CN115896111A - 一种抑制acsl4表达的小干扰rna及其应用 - Google Patents
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Abstract
本发明涉及医药技术领域及神经退行性疾病技术领域,涉及一种抑制ACSL4表达的小干扰RNA及其应用,小干扰RNA的核苷酸序列为:正义链:5’‑GAUGGAUGCUUACAGAUUAtt‑3’;反义链:5’‑UAAUCUGUAAGCAUCCAUCtt‑3’。本发明首次通过设计并合成小干扰RNA来抑制ACSL4表达,显著抑制小胶质细胞介导的神经炎症,并减轻MPTP诱导的帕金森病中多巴胺神经元的死亡率,改善帕金森疾病的运动神经功能。本发明为小干扰RNA的临床应用提供了重要的理论基础,也为治疗帕金森病的药物研发提供了新思路。
Description
技术领域:
本发明涉及医药技术领域及神经退行性疾病技术领域,涉及一种抑制ACSL4表达的小干扰RNA及其应用,利用这个小干扰RNA治疗帕金森疾病。
背景技术:
帕金森病(Parkinson′s disease)是一种常见的中老年神经系统退行性疾病,主要以黑质多巴胺能神经元进行性退变和路易小体形成的病理变化,纹状体区多巴胺递质降低、多巴胺与乙酰胆碱递质失平衡的生化改变,震颤、肌强直、动作迟缓、姿势平衡障碍的运动症状和睡眠障碍、嗅觉障碍、自主神经功能障碍、认知和精神障碍等非运动症状的临床表现为显著特征。目前被广泛接受的治疗策略是早期使用抗帕金森病药物,但抗帕金森病药物治疗时不能突然停药,特别是使用左旋多巴及大剂量多巴胺受体激动剂时,以免发生撤药恶性综合征。且使用抗帕金森病药物之后易感精神疾病,比如焦虑、失眠、精神错乱等。不少研究认为神经炎症是早期导致产生帕金森疾病的原因,目前针对神经炎症进行帕金森治疗的药物还没有报道。
Long-chain acyl-coenzyme A synthetases(ACSLs)是在脂质代谢中发挥重要作用的一个家族蛋白酶。作为ACSL家族的一员,ACSL4表现出优先使用花生四烯酸(AA)作为底物,并通过结合游离AA来促进含AA磷脂的重塑。目前,许多研究揭示代谢相关的酶在神经炎症起始阶段发挥重要调控作用,而ACSL4在神经炎症及帕金森病中的作用未能得到充分阐述。RNA干扰(RNAi)是有效沉默或抑制目标基因表达的过程,该过程通过双链RNA(dsRNA)使得目标基因相应的mRNA选择性失活来实现的。RNA干扰由转运到细胞细胞质中的双链RNA激活。沉默机制可导致由小干扰RNA(siRNA)或短发夹RNA(shRNA)诱导实现靶mRNA的降解,或者通过小RNA(miRNA)诱导特定mRNA翻译的抑制。目前国内外还未发现关于通过设计抑制ACSL4表达的siRNA来抑制神经炎症,进而抑制帕金森金病的发病程度的研究报道。
发明内容:
本发明的目的是为了克服现有技术存在的缺陷,提供一种抑制ACSL4表达的小干扰RNA(siACSL4)及其应用,siACSL4能抑制小胶质细胞介导的神经炎症,进而抑制帕金森病发病进程。
为了实现上述目的,本发明提供一种抑制ACSL4表达的小干扰RNA,(siACSL4),所述siACSL4的核苷酸序列如下所示:
正义链:5’-GAUGGAUGCUUACAGAUUAtt-3’(SEQ ID NO.1);
反义链:5’-UAAUCUGUAAGCAUCCAUCtt-3’(SEQ ID NO.2)。
为了在体内实验对ACSL4的敲减,本发明针对ACSL4的siRNA设计了shRNA,序列为:上链:GATCCGAGGCTTCCTATCTGATTACTCGAGTAATCAGATAGGAAGCCTCTTTTTTG;下链:AATTCAAAAAAGAGGCTTCCTATCTGATTACTCGAGTAATCAGATAGGAAGCCTCG。
本发明还提供所述抑制ACSL4表达的siACSL4及shACSL4在制备治疗帕金森病药物中的应用。
本发明与现有技术相比,有益效果如下:
(1)设计并合成了一种针对ACSL4的小RNAsiACSL4,siACSL4(正义链:5’-GAUGGAUGCUUACAGAUUAtt-3’;反义链:5’-UAAUCUGUAAGCAUCCAUCtt-3’);
(2)针对小胶质细胞BV2,给予siACSL4处理,脂多糖(LPS)刺激后,siACSL4明显降低炎症的发生发展;
(3)针对小胶质细胞BV2,给予siACSL4或对照组处理,脂多糖(LPS)刺激,再与小鼠皮层神经元共培养;ACSL4表达被敲低后胶质细胞的培养上清跟对照相比显著增加了神经元的存活;
(4)设计并制备了干扰ACSL4表达的shACSL4慢病毒
pHBLV-U6-MCS-EF1-mcherry-T2A–PURO(shACSL4),用于验证抑制ACSL4表达的小干扰RNA能够在体内治疗帕金森疾病;
(5)针对C57BL/6小鼠,在黑质区注射shACSL4后构建帕金森病模型小鼠,shACSL4可以有效抑制小胶质细胞的激活,减少炎性因子产生,减轻神经炎症反应;可以有效减轻多巴胺神经元的死亡,改善帕金森小鼠运动神经功能表现。
本发明首次通过设计并合成小干扰RNA(siACSL4)来抑制ACSL4表达,显著抑制小胶质细胞介导的神经炎症,并减轻MPTP诱导的帕金森病中多巴胺神经元的死亡率,改善帕金森疾病的运动神经功能。本发明为小干扰siRNA的临床应用提供了重要的理论基础,也为治疗帕金森病的药物研发提供了新思路。
附图说明:
图1为本发明涉及的实施例1合成的抑制ACSL4表达的小RNA siACSL4在BV2小胶质细胞细胞系上敲减ACSL4表达的效率示意图。
图2为本发明涉及的实施例2siACSL4对小胶质细胞细胞系BV2细胞神经炎症的影响实验结果示意图,其中A为对NFκB-p65磷酸化水平的影响,B为对ACSL4、TNFA、IL-6、IL-1b的mRNA表达水平的影响,C为对TNF-α,IL-6,IL-1β的表达水平的影响。。
图3为本发明涉及的实施例3siACSL4对小胶质细胞上清跟神经元共培养中神经元的存活率影响结果示意图,其中A为神经元-BV2细胞共培养示意图;B、C、D为神经元存活率检测结果。
图4为本发明涉及的抑制ACSL4表达的shACSL4慢病毒的载体图谱。
图5为本发明涉及的实施例5shACSL4对小鼠帕金森病过程中小胶质细胞激活产生的神经炎症的影响实验结果示意图,其中A是shACSL4侵染小胶质细胞,敲减小胶质细中ACSL4表达的效率;B是对ACSL4、TNFA、IL-6、IL-1b的mRNA表达水平的影响;C是对小胶质细胞分支数量和长度的影响
图6为本发明涉及的实施例6shACSL4对小鼠帕金森病过程中多巴胺神经元的存活率影响实验结果示意图,其中sham为未做任何处理的小鼠,shNC为注射了对照病毒的小鼠,shACSL4为注射了ACSL4慢病毒的小。
图7为本发明涉及的实施例7shACSL4对小鼠帕金森病过程中运动神经功能影响实验结果示意图。
具体实施方式:
下面通过具体实施例并结合附图对本发明作进一步说明。
实施例1:
本实施例涉及抑制ACSL4基因表达的小分子干扰序列siACSL4,siACSL4的序列为:
正义链:5’-GAUGGAUGCUUACAGAUUAtt-3’(SEQ ID NO.1);
反义链:5’-UAAUCUGUAAGCAUCCAUCtt-3’(SEQ ID NO.2)。
所述siACSL4序列由上海吉玛基因股份有限公司合成。
本实施例涉及siACSL4对小胶质细胞细胞系BV2细胞中ACSL4表达的敲减效率实验,具体步骤如下:
(1)分组转染:将BV2细胞均匀铺在12孔板中,一共8个小孔,分为2组,实验组(siACSL4)4个小孔转染siACSL4,对照组(siNC)4个小孔转染siNC,转染48h后进行蛋白水平检测实验。将BV2细胞均匀铺在24孔板中,一共4个小孔,分为2组,实验组(siACSL4)2个小孔转染siACSL4,对照组(siNC)2个小孔转染siNC,转染48h后进行RNA水平检测实验;重复做三组实验;
所述siNC序列作为阴性对照,由上海吉玛基因股份有限公司合成,具体序列如下:
正义链:UUCUCCGAACGUGUCACGUTT;
反义链:ACGUGACACGUUCGGAGAATT;
(2)蛋白水平检测实验:使用LPS刺激12孔板实验组和对照组细胞分别0,0.5,1,2h后,在细胞中加入cell lysis收取细胞蛋白,使用western blotting技术检测ACSL4及β-actin水平,结果如图1A所示。从图1A可以看出,实验组ACSL4在蛋白水平的表达被显著敲减。
(3)RNA水平实验:使用LPS刺激24孔板实验组和对照组分别0,4h,对细胞提取RNA进行反转录及荧光定量分析,结果如图1B所示。从图1B可以看出,实验组ACSL4在RNA水平也被明显敲减。
实施例2:
本实施例涉及siACSL4对小胶质细胞细胞系BV2细胞神经炎症的影响实验,具体步骤如下:
(1)分组转染:将BV2细胞均匀铺在12孔板中,一共8个小孔,分为2组,实验组(siACSL4)4个小孔转染siACSL4,对照组(siNC)4个小孔转染siNC,转染48h之后使用脂多糖(LPS)刺激细胞;重复做三组实验。
(2)用LPS刺激细胞0、0.5、1、2小时之后,加入cell lysis收取细胞蛋白,使用western blotting技术检测NFκB-p65磷酸化水平;结果如图2A所示;
(3)用LPS刺激细胞0、4小时之后,加入RA2 lysis收取细胞RNA,反转之后使用荧光定量技术检测ACSL4、TNFA、IL-6、IL-1b的mRNA表达水平;结果如图2B所示;
(4)用LPS刺激0、8小时之后,收取细胞上清,使用ELISA技术检测TNF-α,IL-6,IL-1β的表达水平;结果如图2C所示。
从图2A可以看出,BV2细胞转染siACSL4后,siACSL4显著降低了NFκB-p65磷酸化的表达;从图2B可以看出,BV2细胞转染siACSL4后,siACSL4显著降低了ACSL4、TNFA、IL-6、IL-1b的mRNA表达水平;从图2C可以看出,BV2细胞转染siACSL4后,siACSL4显著降低了TNF-α,IL-6,IL-1β等炎性因子的表达水平。
实施例3:
本实施例涉及siACSL4对小胶质细胞上清跟神经元共培养中神经元的存活率的影响实验,具体步骤如下:
(1)分组转染:将BV2细胞均匀铺在12孔板中,将BV2细胞均匀铺在24孔板中,一共4个小孔,分为2组,实验组(siACSL4)2个小孔转染siACSL4,对照组(siNC)2个小孔转染siNC。两天之后使用脂多糖(LPS)或者PBS刺激细胞;重复做三组实验;
(2)用LPS刺激0、8小时后,收集上清;离心弃去细胞碎片,将上清储存于-80℃条件;
(3)取18个胚胎日龄的C57BL/6J胎鼠,提取胎鼠皮层神经元细胞;
(4)在用NB培养基培养神经元7天时,将神经元培养基(NB)与储存于-80℃的BV2细胞上清体积比1:1混合为条件培养基;使用条件培养基培养神经元24小时,cck8检测细胞死亡率,结果如图3B所示;另外使用免疫荧光技术,用anti-MAP2标记神经元,测量免疫荧光强度检测细胞死亡率,使用Image J技术对结果统计分析,结果如图3C和3D所示。
从图3B可以看出,在脂多糖(LPS)刺激下,siACSL4组敲减ACSL4表达之后的BV2细胞与神经元共培养,与siNC组相比显著提高神经元的存活率。使用活细胞工作站显微镜进行map2的观察及拍摄,最后使用imageJ软件对拍摄的图片进行分析得到定量的数据。从图3C可以看出,无论BV2细胞接受脂多糖(LPS)还是PBS刺激后与神经元共培养,使用siACSL4敲减ACSL4表达的实验组MAP2数目都明显高于siNC组;从图3D可以看出,在脂多糖(LPS)刺激下,siACSL4组敲减ACSL4表达之后的BV2细胞与神经元共培养,MAP2神经元的数目明显高于siNC组。
实施例4:
本实施例设计了抑制ACSL4表达的慢病毒pHBLV-U6-MCS-EF1-mcherry-T2A–PURO(shACSL4),用于验证小干扰RNA能够在体内通过抑制ACSL4表达来治疗帕金森病,shACSL4核苷酸序列如下:
上链:GATCCGAGGCTTCCTATCTGATTACTCGAGTAATCAGATAGGAAGCCTCTTTTTTG;下链:AATTCAAAAAAGAGGCTTCCTATCTGATTACTCGAGTAATCAGATAGGAAGCCTCG,
其中带有下划线的序列为靶向ACSL4的siRNA序列,不带下划线的序列为pHBLV-U6-MCS-EF1-mcherry-T2A–PURO载体中的固有序列,该载体由上海汉恒公司提供。
由于siACSL4适用于细胞上的实验,在动物上需要使用shACSL4,用于敲减小鼠大脑小胶质细胞中ACSL4的表达,但shACSL4最终也将以siRNA的形式在小鼠脑小胶质细胞中敲减ACSL4的表达。如图4A所示为所选用的pHBLV-U6-MCS-EF1-mcherry-T2A-PURO载体图谱。最终如图4B所示步骤做出shACSL4慢病毒成品,之后在动物上使用。
实施例5:
本实施例涉及shACSL4对小鼠帕金森病过程中小胶质细胞激活产生的神经炎症的影响实验,具体为:
(1)使用C57BL/6J小鼠,购买自济南朋悦实验动物繁育有限公司,许可证号:SYXK(鲁)20190003。小鼠饲养于青岛大学生物医学中心实验动物平台,适应性饲养7天后开展实验;
(2)shACSL4病毒由上海汉恒公司进行制备,l-methyl-4-phenyl-l,2,3,6–tetrahydropypridine(MPTP)购买自西格玛(M0896);
(3)12只老鼠分为实验组(shACSL4)和对照组(shNC),使用立体定位注射仪在实验组和对照组小鼠黑质区分别注射shACSL4慢病毒及shNC慢病毒(shNC序列为:TTCTCCGAACGTGTCACGTAA);两周之后对所有进行病毒注射的老鼠进行MPTP注射(20mg/kg),间隔两小时注射,总计注射4次,构建帕金森模型小鼠(MPTP小鼠);在模型制作前、模型制作成功后,检测黑质区的多巴胺神经元存活情况,验证模型制作成功;
(4)在注射MPTP3天之后,杀灭老鼠取脑冰冻切片,使用小胶质细胞标记蛋白抗体(anti-Iba-1),另外取脑进行免疫荧光染色及荧光定量PCR实验评估shACSL4对黑质区域神经炎症的影响;结果如图5所示。
从图5A可以看出,shACSL4被成功注射至小鼠黑质区域并有效的抑制ACSL4的表达;从图5B可以看出,shACSL4显著降低MPTP小鼠脑小胶质细胞激活导致的神经炎症;从图5C可以看出shACSL4增加了小胶质细胞分枝数目及分枝长度。
实施例6:
本实施例涉及shACSL4对小鼠帕金森病过程中多巴胺神经元的存活率影响实验,具体为:
(1)实验组和对照组的实验条件如实施例5所示,另取6只C57BL/6J小鼠作为sham组,该组小鼠不做任何处理;
(2)在注射MPTP7天之后,取脑冰冻切片,使用小胶质细胞标记蛋白抗体(anti-TH)进行荧光染色,标记多巴胺神经元TH+评估shACSL4对帕金森病多巴胺神经元的影响,结果如图6所示。
从图6可以看出,shACSL4明显改善小鼠MPTP后多巴胺存活情况。
实施例7:
本实施例涉及shACSL4对小鼠帕金森病过程中运动神经功能影响实验,具体为:
(1)实验条件如实施例6所示;
(2)小鼠接受MPTP注射前,以及在注射MPTP5天后,使用转棒实验检测小鼠的运动神经功能,评估shACSL4对帕金森病运动神经功能的影响,结果如图7所示。
从图7可以看出,shACSL4能明显改善小鼠MPTP后的运动神经功能。
Claims (2)
1.一种抑制ACSL4表达的小干扰RNA,其特征在于,小干扰RNA为siACSL4,其核苷酸序列如下所示:
正义链:5’-GAUGGAUGCUUACAGAUUAtt-3’;
反义链:5’-UAAUCUGUAAGCAUCCAUCtt-3’。
2.权利要求1所述抑制ACSL4表达的小干扰RNA在制备治疗帕金森病药物中的应用。
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