CN110305951A - miR-129-5p作为靶分子在制备代谢性疾病药物中的应用 - Google Patents
miR-129-5p作为靶分子在制备代谢性疾病药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种miR‑129‑5p作为靶分子在制备代谢性疾病药物中的应用。本发明证明了miR‑129‑5p在脂肪细胞成脂中可抑制脂肪细胞脂滴生成及堆积，尤其是在白色脂肪细胞棕色化以及棕色细胞成脂，证实了miR‑129‑5p有可能成为预测和干预治疗肥胖或其他代谢性疾病的重要靶分子。
Description
技术领域
本发明属于代谢性疾病治疗领域，特别涉及一种miR-129-5p作为靶分子在制备代谢性疾病药物中的应用。
背景技术
miRNA作为小分子非编码RNA，从转录水平调节下游mRNA，通过部分特异性结合于靶mRNA的3’非编码区阻断mRNA编码，从而抑制蛋白质的合成。首次被确认的miRNA是在线虫中的lin-4/let-7通过异性结合其下游靶点mRNA通过上述方式，进而调控线虫的发育过程。据目前的研究显示miRNAs在肺部疾病，心脑血管疾病，与年龄相关的阿尔兹海默症，感染性疾病如败血症，代谢相关性疾病，以及各种肿瘤的发生都有一定的作用。同时随着肥胖以及糖尿病的发病率逐渐增加，对miRNAs在代谢中的作用研究也成为近十年热点研究方向。miRNAs在肥胖发生过程具有重要的作用。且可能通过参与脂肪分化过程进行调节相关代谢过程。白色脂肪细胞以及棕色脂肪细胞miRNAs皆有一定的作用。
目前，miR-129-5p是否参与脂肪细胞分化过程、是否对代谢性疾病有作用仍未明确。
发明内容
本发明所要解决的技术问题是提供一种miR-129-5p作为靶分子在制备代谢性疾病药物中的应用，证明了miR-129-5p在脂肪细胞成脂中可抑制脂肪细胞脂滴生成及堆积，尤其是在白色脂肪细胞棕色化以及棕色细胞成脂，证实了miR-129-5p有可能成为预测和干预治疗肥胖或其他代谢性疾病的重要靶分子。
本发明提供了一种miR-129-5p作为靶分子在制备代谢性疾病药物中的应用。
所述药物以miR-129-5p作为靶分子，配以药学上可接受的辅料或辅助性成分制备成制剂使用。
所述制剂选自注射液、皮下埋植剂、片剂、粉剂、颗粒剂、胶囊、口服液、缓释剂中的一种。
本发明在脂肪分化实验中发现miR-129-5p参与脂肪细胞分化过程，C/EBPα、PPARγ2脂分化标志性调控因子表达降低，成熟脂肪细胞表达标记物UCP1、CIDEA表达降低，从而推断该miRNA可以抑制脂肪细胞分化。
有益效果
本发明证明了miR-129-5p在脂肪细胞成脂中可抑制脂肪细胞脂滴生成及堆积，尤其是在白色脂肪细胞棕色化以及棕色细胞成脂，证实了miR-129-5p有可能成为预测和干预治疗肥胖或其他代谢性疾病的重要靶分子。
附图说明
图1为miR-129-5p类似物抑制雄性小鼠腹部皮下脂肪组织白色脂肪生成示意图；其中，A为成熟脂肪细胞第8天油红O染色结果；B为细胞中相对TG含量；C为mRNA检测；D为用Western blots法测定靶基因蛋白水平表达；E为D中Western blots的灰度值相对定量值。
图2为miR-129-5p类似物过表达抑制白色脂肪细胞分化示意图；其中，A为miR-129-5p在白色脂肪细胞中的表达水平；B-H为采用qRT-PCR检测方法显示成脂过程相关基因的表达水平。
图3为miR-129-5p抑制雄性小鼠腹部皮下脂肪组织基质血管部分(SVF)褐色脂肪生成示意图；其中，A为成熟脂肪细胞第6天油红O染色结果；B为细胞中相对TG含量；C为mRNA检测；D为用Western blots法测定靶基因蛋白水平表达；E为D中Western blots的灰度值相对定量值。
图4为miR-129-5p类似物过表达抑制白色脂肪细胞棕色化示意图；其中，A为miR-129-5p在白色脂肪细胞中的表达水平；B-I为采用qRT-PCR检测方法显示成脂过程相关基因的表达水平。
图5为miR-129-5p抑制雄性小鼠肩胛间脂肪组织间质血管部分(SVF)棕色脂肪生成示意图；其中，A为成熟脂肪细胞第6天油红O染色结果；B为细胞中相对TG含量；C为mRNA检测；D为用Western blots法测定靶基因蛋白水平表达；E为D中Western blots的灰度值相对定量值。
图6为miR-129-5p类似物过表达抑制棕色脂肪细胞分化示意图；其中，A为miR-129-5p在白色脂肪细胞中的表达水平；B-I为采用qRT-PCR检测方法显示成脂过程相关基因的表达水平。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
一、miR-129-5p在体外抑制脂肪组织白色脂肪细胞成脂过程
为了评估miR-129-5p是否在白色脂肪细胞成脂过程中发挥作用，首先从雄性小鼠的皮下腹股沟脂肪组织分离出脂肪原代细胞，并在将其培养至达到100％融合时，于细胞内转入miR-129-5p类似物及阴性对照。之后按照正常白色脂肪细胞诱导成脂过程进行诱导培养。至诱导第8天收取成熟的白色脂肪细胞，观察其形态变化同时检测脂质积累。首先进行了最直观的油红染色和甘油三酯(TG)测定，与阴性对照比较，转导miR-129-5p mimic后，白色脂肪生成明显减少，结果如图1A和1B所示。更重要的是，另外用qPCR技术检测与白色脂肪细胞成脂特异性相关的基因时发现，miR-129-5p过表达明显抑制如PPARγ等成脂转录因子的表达，以及FAS、FABP4等成熟白色脂肪细胞特异性标志物，如图1C所示。同时通过蛋白免疫印记方法检测了白脂特异性性表达的蛋白，展现的是相同的实验结果，如图1D和1E所示，即miR-129-5p可在白色脂肪细胞成脂过程中明显抑制特异性标志基因的表达。这些结果表明，在雄性小鼠腹皮下白色脂肪组织的SVF细胞中，miR-129-5p在白色脂肪细胞成脂过程中有着不可或缺的作用。
二、miR-129-5p体外调节SVF中白色脂肪细胞的分化
为了在体外研究miR-129-5p是否在脂肪细胞参与脂肪细胞分化过程而影响白色脂肪细胞成脂通过在分离雄性小鼠皮下腹股沟脂肪组织SVF后，体外培养脂肪细胞达到100％融合时转导阴性对照和miR-129-5p类似物，构建miR-129-5p过表达模型。然后，按照标准的分化操作步骤诱导细胞向白色成熟脂肪细胞分化。在第0、2、4、6和8天的指定时间点收集细胞。首先，们在体外验证了miRNA类似物的作用。与阴性对照相比，转导miR-129-5p类似物后miR-129-5p水平明显为数千表达，如图2A所示。PPARγ为核受体家族的成员，在脂肪细胞分化过程中起决定性作用，如图2C所示；C/EBP家庭成员包括C/EBPα(图2B)和关键白色脂肪细胞成脂标记基因，如图2D和2E所示。在分化过程中不同时间点被检测到在过表达miR-129-5p时，以上特异性基因被抑制，如图2F-H所示。这表明：miR-129-5p在体外培养的白脂细胞分化过程中起抑制分化的作用。
三、miR-129-5p也在体外抑制多功能干细胞生成棕脂以及白色细胞棕色化
为了明确miR-129-5p特异性介导白色脂肪细胞分化，还进行了在皮下腹股沟白色脂肪组织分离培养脂肪前体细胞，并且将过表达的miR-129-5p类似物与阴性对照转染于达到85％融合白色脂肪前体细胞，24小时后，按照棕色脂肪细胞诱导实验方法进行诱导分化，于第6天后收成熟的褐色脂肪细胞，检测其内成脂情况。出人意料的是油红染色和甘油三酯(TG)测定结果如图3A和3B显示，白色脂肪组织中SVF诱导为成熟的褐色脂肪细胞后，miR-129-5p同样抑制该过程。与此同时，白色组织诱导棕色化涉及的分化过程中特异性基因表达均下调，如图3C-E所示，如UCP1蛋白表达，PRDM16和PPARγ表达均受到抑制，以及PPARα和PGC-1α的表达也被miR-129-5p类似物所下调。以上结果显示，miR-129-5p过表达在白色脂肪分化过程中起调节作用，也明显抑制白色脂肪细胞棕色化过程。
还观察了白色脂肪细胞棕色化中细胞分化过程，证实miR-129-5p在褐色脂肪细胞的分化中发挥了重要作用，如图4A-I所示，皮下腹股沟部位脂肪组织中分离出的白色脂肪组织进行棕色化诱导后，参与棕色细胞分化为成熟脂肪细胞中涉及到特异性转录因子以及成熟标志基因皆在miR-129-5p类似物介导的原代脂肪细胞过表达模型所抑制。由此可以得出结论，miR-129-5p不仅参与了白色脂肪的生成，还可以抑制白色脂肪细胞的棕色化。
在前期研究的基础上，尝试分离雄性小鼠两侧肩胛骨间脂肪组织SVF，并在体外培养脂肪细胞，采用与白色脂肪细胞棕色化细胞诱导一致的方法进行培养。通过转导miR-129-5p类似物，检测肩胛间脂肪组织SVF成熟棕色脂肪细胞油红应染色结果及细胞内TG含量，如图5A和5B所示，跟阴性对照组比较过表达miR-19-5p后发现脂滴含量明显减少。同时，成熟棕色脂肪细胞中与成脂相关的mRNA和蛋白表达下调更为显著如图5C-E所示。
miR-129-5p类似物也抑制了肩胛骨处棕色脂肪组织中SVF分化为成熟棕色脂肪细胞过程，如图6A-I所示，这些结果进一步支持了miR-129-5p过表达介导脂肪组织SVF诱导分化过程被抑制的现象，并且干扰脂肪细胞分化过程的观点。更应该强调的一点是miR-129-5p的调节功能在棕色脂肪细胞成脂及分化中更为重要。
四、结论
以上结果表明，miR-129-5p也参与成脂过程，过表达miR-129-5p可以抑制这一过程。基于miR-129-5p不仅抑制分离皮下腹股沟的白色脂肪组织后，体外培养的脂肪细胞分化过程，抑制白色脂肪细胞成脂；还作用于从肩胛骨棕色脂肪组织分离后培养的棕色脂肪细胞分化过程，从而抑制成熟棕色脂肪细胞成脂。基于以上可以得出结论：miR-129-5p在脂肪分化及成脂中起着不可或缺的作用。
本发明实验同时得出了miR-129-5p参与脂肪细胞分化过程，包括了白色脂肪细胞分化、白色脂肪细胞棕色化、棕色脂肪细胞分化过程，证明了miR-129-5p在脂肪细胞成脂中可抑制脂肪细胞脂滴生成及堆积，尤其是在白色脂肪细胞棕色化以及棕色细胞成脂，证实了miR-129-5p有可能成为预测和干预治疗肥胖或其他代谢性疾病的重要靶分子。
Claims (3)
1.一种miR-129-5p作为靶分子在制备代谢性疾病药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述药物以miR-129-5p作为靶分子，配以药学上可接受的辅料或辅助性成分制备成制剂使用。
3.根据权利要求2所述的应用，其特征在于：所述制剂选自注射液、皮下埋植剂、片剂、粉剂、颗粒剂、胶囊、口服液、缓释剂中的一种。
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