用。因此,如何通过药物要治疗急/慢性肝损伤,防止它进一步进展为肝衰竭,是行业内亟待解决的问题。
外泌体(Exosomes)属于胞外囊泡(Extracellular vesicles,EVs)的一个亚群,是细胞所分泌的微小膜泡,它来源于内体,具有脂质双层膜结构,直径大约为30-150nm。外泌体广泛存在于血液、唾液、尿液、脑脊液等各种体液中,它通过向受体细胞运送MiRNAs、mRNAs和蛋白质,在细胞间的通讯中发挥关键作用。近期有研究发现,利用干细胞来源的外泌体治疗损伤后的肝脏,表现出与细胞移植治疗类似的、能促进受损的肝细胞再生修复的特性。与细胞移植治疗相比,外泌体治疗具有安全性更好、可以修饰改造、可以长期保存等显著优势,在重症肝脏疾病的治疗方面显示出了很好的转化和临床应用前景。
近年来的研究表明,不同细胞来源的外泌体对肝脏疾病具有良好的治疗效果,其中间充质干细胞来源的外泌体(MSC-Exos)治疗肝损伤的研究较多,如有研究报道人脐带来源的MSC-Exos减轻了CCl4诱导的肝纤维化小鼠肝脏的炎症和胶原沉积,从而减轻了肝纤维化进程。人脂肪来源的MSC-Exos通过抑制PI3K/Akt/mTOR通路和重塑胆碱代谢来改善肝纤维化;也有研究报道骨髓来源的MSC-Exos治疗后可显著降低纤维化标志物Col1α1、Col3α1、TGF-β1和p-Smad2的表达水平,从而抑制肝纤维化进程;人绒毛膜来源的MSC-Exos通过递送MiRNA-125b,可以抑制Hh信号的异常激活,从而抑制肝纤维化进程;人月经血来源的MSC-Exos可显著下调肝脏组织的Col1α1、Vimentin、α-SMA的表达水平,明显减轻小鼠慢性肝损伤;在毒物诱导的肝损伤模型中,骨髓来源的MSC-Exos主要通过激活肝细胞的增殖和再生,从而保护受损的肝脏;新生儿脐带来源的MSC-Exos富含谷胱甘肽过氧化物酶1(GPX1),可以减少H2O2诱导的氧化应激和CCl4导致的细胞凋亡,从而防止肝脏氧化损伤;人月经血中间充质肝细胞来源的MSC-Exos通过减少肝单核细胞的数量,激活Caspase-3阳性的细胞来预防D-Gal/LPS所诱导的小鼠急性肝损伤;另外,骨髓来源的MSC-Exos可通过OTUB1所介导的去泛素化,从而增强SLC7A11蛋白的稳定性,防止CCl4所诱导的肝细胞死亡。以上这些研究结果表明间充质干细胞来源的外泌体可以通过外泌体所负载的MiRNAs或蛋白质来治疗急/慢性肝损伤,有很好的临床转化前景。
虽然上述干细胞来源、脂肪来源、骨髓来源等来源的外泌体被报道能在肝损伤中发挥修复作用,但上述细胞扩增时间长,限制了其临床应用。
脐血中含有造血干细胞、间充质干细胞、内皮祖细胞等丰富的干细胞资源,主要用于血液系统疾病、神经系统疾病、代谢性疾病等的造血干细胞移植治疗。
有报道显示脐带血血浆外泌体(CBP-Exos)可在骨分化和组织修复、促进皮肤伤口愈合等方面有作用,也能改善老年骨质疏松小鼠得骨丢失和促进成骨细胞分化。但关于脐带血血浆外泌体对肝损伤的修复作用还未见报道。
发明内容
本发明要解决的技术问题为:现有研究仅发现干细胞来源的外泌体在肝损伤修复中具有积极作用,但这些细胞来源的外泌体细胞扩增时间长,不适宜产业化应用于肝损伤的修复中的问题。
本发明解决上述技术问题的技术方案为:提供了一种MiR-410-3p在制备预防或治疗肝损伤药物中的用途。
其中,上述用途中,所述的MiR-410-3p为血浆来源的外泌体中的MiR-410-3p。
进一步的,上述用途中,所述的血浆来源是指人脐带血血浆。
其中,上述用途中,所述的MiR-410-3p的核苷酸序列如SEQ ID NO:1所示。
SEQ ID NO:1人脐带血血浆外泌体中的MiR-410-3p的核苷酸序列
5’aatataatatagatggcctgt 3’。
其中,上述用途中,所述肝损伤是指急性肝损伤或慢性肝纤维化。
其中,上述用途中,所述的MiR-410-3p用量为0.2nmol/g体重小鼠。
本发明还提供了一种预防或治疗肝损伤的药物,包含MiR-410-3p。
进一步的,上述预防或治疗肝损伤的药物中,所述的MiR-410-3p为人脐带血血浆外泌体中的MiR-410-3p。
与现有技术相比,本发明的有益效果为:
本发明首次发现了人脐带血血浆来源的外泌体对肝脏损伤的修复作用,并进一步分离鉴定CBP-Exos与OBP-Exos在H2O2所诱导的肝细胞损伤的保护作用以及对CCl4诱导的小鼠肝损伤的疗效,并通过比较CBP-Exos和OBP-Exos之间差异表达的MiRNAs,发现了CBP-Exos所携带的MiR-410-3p能通过激活线粒体介导的抗凋亡信号通路Bim-Bcl2-CytoC,从而抑制氧化应激导致的肝损伤和肝细胞凋亡过程,在肝损伤治疗中具有重要的应用前景。因此,本发明提供了一种MiR-410-3p在制备预防或治疗肝损伤药物中的用途,还提供了一种包含MiR-410-3p的预防或治疗肝损伤的药物,为急慢性肝损伤修复提供了一种新的方式,并且本发明的MiR-410-3p来自人脐带血血浆,取样简单安全,还可进行产业化提取,使用方便,容易保存,具有很好的应用价值。
附图说明
图1所示为脐带血血浆外泌体(CBP-Exos)的分离与鉴定;其中,A.CBP-Exos的制备步骤;B.CBP-Exos的Western Blot鉴定和纳米粒径分析;C.CBP-Exos的形态鉴定。
图2所示为CBP-Exos改善急性肝损伤(ALI)小鼠肝脏的结构和功能。其中,A.Control组(n=3)、CCl4组(n=4)、CCl4+CBP-Exos组(n=4)小鼠在建模后第1天、第2天、第3天、第5天的血清生化指标ALT和AST含量的检测;从左至右依次为:Control组、CCl4组、CCl4+CBP-Exos组的结果;B.各分组小鼠在建模后第1天、第2天、第3天、第5天的肝组织H&E染色。
图3所示为CBP-Exos改善ALI小鼠肝脏的凋亡和氧化应激水平。其中,A为免疫组织化学染色检测各组小鼠肝组织内cleaved-caspase3的表达;B为MDA检测各组小鼠肝组织内脂质过氧化物MDA的表达水平,n=5。
图4所示为CBP-Exos抑制小鼠原代肝细胞凋亡和氧化应激。其中,A为荧光显微镜检测对照组、H2O2组、CBP-Exos组和OBP-Exos组的ROS水平,n=3;B为Tunel染色检测对照组、H2O2组、CBP-Exos组和OBP-Exos组的凋亡水平,n=3。
图5所示为CBP-Exos与OBP-Exos中MiRNAs的差异富集分析及RT-qPCR鉴定。其中,A为CBP-Exos和OBP-Exos中差异MiRNAs的火山图分析,n=3;B为CBP-Exos和OBP-Exos中差异MiRNAs及其靶基因mRNA在肝损伤凋亡信号通路中的MiRNA-mRNA调控网络分析,n=3;C为CBP-Exos中高富集的前十位差异MiRNAs的热图分析,n=3;D为RT-qPCR验证差异表达MiRNAs(MiR-24-3p、MiR-150-5p、MiR-181a-5p、MiR-191-5p、MiR-486-5p、MiR-410-3p)在CBP-Exos和OBP-Exos中的表达量,n=3。
图6所示为差异表达MiRNAs抑制小鼠肝细胞氧化应激和凋亡的功能筛选。
其中,A为荧光镜检H2O2所诱导的AML12肝细胞中分别转染MiR-24-3p mimic、MiR-150-5pmimic、MiR-181a-5p mimic、MiR-191-5p mimic、MiR-486-5p mimic和MiR-410-3pmimic的ROS水平变化,n=3;B为流式分析H2O2所诱导的AML12肝细胞中分别转染MiR-24-3pmimic、MiR-150-5p mimic、MiR-181a-5p mimic、MiR-191-5p mimic、MiR-486-5p mimic和MiR-410-3pmimic的凋亡情况,n=3。
图7所示为CBP-Exos中发挥肝保护作用的MiRNA鉴定。其中,A为CBP-Exos处理H2O2所诱导的AML12肝细胞中转染MiR-150-5p inhibitor、MiR-486-5p inhibitor和MiR-410-3pinhibitor的ROS水平检测,n=3;B为CBP-Exos处理H2O2所诱导的AML12肝细胞中转染MiR-150-5p inhibitor、MiR-486-5p inhibitor和MiR-410-3p inhibitor的凋亡水平检测。
图8所示为MiR-410-3p治疗ALI小鼠的疗效评估。其中,A为对照组(n=3)、CCl4组(n=3)、CCl4+CBP-Exos组(n=3)、CCl4+NC agomir组(n=5)、CCl4+MiR-410-3p agomir组(n=5)小鼠血清中ALT和AST含量的检测;B为对照组、CCl4组、CCl4+CBP-Exos组、CCl4+NCagomir组、CCl4+MiR-410-3p agomir组小鼠肝脏组织HE染色;C为对照组、CCl4组、CCl4+CBP-Exos组、CCl4+NC agomir组、CCl4+MiR-410-3p agomir组小鼠肝脏组织免疫组化F4/80染色。
具体实施方式
本发明首次发现了人脐血血浆来源的外泌体在肝损伤修复中具有积极作用。目前,大多研究聚焦于使用间充质干细胞来源的MSC-Exos开展肝损伤治疗研究。此外,也有部分研究使用人肝干细胞,肝细胞,羊膜上皮细胞来源的外泌体开展治疗研究。
脐带血作为最年轻的血液,含有造血干细胞、间充质干细胞、内皮祖细胞等丰富的干细胞资源,广泛用于治疗各种疾病,但很少有研究挖掘脐血血浆来源的外泌体在疾病治疗当中的作用。我们的研究结果丰富了脐带血血浆外泌体的临床应用潜力,提示可将其用于肝脏疾病的治疗,为UCB银行资源的充分利用提供了新的方向。目前工业化简单易操作的外泌体提取方式正在日新月异的改进,未来技术完善时可在造血干制备过程中将血浆收集后进行外泌体产业化提取,通过冷冻技术将CBP-Exos制备成冻干粉,即可长期保存,需要时即可使用,具有潜在的临床转化价值。
下面将通过实施例对本发明的具体实施方式做进一步的解释说明,但不表示将本发明的保护范围限制在实施例所述范围内。
实施例所用到的仪器和试剂均为普通市售产品。本申请中所用到的人脐带血血样经捐赠者同意,取自四川省妇幼保健院的健康产妇捐赠者(产妇年龄:22-35周岁)。老年人血浆来源的外泌体是取自年龄65-85岁的健康志愿者捐赠的正常血液。实验已获四川大学华西医院医学伦理委员会批准。
实施例1分离人脐带血血浆CBP-Exos并进行鉴定
(1)分离CBP-Exos。步骤如下:
使用抗凝采血袋收集人脐带血样(50-60ml/份),以柠檬酸磷酸盐葡萄糖作为抗凝剂;将脐带血样本转移到50ml离心管中,25ml/管,4℃,1000g离心20min收集血浆,弃沉淀;随后,4℃,3000g离心10min,收集血浆,弃沉淀;在冷冻离心机中4℃,12000g离心30min,收集血浆,弃沉淀;将血浆稀释6倍,4℃,在超高速离心机中120000g过夜离心,用30ml PBS重悬,0.22μm滤器过滤;在超高速离心机中4℃,100000g离心2h,用100μl PBS重悬,存储在-80℃或用于下游实验。(短期存储于4℃,长期存储于-80℃,切忌反复冻融)。如图1A所示。
(2)CBP-Exos的Western Blot鉴定和纳米粒径分析
如图1B所示,我们使用免疫印迹试验检测到CBP-Exos高表达外泌体标记蛋白Alix和CD63的表达。另外,使用ZetaView进行纳米粒径分析观察到CBP-Exos处于30-150nm之间。
(3)CBP-Exos的形态鉴定
如图1C所示,我们使用透射电子显微镜TEM观察了CBP-Exos呈现出外泌体典型的杯托状结构。
实施例1的结果表明我们分离得到了人脐带血血浆来源的外泌体,即CBP-Exos。
实施例2CBP-Exos治疗CCl4诱导的急性肝损伤(ALI)小鼠
具体的操作步骤如下:
(1)急性肝损伤(ALI)小鼠建模
将CCl4与橄榄油按体积比1:5配制成工作液,现用现配;选取8-10周龄,体重在20-25g的雄性小鼠随机分组,模型组腹腔注射3μL/g的CCl4工作液每天注射一次,待血生化指标迅速升高,肝组织坏死时,提示建模成功;空白对照组腹腔注射等体积的橄榄油;治疗组在CCl4建模同时以及建模后1天给予尾静脉CBP-Exos输注,治疗剂量为10μg/g体重。
(2)测定各组小鼠在建模后第1天、第2天、第3天、第5天的血清生化指标ALT和AST含量
分别在注射CCl4工作液的第1天、第2天、第3天、第5天通过眼球取血法收集小鼠血液于1.5ml EP管中,4℃放置过夜,4℃3000rpm离心15分钟,取上层血清进行谷丙转氨酶ALT和谷草转氨酶AST的检测,ALT和AST在国家新药安全性评价中心(成都华西海圻医药科技有限公司)GLP车间进行。
ALT和AST的结果的实验结果如图2A所示。ALT和AST是反应肝脏损伤的主要指标,当两者升高时,提示肝脏受损;两者的数值越高,说明受损越严重。图2A的结果显示:建模第二天的小鼠血清中,经CBP-Exos输注的小鼠血清中ALT和AST含量显著高于对照组。由此可见,经CBP-Exos输注的小鼠从第二天起,就已经成功建立肝损伤模型。
(3)各组小鼠在建模后第1天、第2天、第3天、第5天的肝组织H&E染色
分别在注射CCl4工作液的第1天、第2天、第3天、第5天,处死小鼠后收集小鼠肝组织,用PBS清洗后使用4%多聚甲醛固定;固定48h后进行石蜡包埋肝组织;进行肝组织切片和H&E染色(如图2B所示)。
H&E染色结果显示:正常小鼠肝脏呈条索状,CCl4建模后1d肝脏结构开始紊乱,有少量出血点,CBP-Exos治疗后的肝组织结构基本维持正常,未观察到出血点;建模后2d时肝坏死区域显著增加,出血点增加,有大量的炎性细胞浸润,CBP-Exos治疗后肝坏死区域面积显著减少,炎性浸润减少;建模后3d肝脏结构有所恢复,但仍存在较多的坏死区域面积,CBP-Exos治疗后的肝组织结构恢复呈条索状,观察到少量坏死区域;建模后5d肝组织结构几乎完全恢复正常,肝组织内有少量炎性细胞浸润,CBP-Exos治疗后的肝组织结构完全恢复,肝组织内没有观察到炎性细胞浸润。以上结果共同表明,CBP-Exos治疗可显著减少ALI小鼠肝坏死区域面积、炎性细胞浸润和出血点,并恢复肝组织条索状结构。
实施例3CBP-Exos改善ALI小鼠肝脏的凋亡和氧化应激水平
具体的操作步骤如下:
(1)免疫组织化学染色检测各组小鼠肝组织内cleaved-caspase3的表达
分别在注射CCl4工作液的第1天、第2天、第3天,处死小鼠后收集小鼠肝组织,用PBS清洗后使用4%多聚甲醛固定;固定48h后进行石蜡包埋肝组织;进行肝组织切片和cleaved-caspase3的免疫组织化学染色(图3A所示)。
cleaved-caspase3的免疫组织化学染色结果显示:我们对小鼠肝脏石蜡切片进行细胞凋亡标记物cleaved-caspase3的免疫组化染色,由于建模后第五天的小鼠肝组织结构基本恢复正常。因此,我们只选取了建模后1d、2d、3d这几个时间点进行IHC染色,结果显示建模后1d,建模组和治疗组均呈现少量cleaved-caspase3阳性细胞;建模后2d,CCl4组出现大量cleaved-caspase3阳性细胞区域,而CBP-Exos治疗组阳性区域面积显著减少;建模后3d,CCl4组阳性细胞区域相比2d时有所降低,而CBP-Exos治疗组几乎观察不到cleaved-caspase3阳性细胞区域。
上述实验结果共同说明CBP-Exos治疗可显著降低肝组织凋亡水平。
(2)检测各组小鼠肝组织内脂质过氧化物MDA的表达水平
在建模后第2天牺牲小鼠后收集小鼠肝组织,用PBS清洗后在液氮中碾碎组织;收集20mg肝组织粉末,加入适量蛋白裂解液,混匀后使用超声破碎仪进行进一步裂解,4℃超声5分钟;超声后4℃,12000rpm离心15分钟,取上清使用AmpliteTM荧光丙二醛(MDA)定量试剂盒检测肝组织内MDA的水平。结果如图3B所示。
结合前面的疗效评估结果,我们发现在不同采样点(1d、2d、3d、5d)的疗效评估中,建模后2d是肝损伤最为严重的时间点,其各项典型病症在CBP-Exos治疗后有显著恢复。因此,在后续的实验中我们均采用建模后2d为采样点进行研究。由于肝脏内游离的ROS水平在离体后急剧变化不利于检测,因此,我们采用脂质过氧化物丙二醛MDA的测定来反映肝组织内氧化应激水平。丙二醛(MDA)是脂质过氧化过程中的天然副产物之一,MDA的量化可以评估病理生理过程中氧化应激的水平,是反映氧化应激水平的可靠生物标志物。MDA检测结果表明,与对照组相比(76.1μmol/g),CCl4组肝组织MDA水平显著升高(121.3μmol/g),CBP-Exos治疗极显著的降低了肝组织内MDA的积累水平(50.7μmol/g)(P<0.001)。以上研究表明CBP-Exos治疗可以降低肝组织凋亡水平和氧化应激水平,具有抗氧化损伤的肝保护功能。
实施例4 CBP-Exos和老年人血浆来源的外泌体(OBP-Exos)对过氧化氢(H2O2)诱导的小鼠原代肝细胞氧化应激的影响
由于药物(如CCl4)诱导所产生的氧化应激信号是导致急性肝损伤和肝脏毒性的主要因素。我们通过H2O2诱导肝细胞氧化应激来模拟体内CCl4诱导的肝损伤模型,以明确CBP-Exos在体外对氧化损伤肝细胞的保护作用。
(1)荧光显微镜检测对照组、H2O2组、CBP-Exos组和老年人OBP-Exos组的原代肝细胞ROS水平
我们采用原位灌注法,分离了小鼠原代肝细胞(Primary hepatocytes,PHs)。将分离得到的小鼠原代肝细胞接种到提前用鼠尾胶原包被的孔板中,3h后原代肝细胞贴壁,分别加入CBP-Exos和OBP-Exos与原代肝细胞共孵育;共孵育16h后使用200nM H2O2诱导小鼠原代肝细胞产生氧化应激;诱导5h后使用DCFH-DA检测细胞内ROS水平。DCFH-DA,2,7-二氯荧光素二乙酸酯,又名活性氧ROS荧光探针(2.7-二氯荧光素二氢二乙酸酯,是一种可渗透细胞的荧光探针,用于检测活性氧(ROS)和一氧化氮(·NO)以及确定总体氧化应激程度。
荧光镜检结果如图4A所示,结果显示H2O2所诱导的小鼠原代肝细胞内ROS水平显著增加(P<0.01),而在加入CBP-Exos处理后ROS水平显著降低(P<0.05)。另外,考虑到后续机制研究,在此我们使用了老年人血浆来源的外泌体OBP-Exos作为对照,探究OBP-Exos对肝细胞内氧化应激的影响,结果显示OBP-Exos处理组与H2O2组一致,胞内ROS水平与Control组相比显著增加(P<0.01),说明OBP-Exos不能降低H2O2诱导后的原代肝细胞内ROS水平。说明CBP-Exos可抑制H2O2所诱导的肝细胞内的氧化应激水平。
(2)Tunel染色检测对照组、H2O2组、CBP-Exos组和OBP-Exos组的原代肝细胞凋亡水平
小鼠原代肝细胞贴壁后分别加入CBP-Exos和OBP-Exos与原代肝细胞共孵育;共孵育16h后使用200nM H2O2诱导小鼠原代肝细胞产生氧化应激;诱导5h后使用Tunel染色法检测细胞凋亡。
Tunel染色法的操作步骤如下:
1)对于待检测的贴壁细胞爬片,PBS洗涤一次,然后用4%多聚甲醛固定细胞30min,PBS洗涤3次,3min/次;
2)用含0.3% Triton X-100的PBS进行细胞通透打孔,室温孵育10min,PBS洗涤3次,3min/次;
3)配制Tunel检测液:如表1所示。
表1反应体系组成
试剂 | 1个样本 |
TdT酶 | 5μL |
荧光标记液 | 45μL |
Tunel检测液 | 50μL |
4)取出细胞爬片,正面朝上放在载玻片上,加入50μL Tunel检测液,于湿盒中37℃避光孵育60min;
5)将细胞爬片放回孔板中,PBS洗涤3次,3min/次;使用DAPI进行细胞核染色,室温避光孵育15min,PBS洗涤3次,3min/次;
6)使用干净的做好标记的载玻片,滴加一滴抗荧光淬灭封片剂,小心将爬片取出,吸干多余水分,细胞面朝下倒扣在滴加了抗荧光淬灭封片剂的载玻片上,让封片剂自然扩散,避免产生气泡;
7)静置5min后进行图像采集,若不能及时拍照,则可置于-20℃保存,仍需尽快采集图像,避免荧光信号丢失。
我们进行了Tunel染色实验。其原理是细胞凋亡过程中基因组DNA会发生断裂,暴露出的3’-OH可以在末端脱氧核苷酸转移酶的催化下加上荧光素(FITC)标记的dUTP,细胞凋亡程度越高,荧光强度越强、阳性细胞数越多,从而可以通过荧光显微镜检测细胞的凋亡水平。Tunel染色结果如图4B所示,结果表明:H2O2诱导后Tunel阳性细胞数显著增加,而CBP-Exos处理后Tunel阳性细胞数显著减少,OBP-Exos处理组与H2O2组一致。这些结果表明体外CBP-Exos处理可以显著降低H2O2所诱导的氧化损伤模型下的肝细胞凋亡,而OBP-Exos无效果。
实施例5CBP-Exos与老年人血浆来源的外泌体(OBP-Exos)中MiRNAs的差异富集分析及RT-qPCR鉴定
具体的操作步骤如下:
(1)CBP-Exos和OBP-Exos中差异MiRNAs的火山图分析
由实施例3、4可知,CBP-Exos对急性肝损伤小鼠的肝保护作用。为了进一步的明确CBP-Exos内容物中发挥肝保护作用的关键分子,我们又进行了下述实验。
我们提取了脐带血血浆和对照组老年人血浆来源的外泌体,采用MiRNAs高通量测序技术分析了CBP-Exos和OBP-Exos中差异表达的MiRNAs。由于火山图可以方便直观地展示两个样本间差异表达的MiRNAs分布情况,我们对CBP-Exos和OBP-Exos之间差异表达的MiRNAs进行火山图分析,图中MiRNAs的差异倍数在2倍以上,p值小于0.05的被视为有统计学差异。
实验结果如图5A所示,结果表明,一共有34个差异表达的MiRNAs,其中16个MiRNAs在CBP-Exos中显著上调,18个MiRNAs在CBP-Exos中显著下调。我们进一步在CBP-Exos中显著上调的这18个MiRNAs进行筛选:①选择人和小鼠同源的差异MiRNAs;②选择火山图右上角的数据点(由于差异倍数越大的基因t检验越显著,所以左上角和右上角的数据点往往更具有生物学研究意义),最终筛选出的MiRNA为MiR-410-3p。)
(2)CBP-Exos和OBP-Exos中差异MiRNAs及其靶基因mRNA在肝损伤凋亡信号通路中的MiRNA-mRNA调控网络分析
由于在前面的表型研究中发现CBP-Exos具有肝保护作用,我们接下来对差异表达的MiRNAs及其靶基因mRNA进行肝损伤凋亡通路相关的MiRNA-mRNA调控网络分析。
调控网络分析结果如图5B所示,由图可见,MiR-24-3p(差异倍数2倍以上)也可能参与氧化应激、细胞凋亡信号通路。
(3)CBP-Exos中高富集的前十位差异MiRNAs的热图分析
为筛选出CBP-Exos中富集程度高且可能发挥肝保护作用的功能性MiRNAs,我们进一步分析了CBP-Exos中高富集的前十位差异MiRNAs并绘制热图进行筛选。
结果如图5C所示,可见,我们最终筛选出4个可能参与肝损伤途径并与小鼠同源的MiRNAs,分别为MiR-181a-5p、MiR-150-5p、MiR-191-5p和MiR-486-5p。
(4)RT-qPCR验证差异表达MiRNAs(MiR-24-3p、MiR-150-5p、MiR-181a-5p、MiR-191-5p、MiR-486-5p、MiR-410-3p)在CBP-Exos和OBP-Exos中的表达量
根据上述差异分析结果,我们一共筛选出人和小鼠同源的6个可能发挥肝保护作用的差异表达MiRNAs(MiR-24-3p、MiR-150-5p、MiR-181a-5p、MiR-191-5p、MiR-486-5p、MiR-410-3p),为进一步验证这些差异表达的MiRNAs在CBP-Exos和OBP-Exos中的表达水平,我们将提取的外泌体RNA进行MiRNAs的RT-qPCR验证,结果如图5D所示,这6个差异表达MiRNAs在CBP-Exos中的表达量均显著高于OBP-Exos。
实施例6差异表达MiRNAs抑制小鼠肝细胞氧化应激和凋亡的功能筛选
(1)ROS水平检测
为明确筛选出的这6个差异表达MiRNAs的功能,我们使用MiRNAmimic进行鉴定,MiRNA mimic是MiRNA的模拟物,它是针对内源性MiRNA成熟体结构设计合成的双链小RNA,可在DICER酶作用下形成单链MiRNA,从而增加MiRNA丰度,用于功能获得性研究。我们合成了这6个MiRNAs的mimic,其序列在下述序列的基础上,采用“u”代替“t”,序列如下所示:
SEQ ID NO:2、SEQ ID NO:3
MiR-24-3p:3'gacaaggacgacttgactcggt 5';3'accgagtcaagtcgtccttgtc 5'。
SEQ ID NO:4、SEQ ID NO:5
MiR-150-5p:3'gtgaccatgttcccaaccctct 5';3'agagggttgggaacatggtcac 5'。
SEQ ID NO:6、SEQ ID NO:7
MiR-181a-5p:3'tgagtggctgtcgcaacttacaa 5';3'ttgtaagttgcgacagccactca5'。
SEQ ID NO:8、SEQ ID NO:9
MiR-191-5p:3'gtcgacgaaaaccctaaggcaac 5';3'gttgccttagggttttcgtcgac 5'。
SEQ ID NO:10、SEQ ID NO:11
MiR-486-5p:3'gagccccgtcgagtcatgtcct 5';3'aggacatgactcgacggggctc 5'。
SEQ ID NO:12、SEQ ID NO:13
MiR-410-3p:3'tgtccggtagacacaatataa5';3'ttatattgtgtctaccggaca5'。
以NC mimic(锐博公司,miR1N0000001-1-5micrON mimic NC#22,5nmol)合成为阴性对照。接下来,我们观察了MiRNAs mimic对H2O2所诱导肝细胞氧化应激水平的影响,在MiRNAs mimic(100nM)转染AML12肝细胞24h后,使用200nM H2O2诱导肝细胞产生氧化应激,2h后使用DCFH-DA检测细胞内ROS水平变化。
结果如图6A所示,结果表明,与NC组相比,MiR-150-5p、MiR-486-5p和MiR-410-3p均显著降低了细胞内ROS水平。
(2)流式检测细胞凋亡
MiRNAs mimic转染AML12肝细胞24h后,使用200nM H2O2诱导肝细胞产生氧化应激,2h后使用流式细胞术检测细胞凋亡水平。
结果如图6B所示,结果表明,与NC组的凋亡细胞数相比,MiR-150-5p、MiR-181a-5p、MiR-486-5p均不同程度地减少了肝细胞凋亡,但只有MiR-410-3p具有显著抑制细胞凋亡的效果(P<0.05)。
实施例7CBP-Exos中发挥肝保护作用的MiRNA鉴定
根据上述MiRNAs mimic作用于损伤AML12肝细胞和原代肝细胞的功能验证结果,表明MiR-410-3p具有显著抑制损伤肝细胞氧化应激和凋亡的功能,而MiR-150-5p、MiR-486-5p可能具有肝保护作用。为确认我们筛选出的这3个MiRNAs是否在CBP-Exos发挥肝保护功能中起决定性作用,我们利用MiRNA inhibitor进行鉴定。我们分别合成了这3个MiRNAs相应的MiRNA inhibitor,核苷酸序列在下述序列的基础上,采用“u”代替“t”,序列如下所示:
SEQ ID NO:14
MiR-150-5p:3'agagggttgggaacatggtcac 5'。
SEQ ID NO:15
MiR-486-5p:3'aggacatgactcgacggggctc 5'。
SEQ ID NO:16
MiR-410-3p:3'ttatattgtgtctaccggaca5'。
同时以NC inhibitor(锐博公司,miR2N0000001-1-5micrOFF inhibitor NC#22,5nmol)作为阴性对照。
(1)ROS水平检测
将CBP-Exos与AML12肝细胞预孵育,同时分别转染这3个MiRNAs的inhibitor,以及阴性对照NC inhibitor,转染24h后使用200nM H2O2诱导肝细胞产生氧化应激,2h后使用荧光显微镜检测细胞内ROS水平。
结果如7A所示,结果显示,与NC inhibitor组相比,CBP-Exos+NC inhibitor组、CBP-Exos+MiR-150-5p inhibitor组和CBP-Exos+MiR-486-5p inhibitor组均显著降低了细胞内的ROS水平(P<0.05);而CBP-Exos+MiR-410-3p inhibitor组和NC inhibitor组无差异;说明只有在抑制MiR-410-3p的功能后,CBP-Exos降低损伤肝细胞内ROS水平的作用才会消失。
(2)流式检测细胞凋亡
将CBP-Exos与AML12肝细胞预孵育,同时分别转染这3个MiRNAs的inhibitor(100nM),以及阴性对照NC inhibitor(100nM),转染24h后使用200nM H2O2诱导肝细胞产生氧化应激,2h后使用Tunel染色法检测细胞凋亡水平。
结果如图7B所示,结果表明,与NC inhibitor组相比,CBP-Exos+NC inhibitor、CBP-Exos+MiR-150-5p inhibitor和CBP-Exos+MiR-486-5p inhibitor组均显著降低了细胞凋亡(P<0.05);而CBP-Exos+MiR-410-3p inhibitor组不能降低凋亡细胞数,与NCinhibitor组没有显著差异;说明只有抑制MiR-410-3p功能后,CBP-Exos抑制肝细胞凋亡的功能才会消失。
实施例8MiR-410-3p agomir能恢复ALI小鼠肝脏的正常结构和功能
我们在实施例6和7中证实了MiR-410-3p具有抑制肝细胞氧化应激和凋亡的肝保护作用,为了进一步验证MiR-410-3p是否在体内也能发挥肝保护作用,我们继续在ALI小鼠中进行了研究。我们采用了MiR-410-3p agomir来探究MiR-410-3p对ALI小鼠的治疗效果,同时以NC agomir作为对照(锐博公司,miR4N0000001-4-5micrON agomir NC#22,in vivo,5nmol)。MiR-410-3p agomir比体外研究中使用的MiR-410-3p mimic具有更高的稳定性,并且在复杂的环境中能保持更长时间的活性,同时具有更高的细胞透过率,在没有转染试剂的情况下,也可以克服细胞膜等障碍进入靶细胞中发挥作用,更适用于动物体内实验。
(1)各分组小鼠在建模后第2天的血清生化指标ALT和AST含量的检测
将CCl4与橄榄油按体积比1:5配制成工作液,现用现配;选取8-10周龄,体重在20-25g的雄性小鼠随机分组,模型组腹腔注射3μL/g的CCl4工作液;空白对照组腹腔注射等体积的橄榄油;MiR-410-3p agomir治疗组在CCl4建模同时给予尾静脉CBP-Exos输注5nmolMiR-410-3p agomir/只进行治疗,在建模后2d进行疗效评估。建模后第2天通过眼球取血法收集小鼠血液于1.5ml EP管中,4℃放置过夜,4℃3000rpm离心15分钟,取上层血清进行谷丙转氨酶ALT和谷草转氨酶AST的检测。
结果如图8A所示,小鼠血清ALT、AST水平检测结果显示,MiR-410-3p治疗可显著降低ALI小鼠的血生化ALT、AST的水平,恢复肝功能,并且优于CBP-Exos治疗组,原因可能是CBP-Exos更为复杂,CBP-Exos治疗后个体差异较大,导致显著性差异不如MiR-410-3p组。
(2)各分组小鼠在建模后第2天的肝组织H&E染色
在建模后第2天牺牲小鼠,收集小鼠肝组织,用PBS清洗后使用4%多聚甲醛固定;固定48h后进行石蜡包埋肝组织;进行肝组织切片和H&E染色。
结果如图8B所示,H&E染色结果表明,MiR-410-3p可显著恢复肝组织结构、减少肝坏死区域面积和炎性细胞浸润,与CBP-Exos治疗的效果相当。
(3)各分组小鼠在建模后第2天的肝组织F4/80免疫组化染色
在建模后第2天牺牲小鼠,收集小鼠肝组织,用PBS清洗后使用4%多聚甲醛固定;固定48h后进行石蜡包埋肝组织;进行肝组织切片和F4/80免疫组化染色。
为评估肝组织内炎性水平,我们对巨噬细胞标记物F4/80进行了免疫组化染色,结果如图8C所示,表明MiR-410-3p治疗后可显著减少巨噬细胞标记物F4/80的表达,提示MiR-410-3p治疗后炎症有所减轻。
综上所述,本发明首次发现人脐带血血浆来源的外泌体在肝脏损伤修复中的作用,并进一步发现人脐带血血浆来源的外泌体发挥关键作用的因子是MiR-410-3p,根据本发明的发现,可开发出MiR-410-3p在制备预防或治疗肝损伤药物中的新用途,为肝损伤修复提供了新的思路,并且提供了可产业化实施的条件,具有显著的进步。
Claims (9)
1.MiR-410-3p在制备预防或治疗肝损伤药物中的用途。
2.根据权利要求1所述的MiR-410-3p在制备预防或治疗肝损伤药物中的用途,其特征在于:所述的MiR-410-3p来自血浆外泌体。
3.根据权利要求2所述的MiR-410-3p在制备预防或治疗肝损伤药物中的用途,其特征在于:所述的血浆外泌体为脐带血血浆外泌体。
4.根据权利要求3所述的MiR-410-3p在制备预防或治疗肝损伤药物中的用途,其特征在于:所述的脐带血血浆外泌体为CBP-Exos。
5.根据权利要求1-4任一项所述的MiR-410-3p在制备预防或治疗肝损伤药物中的用途,其特征在于:所述的MiR-410-3p的核苷酸序列如SEQ ID NO:1所示。
6.根据权利要求1-5任一项所述的MiR-410-3p在制备预防或治疗肝损伤药物中的用途,其特征在于:所述肝损伤是指急性肝损伤或慢性肝纤维化。
7.根据权利要求1-5任一项所述的MiR-410-3p在制备预防或治疗肝损伤药物中的用途,其特征在于:所述的MiR-410-3p用量为0.2nmol/g体重小鼠。
8.本发明还提供了一种预防或治疗肝损伤的药物,其特征在于:包含权利要求1-7任一项中所述的MiR-410-3p。
9.根据权利要求8所述的预防或治疗肝损伤的药物,其特征在于:所述的MiR-410-3p为人脐带血血浆外泌体中的MiR-410-3p。
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CN111621558A (zh) * | 2020-06-10 | 2020-09-04 | 南通大学 | 与血脑屏障破坏程度相关的血清外泌体miR-410-3p的应用及其检测方法 |
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CN111621558A (zh) * | 2020-06-10 | 2020-09-04 | 南通大学 | 与血脑屏障破坏程度相关的血清外泌体miR-410-3p的应用及其检测方法 |
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