CN111593114A - miR-122及其抑制剂在预防/治疗放射性脑损伤中的应用 - Google Patents
miR-122及其抑制剂在预防/治疗放射性脑损伤中的应用 Download PDFInfo
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Abstract
本发明提供了一种miR‑122及其抑制剂在预防/治疗放射性脑损伤中的应,通过实验研究发现,miR‑122‑5p在照射后的小鼠海马及临床鼻咽癌放疗病人中的表达显著上调;而通过miR‑122干预后,在RBI动物模型中不仅能缓解神经炎症损伤,改善行为认知等;且体外细胞放射性损伤模型中miR‑122的抑制能调控小胶质细胞趋于M2型极化。
Description
技术领域
本发明涉及生物医学领域,具体涉及miR-122及其抑制剂在预防/治疗放射性脑损伤中的应用。
背景技术
放射性脑损伤(Radiation-induced Brain Injury,RBI)是头颈部肿瘤尤其是鼻咽癌(Nasopharyngeal Carcinoma,NPC)放射治疗下的主要剂量限制性不良事件,也是放射治疗最为严重的并发症之一,从广义上来说,放射性脑损伤是放射治疗后神经细胞和颅内血管受损后出现的一系列病理生理改变,可有影像学可见的脑部病灶。最常表现为广泛脑水肿引起的进行性颅内压增高、脑疝形成,并在疾病后期出现认知功能障碍、癫痫和严重痴呆等中枢神经系统(Central nervous system,CNS)损害。一旦发生将不可逆,目前尚无有效治疗方法。
RBI的发病机制以神经元凋亡、神经炎症、小胶质细胞和星形胶质细胞的极化最受关注。但目前治疗只依赖部分脑保护治疗药物及其它对症支持治疗,包括胞二磷胆碱、神经节苷脂、注射用鼠神经生长因子、维生素B12等。但对于肿瘤放射治疗后产生神经系统损伤的患者,不建议长期大剂量使用维生素B12。因其可通过叶酸促进细胞分裂,文献报道具有诱发肺癌风险。而研究表明,注射用鼠神经生长因子有减轻动物血脑屏障受损、修复微血管等作用,提示其能促进放射性脑损伤恢复,但其主要采用注射用药方式,且仅改善了脑水肿。
因此,找寻RBI发生发展通路中的可能靶点进行精准调控以及选择何种方式能快速直接地穿过血脑屏障,从而使药物进入到中枢神经系统对疾病进行干预是主要困难。
小胶质细胞激活及M1/M2表型转换与RBI、脑出血,帕金森病,阿尔兹海默病,多发性硬化等疾病发生及转归密切相关。小胶质细胞是CNS特有的免疫细胞,激活后的小胶质细胞通常处于经典的促炎型(M1)和抗炎修复型(M2)的动态平衡之中,其状态的转变与其所处的局部微环境及病理刺激有关。在损伤状态或炎性因素刺激下,激活的小胶质细胞胞体增大、突起变短、细胞形态呈圆形或杆状;此时小胶质细胞通常趋于M1样极化,产生促炎细胞因子,具有较强的微生物灭杀特征,但M1样过度活化会通过释放促炎因子及神经毒性介质造成细胞毒性,进而引发神经损伤的恶性循环。而M2型通常细胞突起变长或减少消失、细胞形态呈阿米巴状,并具有吞噬功能,可以抑制炎症并参与促进组织修复。照射刺激常引起CNS中的氧化应激,导致小胶质细胞向M1型过度激活,导致炎症因子如IL-1β、IL-6、TNF-α等被大量释放,从而介导RBI的发生。
而MiRNA(microRNA)作为经典的短链非编码单链RNA,通过与靶基因3'UTR结合,从而调控单个通路中的多种基因或多种交互通路,是CNS生理和病理进程的关键调节剂,是小胶质细胞活化和极化以及正常和病变中枢神经系统中巨噬细胞分化的普遍调节因子。小胶质细胞参与血脑屏障稳态维持,小胶质细胞极化相关的炎症转化及级联损伤是RBI病理进程的关键环节。目前,虽然围绕RBI中小胶质细胞相关通路的干预研究不断开展,但miRNA对RBI的直接预防或治疗用途还未被探索。
发明内容
针对现有技术中存在的问题,本发明的目的在于提供一种miR-122及其抑制剂在预防/治疗放射性脑损伤中的应用,以解决当前常用的放射性脑损伤药物存在副作用、治疗不理想的问题。
为了实现上述目的,本发明采用以下技术方案予以实现。
(一)miR-122在制备用于预防放射性脑损伤的药物中的应用。
优选的,所述放射性脑损伤为鼻咽癌放疗引起的放射性脑损伤。
(二)miR-122在制备用于治疗放射性脑损伤的药物中的应用。
优选的,所述用于治疗放射性脑损伤的药物还包括糖皮质激素、贝伐珠单抗、胞二磷胆碱、神经节苷脂、艾地苯醌、超氧化物歧化酶和维生素E中的至少一种。
(三)miR-122抑制剂在制备用于预防放射性脑损伤的药物中的应用。
(四)miR-122抑制剂在制备用于治疗放射性脑损伤的药物中的应用。
优选的,所述miR-122抑制剂为AntagomiR-122。
优选的,所述AntagomiR-122减少放射导致的小胶质细胞相关炎症因子的释放。
优选的,所述AntagomiR-122促进被照射后的小胶质细胞向M2型极化。
相比于现有技术,本发明的有益效果在于:
1)本发明的实验研究发现,miR-122-5p在照射后的小鼠海马及临床鼻咽癌放疗病人中的表达显著上调;而通过miR-122干预后,在RBI动物模型中不仅能缓解神经炎症损伤,改善行为认知等;且体外细胞放射性损伤模型中miR-122的抑制能调控小胶质细胞趋于M2型极化。
2)本发明的实验还表明,TNS1与miR-122具有潜在的结合作用。而现有的研究证明,TNS1参与调控人乳腺癌细胞生物学行为及放疗抵抗,也参与肿瘤细胞外基质重塑、细胞极化,迁移和侵袭过程;并且与创伤性脑损伤模型中的神经功能恢复、局灶性脑缺血性损伤等发生发展密切相关。因此,结合胶质细胞及神经炎症相关通路预测并检测筛选出miR-122的潜在下游靶点TNS1,miR-122干预能调控小胶质细胞向M2型极化从而缓解RBI中炎症级联损伤,来预防或治疗放射性脑损伤疾病。
3)由于所述用于预防放射性脑损伤的药物包括有效剂量的miR-122抑制剂,特别是AntagomiR-122,因此,所述药物能够抑制体内的miR-122发挥作用,从而降低炎症因子的表达,达到预防或治疗放射性脑损伤的目的。
附图说明
图1为RBI模型小鼠及NPC放疗后患者的差异表达miRNA的筛选图;其中,图1A为小鼠行为学测试水迷宫数据及小鼠脑组织切片海马区组织的HE染色结果图;图1B~C为小鼠海马miRNA二代测序及RT-qPCR验证结果图;图1D为鼻咽癌患者放疗前后的S100B、miR-122和IL-1β的表达水平变化图。
图2为Antagomir-122鼻腔给药改善照射后行为认知图;其中,图2A为小鼠行为学测试水迷宫数据图;图2B为小鼠水迷宫测试的活动轨迹图;图2C为小鼠水迷宫测试的穿越平台次数图;图2D为小鼠水迷宫测试的目标象限活动时间图;图2E为小鼠水迷宫测试的目标象限路程占比百分图。
图3为Antagomir-122鼻腔给药改善照射后病理损伤图。
图4为Antagomir-122鼻腔给药改善照射后小胶星胶及神经元炎症损伤图;其中,图4A为小鼠海马区RT-qPCR的数据图;图4B为小鼠海马区TNF-α的表达水平图;图4C为小鼠海马区S100B的表达水平图;图4D为小鼠海马区Iba-1/DAPI图;图4E为小鼠海马区GFAP/DAPI图;图4F为小鼠海马区TUBBLIN/DAPI图。
图5为体外放射性损伤细胞模型干预通过调控小胶质细胞极化改善小胶质细胞炎症级联损伤图;其中,图5A为小胶质细胞体外转染Antagomir-122及450nm处吸光度值数据图;图5B为IL-1β和IL-6炎症因子水平表达数据图;图5C为人神经母细胞瘤细胞SH-SY5Y中转染Antagomir-122后miR-122相对表达的转染效率;图5D为小胶质细胞BV2和神经元细胞SH-SY5Y共培养之后神经元细胞采用流式细胞术Tunel染色标记出的晚期凋亡和坏死的情况;图5E为小胶质细胞BV2和神经元细胞SH-SY5Y共培养之后神经元细胞采用流式细胞术Tunel染色标记出的早期凋亡情况;图5F为小胶质细胞BV2和神经元细胞SH-SY5Y共培养之后的流式凋亡展示图。
图6为miR-122轴调控小胶质细胞极化下游靶点筛选图;其中,图6A为转录组分析mRNA测序图;图6B为TNS1与miR-122序列图;图6C为TNS1与miR-122构建双荧光素酶载体图;图6D为蛋白免疫印迹验证图。
具体实施方式
为使本发明的技术方案和优点更加清楚,下面将结合具体实施方式和说明书附图,对本发明及其有益效果作进一步详细的描述,但本发明的实施方式不限于此。
专有名词解释:
AntagomiR-122为micrOFF hsa-miR-122-5p antagomir进行干预,Antagomir主要在3个方面做了修饰:①RNA骨架的每个核苷酸都在呋喃糖的2位碳羟基上连接1个甲基;②核苷酸两端都被磷硫酰修饰;③在核酸的3’端连上一个功能性的胆固醇基团。其中,前2个修饰大大提高了它对于核酸酶的耐性,增长了这些分子在细胞内的半衰期。
而AntagomiR具有的优势有:micrOFF miRNA antagomir是经过特殊化学修饰的miRNA拮抗剂,通过与体内成熟的miRNA强竞争性结合,阻止miRNA与其靶基因mRNA的互补配对,抑制miRNA发挥作用。miRNA antagomir均为miRNA成熟链的反向互补序列,全链进行甲基化修饰,在5’端和3’端分别有2个和4个碱基硫代修饰,并在3’端连接有高亲和性胆固醇修饰。与普通抑制剂相比,micrOFF miRNA antagomir在动物实验和细胞实验中具有更高的稳定性和抑制效果,更易通过细胞膜、组织间隙而富集于靶细胞。而本发明实施例中采用的AntagomiR-122为广州锐博公司合成。
鼻脑通路给药:主要是以无创方式绕过血脑屏障,鼻内途径可沿着嗅觉将药物从鼻腔直接运输到大脑和三叉神经。鼻内途径由两种途径组成,一是细胞内途径,二是细胞外途径。细胞内途径从嗅觉感觉细胞的内吞作用开始,然后轴突转运到嗅球中的突触裂隙,药物被胞吐。嗅觉神经元会重复进行这种突触过程,从而将药物分配到其他大脑区域。在细胞外机制中,药物首先穿过脑脊液直接转运到脑脊髓液中,跨鼻上皮细胞间隙,然后穿过神经周围间隙到达大脑的蛛网膜下腔。
S100B:一种钙离子结合蛋白,在脑中主要由神经胶质细胞分泌。
Iba1:一个17kDa的钙结合蛋白,离子钙结合衔接分子1(Iba1)是小胶质细胞和巨噬细胞特异性的钙结合蛋白,参与激活的小胶质细胞的细胞膜皱褶形成和吞噬作用,通常作为中枢神经系统中的小胶质细胞标记物。
GFAP:胶质纤维酸性蛋白(glial fibrillary acidic protein),是星形胶质细胞活化的标志物。
β-tubulinⅢ:神经元细胞骨架微管蛋白。
实施例1
如图1所示,miR-122-5p表达量与放射性脑损伤的关系:
通过30Gy的照射剂量构建了RBI模型小鼠(见图1A),然后对其海马进行了miRNA二代测序。测试结果提示miR-122在RBI小鼠海马中表达异常升高,且RT-qPCR验证结果与基因测序结果一致(见图1B~C)。
随后,为进一步分析miR-122表达量与RBI严重程度的关联性,本发明人对28名鼻咽癌患者放疗前、放疗第2周、放疗第4周、和放疗结束后1个月的血清中S100B、miR-122和IL-1β表达量进行了检测。结果显示放疗后鼻咽癌患者的S100B、miR-122和IL-1β水平随放疗的剂量增加逐渐升高,且均比放疗前的表达量要高(见图1D)。
由测试结果可知miR-122-5p在RBI模型小鼠海马及鼻咽癌放疗病人血清中显著上调,提示miR-122与RBI疾病发生/发展相关联。
实施例2
如图2~4所示,在实施例1的基础上,通过Antagomir-122鼻腔给药进行动物水平干预,同样采用水迷宫对小鼠行为进行测试,其中,鼻腔给药的具体步骤为:将Antagomir-122溶解于1mL无RNase的水中,将小鼠用简单的鼠固定器固定并仰卧,然后使用移液器滴加24ul Antagomir-122(40nmol/mL)溶液,左右鼻孔交替注射(1ul/次),每次注射间隔3-5min。经鼻内递送24小时后取海马组织,并使用实时荧光定量聚合酶链式反应(RT-qPCR)进一步分析经鼻内递送Antagomir-122后海马中miR-122的动态变化。其中,可采用TRIzol抽提组织总RNA,实时荧光定量聚合酶链式反应可用SYBR RT-PCR试剂盒(Takara),在LightCyclerl(Roche)罗氏实时荧光定量PCR仪上完成,MiRNA的相对定量使用2-ΔΔCt方法进行计算,以U6作为内参,引物序列如下:
MiR-122-5p:
前:5'-CGCGTGGAGTGTGACAATGG-3';
后:5'-AGTGCAGGGTCCGAGGTATT-3';
内参U6:
前:5'-ATTGGAACGATACAGAGAAGATT-3';
后:5'-GGAACGCTTCACGAATTTG-3'。
测试结果如下:
1)行为认知:如图2所示,通过水迷宫测试,miR-122干预组与照射组相比,小鼠找寻平台潜伏期显著缩短(见图2A);撤去平台后,miR-122干预后小鼠穿越平台次数增多,说明其空间学习记忆能力较好(见图2C);撤去平台后,miR-122干预显著改善了小鼠活动轨迹,找寻平台及平台附近活动轨迹增多,且在平台所在象限探索时长及路程显著增加,表明其空间记忆能力较好(见图2B,D,E)。由此可见,miR-122干预能改善小鼠空间记忆及认知障碍。
2)病理损伤:如图3所示,通过脑组织切片HE染色,miR-122干预可改善照射后神经元面积缩小、胞浆染色变暗、海马核固缩和间质水肿等病理损伤。
3)小胶质细胞、星形胶质细胞及神经元炎症损伤:如图4所示,miR-122干预显著降低了小鼠海马区miR-122的相对表达(见图4A);且显著降低RBI相关炎症指标肿瘤坏死因子-α(TNF-α)水平及血脑屏障破坏后胶质细胞损伤特异性蛋白S100B水平(见图4B~C);而通过Iba-1与DAPI分别标记小胶质细胞及细胞核,发现,照射后小胶质细胞过度活化,经miR-122干预后小胶质细胞突起长度较照射后增加(见图4D);再通过GFAP与TUBBLIN分别标记星形胶质细胞与神经元细胞骨架,照射后星胶过度活化,而神经元骨架蛋白明显减少,表明照射后神经元骨架破坏,经miR-122干预后,GFAP标记的星胶面积与荧光强度显著降低,并改善了照射后的cortex and hippocampus(即皮层和海马区域中免疫荧光标记的神经元骨架破坏),见图4E~F。由此可见,miR-122干预能够减轻小胶质细胞、星形胶质细胞及神经元细胞的炎症损伤。
当将Antagomir-122实际应用于临床试验时,Antagomir-122的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定,所述的因素包括但不限于:Antagomir-122的药代动力学参数例如生物利用率、代谢、半衰期等;患者所要治疗的疾病、患者的体重、患者的免疫状况、给药的途径等。
具体的,Antagomir-122还可以与常见的放射性损伤预防和治疗药物相配合使用,常见的放射性损伤预防和治疗药物包括糖皮质激素、贝伐珠单抗、胞二磷胆碱、神经节苷脂、艾地苯醌、超氧化物歧化酶和维生素E中的至少一种。此外,Antagomir-122还可以与其它药物和治疗手段(例如高压氧治疗、手术疗法)联合,用于放射性脑损伤的预防和治疗。
实施例3
如图5所示,miR-122-5p抑制剂体外抑制放射性细胞损伤:
对小胶质细胞BV2进行了AntagomiR-122转染后,进行10Gy的照射并对小胶质细胞的形态、吞噬功能以及相关炎症因子进行分析,其中,图5A为采分别采用50nmol和100nmol转染效率的数据;图5C为采用的100nmol转染效率的数据。结果表明,miR-122干预后改善了照射导致的细胞活力下降(见图5A);miR-122干预后显著减轻了照射导致的IL-1β和IL-6炎症因子水平(见图5B),其中,图5B中,免疫荧光Iba-1标记小质细胞胶,DAPI染细胞核,beads是加入细胞中用于评估小胶质细胞吞噬活力的荧光微球,merge代表汇总重叠图,由该图可看出AntagomiR-122能够减少放射导致的小胶质细胞相关炎症因子释放,且使得照射后的小胶质细胞突起变长,提示小胶质细胞向M1极化减少,且吞噬能力得到增强,说明由M1样极化向M2型极化转换;此外,miR-122干预后改善了照射后与小胶质细胞共培养的神经元细胞SH-SY5Y凋亡(见图5C-F)。由此可推测出,miR-122-5p抑制剂通过调控小胶质细胞极化进而干预RBI的发生/发展。
实施例4
如图6所示,miR-122-5p抑制剂通过调节小胶质细胞极化体外抑制放射性细胞损伤的下游靶点筛选:
通过转录组分析mRNA(message RNA)测序筛选了正常、照射及miR-122鼻腔给药干预各组小鼠的海马组织进行分析。结果表明,TNS1在照射后被显著下调,且该下调被miR-122的干预逆转了(见图6A)。而通过TNS1与miR-122序列和构建双荧光素酶载体表明(见图6B~C),TNS1与miR-122具有潜在的结合作用,其中,TNS1-WT与TNS1-MUT分别代表TNS1野生型与突变型,相对荧光强度正是验证了两者存在结合关系。再通过蛋白免疫印迹验证(见图6D),蛋白免疫印迹同样也支持TNS1可能是miR-122的下游靶点,其中,miR-122mimics为miR-122模拟物,miR-122inhibitor为miR-122抑制剂,control组为正常对照组,以α-Tubulin作为内参。
由上述的实验结果可知,miR-122与RBI疾病发生/发展相关联,通过miR-122干预,能有效改善行为认知、病理损伤和小胶质细胞、星形胶质细胞及神经元炎症损伤。而TNS1作为miR-122的下游靶点,通过miR-122-5p抑制剂可以调控小胶质细胞向M2型极化,进而干预RBI的发生/发展。本发明不仅揭示了miR-122-5p在预防/治疗放射性脑损伤中的新用途,也为miR-122-5p、乃至miRNA的研究和开发利用提供了新的思路和途径。
此外,相比于现有技术,本发明采用的miR-122抑制剂副作用小,且可以有效预防/治疗放射性脑损伤,为本领域提供了一种新颖的放射性脑损伤预防剂和/或治疗剂,具有一定的临床应用前景。
根据上述说明书的揭示和教导,本发明所属领域的技术人员还能够对上述实施方式进行变更和修改。因此,本发明并不局限于上述的具体实施方式,凡是本领域技术人员在本发明的基础上所作出的任何显而易见的改进、替换或变型均属于本发明的保护范围。此外,尽管本说明书中使用了一些特定的术语,但这些术语只是为了方便说明,并不对本发明构成任何限制。
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上海艾博思生物科技有限公司
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Claims (9)
1.miR-122在制备用于预防放射性脑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,所述放射性脑损伤为鼻咽癌放疗引起的放射性脑损伤。
3.miR-122在制备用于治疗放射性脑损伤的药物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述用于治疗放射性脑损伤的药物还包括糖皮质激素、贝伐珠单抗、胞二磷胆碱、神经节苷脂、艾地苯醌、超氧化物歧化酶和维生素E中的至少一种。
5.miR-122抑制剂在制备用于预防放射性脑损伤的药物中的应用。
6.miR-122抑制剂在制备用于治疗放射性脑损伤的药物中的应用。
7.根据权利要求5或6所述的应用,其特征在于,所述miR-122抑制剂为AntagomiR-122。
8.根据权利要求7所述的应用,其特征在于,所述AntagomiR-122减少放射导致的小胶质细胞相关炎症因子的释放。
9.根据权利要求7所述的应用,其特征在于,所述AntagomiR-122促进被照射后的小胶质细胞向M2型极化。
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