CN107513571B - miRNA的应用 - Google Patents
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- CN107513571B CN107513571B CN201710915859.9A CN201710915859A CN107513571B CN 107513571 B CN107513571 B CN 107513571B CN 201710915859 A CN201710915859 A CN 201710915859A CN 107513571 B CN107513571 B CN 107513571B
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Abstract
本发明涉及分子生物学领域，特别涉及miRNA的应用。本发明获得了15种miRNAs非牙源性组织来源的间充质干细胞和牙源性组织来源的间充质干细胞被区分表达。其中，对比于牙源性组织来源的间充质干细胞，非牙源性组织来源的间充质干细胞中9种高表达，6种低表达。候选miRNAs(hsa‑miR‑615‑3p，hsa‑miR‑196b‑5p)的抑制可以提高WJCMSCs的牙本质分化潜能。此外，获得了在起源于间充质干细胞的口腔组织中高度表达的七个关键目标基因，包括TFAP2C、SEMA6D、PDGFRA、PAX9、NR4A3、SATB2和ZNF367。
Description
技术领域
本发明涉及分子生物学领域，特别涉及miRNA的应用。
背景技术
牙齿缺损和缺失是普遍而且经常发生的疾病，会影响咀嚼效率、语言功能、消化功能，甚至是心理健康。现在的治疗属于非生物疗法，很难重建天然牙的正常结构和功能。随着生物技术的发展，间充质干细胞联合组织工程学技术促使口腔组织再生的应用成为前沿且重要的科学问题。
间充质干细胞，是可以从骨髓的间质组织中分离的产后干细胞，具有自我更新的能力，还有分化为多种组成骨骼、牙本质、脂肪组织、软骨、腱和骨骼肌的间充质细胞家系的潜能。现今，用于牙体组织再生的间充质干细胞可以被分成两类，一类是牙源性组织来源的间充质干细胞。这些从口腔组织中分离出的间充质干细胞，是基于其具有分化成成牙本质细胞潜力的干细胞特性，包括牙周膜干细胞(PDLSCs)、牙髓干细胞(DPSCs)和根尖乳头干细胞(SCAPs)等等。另一类是非牙源性组织来源的间充质干细胞，例如起缘于脐带(WJCMSCs)、骨髓(BMSCs)、脂肪组织(ADSCs)的干细胞等等。这些间充质干细胞似乎具有以下优点：充足的供应，自体移植和以备将来使用的细胞冷冻保存。不幸的是，与牙源性组织来源的间充质干细胞相比，这些非牙源性组织来源的间充质干细胞与口腔组织联系不紧密，而这阻止了这些间充质干细胞牙源性分化的倾向。然而，促进间充质干细胞分化为成牙本质细胞是提高间充质干细胞介导口腔组织再生的关键问题。所以，牙本质发生分化调控机制的研究是促进间充质干细胞再生口腔组织所必须的。如今，已经发现了数条涉及牙源性分化调控的信号通路，包括Wnt/β-catenin、Notch、BMP和MAPK信号通路。但是，间充质干细胞如何分化成成牙本质细胞仍然不明，这限制了口腔组织再生的研究和临床应用。
miRNAs是18-22个核苷酸长、未编码的RNA，由RNA聚合酶II转录。它们通过结合特异信使RNA的3号未翻译区域调控它们靶基因的表达，同时引起信使RNA的降解或翻译抑制。近期的研究清楚地描述了大多数(>80％)miRNA通过降低信使RNA的稳定性在转录水平影响它们的同源靶基因。MiRNAs涉及数个中心生物进程，例如细胞发育、增生、分化和凋亡。因此，评估非牙源性组织来源的间充质干细胞和牙源性组织来源的间充质干细胞的不同miRNA序列可以增加对牙本质发生分化和miRNA-mRNA相互作用的理解，同时也能发现这些调控机制的新方向。
在最近的研究中，通过miRNAs芯片检测和进一步的生物信息学分析研究了牙源性组织来源的间充质干细胞(牙周膜干细胞、牙髓干细胞和根尖乳头干细胞)和非牙源性组织来源的间充质干细胞(脐带干细胞、骨髓干细胞和脂肪干细胞)在miRNA表达序列中的区别。这些结果将为阐明间充质干细胞的牙本质发生分化机制提供有用的信息，同时有助于发现促进间充质干细胞介导的口腔组织再生的关键miRNAs和必要的候选目标基因。间充质干细胞由于其牙本质发生潜能，是口腔组织再生与修复最有前景的细胞种类。间充质干细胞的分化被细胞外环境的物理和分子信号精确调控，涉及到复杂的转录与转录前水平的通路调控。但是，目前对于miRNAs在间充质干细胞的牙本质发生分化的调控中所扮演的角色依旧不太清楚。
发明内容
有鉴于此，本发明提供了miRNA的应用。本发明获得了两种促进间充质干细胞介导口腔组织分化的潜在目标miRNAs(hsa-miR-615-3p、hsa-miR-196b-5p)。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了miRNA的差异表达在影响不同来源的间充质干细胞的成牙本质或成骨分化中的应用；
所述miRNA选自hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p、hsa-miR-10a-5p、hsa-miR-31-3p、hsa-miR-3613-3p、hsa-miR-378a-5p、hsa-miR-31-5p、hsa-miR-8071、hsa-miR-3619-5p、hsa-miR-887-3p、hsa-miR-6849-5p、hsa-miR-6807-5p或hsa-miR-4788。
在本发明的一些具体实施方案中，所述不同来源选自非牙源性组织或牙源性组织；
所述非牙源性组织选自脐带、骨髓或脂肪；
所述牙源性组织选自牙周膜、牙髓或根尖乳头。
在本发明的一些具体实施方案中，所述差异表达为高表达或低表达；所述影响为抑制或促进；其中：
miRNA的高表达抑制间充质干细胞的成牙本质或成骨分化；所述miRNA选自hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p、hsa-miR-10a-5p、hsa-miR-31-3p、hsa-miR-3613-3p、hsa-miR-378a-5p或hsa-miR-31-5p。
在本发明的另一些具体实施方案中，miRNA的低表达促进间充质干细胞的成牙本质或成骨分化；所述miRNA选自hsa-miR-8071、hsa-miR-3619-5p、hsa-miR-887-3p、hsa-miR-6849-5p、hsa-miR-6807-5p或hsa-miR-4788。
本发明还提供了miRNA的差异表达在辨别不同来源的间充质干细胞中的应用；所述miRNA选自hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p、hsa-miR-10a-5p、hsa-miR-31-3p、hsa-miR-3613-3p、hsa-miR-378a-5p、hsa-miR-31-5p、hsa-miR-8071、hsa-miR-3619-5p、hsa-miR-887-3p、hsa-miR-6849-5p、hsa-miR-6807-5p或hsa-miR-4788。
在本发明的一些具体实施方案中，所述不同来源选自非牙源性组织或牙源性组织；
所述非牙源性组织选自脐带、骨髓或脂肪；
所述牙源性组织选自牙周膜、牙髓或根尖乳头；
miRNA高表达于牙源性组织；所述miRNA选自hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p、hsa-miR-10a-5p、hsa-miR-31-3p、hsa-miR-3613-3p、hsa-miR-378a-5p或hsa-miR-31-5p。
在本发明的另一些具体实施方案中，miRNA高表达于非牙源性组织；所述miRNA选自hsa-miR-8071、hsa-miR-3619-5p、hsa-miR-887-3p、hsa-miR-6849-5p、hsa-miR-6807-5p或hsa-miR-4788。
本发明还提供了hsa-miR-615-3p和/或hsa-miR-196b-5p的抑制和/或消除在促进牙本质发生标记物(DMP 1和DSPP)的表达上调中的应用。
此外，本发明还提供了hsa-miR-615-3p和/或hsa-miR-196b-5p的抑制和/或消除在促进间充质干细胞的成牙本质或成骨分化中的应用。作为优选，所述间充质干细胞为非牙源性组织的间充质干细胞。
在本发明的一些具体实施方案中，hsa-miR-615-3p抑制序列，如SEQ ID No.2所示，5’-AAGAGGGAGACCCAGGCTCGGA-3’；hsa-miR-196b抑制序列，如SEQ ID No.3所示，5’-CCCAACAACAGGAAACTACCTA-3’。
在本发明的一些具体实施方案中，所述miRNA的相关调控基因选自锌指蛋白367(ZNF367)、核受体亚家族4组A成员3(NR4A3)、SATB同源盒2(SATB2)、配对盒9(PAX9)、血小板生长因子受体α(PDGFRA)、转录因子AP-2γ(TFAP2C)或脑信号蛋白6D(SEMA6D)。
本发明提供了miRNA的差异表达在影响不同来源的间充质干细胞的成牙本质或成骨分化中的应用；所述miRNA选自hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p、hsa-miR-10a-5p、hsa-miR-31-3p、hsa-miR-3613-3p、hsa-miR-378a-5p、hsa-miR-31-5p、hsa-miR-8071、hsa-miR-3619-5p、hsa-miR-887-3p、hsa-miR-6849-5p、hsa-miR-6807-5p或hsa-miR-4788。实验表明，共有15种miRNAs非牙源性组织来源的间充质干细胞和牙源性组织来源的间充质干细胞被区分表达。其中，对比于牙源性组织来源的间充质干细胞，非牙源性组织来源的间充质干细胞中9种高表达，6种被低表达。实验结果还表明候选miRNAs(hsa-miR-615-3p，hsa-miR-196b-5p)的抑制可以提高WJCMSCs的牙本质分化潜能。生物信息分析分辨出这些miRNAs的目标基因的数条可能与间充质干细胞的牙本质分化潜能相联系的通路，例如MAPK信号通路、T细胞受体和Wnt信号通路等等，还分辨了在起源于间充质干细胞的口腔组织中高度表达的七个关键目标基因，包括TFAP2C、SEMA6D、PDGFRA、PAX9、NR4A3、SATB2和ZNF367。
本发明的结果使得对miRNAs的分子机制、候选靶基因和它们的协同潜在间充质干细胞牙本质分化有了更好的理解。并且分离了两种可能是促进间充质干细胞介导口腔组织分化的潜在目标miRNAs(hsa-miR-615-3p、hsa-miR-196b-5p)。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示间充质干细胞中5个被选择miRNAs的表达；与根尖牙乳头干细胞、牙周膜干细胞和牙髓干细胞相比，实时反转录PCR结果表明hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p和hsa-miR-10a-5p在脐带干细胞、骨髓干细胞、脂肪干细胞中高度表达；U6为内部控制；数值是由每种细胞三份不同的细胞样本计算得来；All errorbars represent the s.d.(n＝3)；**P≤0.01；
图2示hsa-miR-615-3p敲低提高了脐带干细胞的成牙本质和成骨分化；其中(A)实时反转录PCR结果表明脐带干细胞中hsa-miR-615-3p敲低；(B)碱性磷酸酶活性检测表明miR-615-3p敲低提高了脐带干细胞的碱性磷酸酶活性；(C)茜素红染色结果表明miR-615-3p敲低提高了矿化；(D-F)实时反转录PCR结果表示hsa-miR-615-3p敲低上调了脐带干细胞中DSPP(D)、DMP 1(E)和BSP(F)的表达；GAPDH作为内部控制；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；*P≤0.05；**P≤0.01；
图3示hsa-miR-196b-5p敲低提高了脐带干细胞成牙本质和成骨分化；其中(A)实时反转录PCR结果表明脐带干细胞中hsa-miR-196b-5p敲低；(B)碱性磷酸酶活性检测表明hsa-miR-196b-5p敲低提高了脐带干细胞的碱性磷酸酶活性；(C)茜素红染色结果表明hsa-miR-196b-5p敲低提高了矿化；(D-F)实时反转录PCR结果表示hsa-miR-196b-5p敲低上调了脐带干细胞中DSPP(D)、DMP 1(E)和BSP(F)的表达；GAPDH作为内部控制；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；*P≤0.05；**P≤0.01；
图4示实时PCR测定非牙源性间充质干细胞和牙源性间充质干细胞的基因表达；与在脐带干细胞、骨髓干细胞、脂肪干细胞相比，实时反转录PCR结果表明ZNF367、NR4A3、SATB2、PAX9、PDGFRA、TFAP2C和SEMA6D的mRNA表达在根尖牙乳头干细胞、牙周膜干细胞和牙髓干细胞更高；GAPDH作为内部控制；数值是由每种细胞三份不同的细胞样本计算得来；Allerror bars represent the s.d.(n＝3)；**P≤0.01。
具体实施方式
本发明公开了miRNA的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
间充质干细胞分化是受物理和分子信号精密调控和构建的，这在近几年来一直备受关注。在研究的干细胞群中，牙间充质干细胞最有可能在体内或体外再生和发育成典型的牙齿状结构。然而，牙间充质干细胞的供应是有限的，因此更容易获得的间充质干细胞和其牙本质生成分化调控需要被研究用作牙齿再生的种子细胞。
本发明提供了在三种牙源性组织来源的间充质干细胞(包括牙周膜干细胞、牙髓干细胞和根尖乳头干细胞)和三种非牙源性组织来源的间充质干细胞(包括脐带间充质干细胞、骨髓间充质干细胞和脂肪间充质干细胞)中的miRNAs序列图，并确定了15种差异表达的miRNAs。本发明的实验结果提供了一些间充质干细胞miRNAs表达的差异序列，且表明这些miRNAs是间充质干细胞牙本质发生分化的关键候选miRNAs。接下来，选择了两种候选miRNAs(miR-615-3p和miR-196b-5p)来研究它们在非牙源性组织来源的间充质干细胞牙本质发生分化的功能。结果显示miR-615-3p和miR-196b-5p的敲除促进了脐带间充质干细胞的ALP活性和体外矿化。牙本质发生标记物(DMP1和DSPP)以及成骨发生标记物(BSP)与间充质干细胞的牙本质发生分化有关联。miR-615-3p和miR-196b-5p的抑制引起DSPP、DMP1和BSP的表达上调表明了这两种miRNAs可能在间充质干细胞的牙本质发生分化中起着重要的作用。
接着，通过进一步的生物信息分析扫描了这些miRNAs的靶基因和重要通路。首先，确定了总共372个靶基因和MAPK信号通路、T细胞受体信号通路、Wnt信号通路很大程度上在功能调控中有着重要的作用。大量的研究表明许多关键信号通路涉及调控间充质干细胞的谱系定型，包括Wnt、Notch、BMP和MAPK信号通路。MAPK和Wnt信号通路已经被体内和体外的研究报导了可以调控牙间充质干细胞的分化和牙本质发生。例如，p38-MAPK信号通路调控hDPCs的ALP表达，并通过p38磷酸化和增进的核转移在第三期牙本质生成的成牙本质细胞刺激时激活。Wnt信号通路是牙齿发育和干细胞自我更新的一条关键通路，已经被发现涉及DPSCs的成牙本质细胞样分化。总的来说，上述实验结果表明这些靶基因涉及Wnt和MAPK信号通路，而且它们调控的miRNAs可能在间充质干细胞的牙本质发生分化中扮演重要的角色。
此外，本发明确定了7个在牙源性间充质干细胞中高度表达的候选靶基因(包括TFAP2C、SEMA6D、PDGFRA、PAX9、NR4A3、SATB2和ZNF367)。Sema6D属于Semaphorin基因家族，是BMP信号的一个靶基因，同时含有在牙源性间充质干细胞中下调的miR-31-5p结合位点。以前的报导发现SEMA6D和其他Semaphorin基因在神经系统轴突引导的发育中起着重要的作用。在压碎组织再生中，Sema6D可能涉及血管发生和牙髓神经再生。转录因子激活蛋白2(AP2)在调控颅骨闭合和颅面发育骑着关键作用，而且，小鼠敲出AP2后削弱了骨发育和形成。Pdgfra被发现可以导致条纹鱼颅面异常发育包括腭裂。以前来自Pdgfra敲除小鼠的证据支持了它在腭和牙齿发育中扮演的重要角色。同样的，Pdgfra似乎在牙本质发生时通过自分泌机制调控牙齿发育的大小和阶段。PAX基因编码了一族进化上保守的转录因子，是细胞迁移和组织图式发育的重要调控者。明确的是，PAX9在胚胎发生，特别是骨骼发生、牙齿形成、腭发生和神经管发育中扮演关键角色。PAX9被确定为一种参与牙本质发生的广泛研究的基因。它通过骨形态发生蛋白4(Bmp4)和Msx1表达的上调促进牙齿发育。PAX9基因突变导致人和小鼠牙齿缺失。NR4A组成3个高度同源的蛋白质(NR4A1、NR4A2和NR4A3)。这提示NR4A可能作为增生、凋亡和分化的稳态调节器。SATB2是负责染色质重组的同源异形盒基因，与RUNX2有协同作用。SATB2敲除的小鼠显示出颅面发育异常和成骨细胞分化的缺陷。之前的研究揭露了SATB2在BMSCs的位点特异性特征中扮演必要的角色，例如无茎的、防衰老的能力和成骨细胞分化。文献已经确定miR-31可以调控SATB2，形成一个发育和成骨细胞分化的调节链——RUNX2-miR-31-SATB2。ZNF367(也叫ZFF29和CDC14B)是锌指蛋白家族的一员，有独特的Cys2His2锌指基序，在胚胎或胎儿的红色组织中表达。合起来看，这些证据暗示了这些基因可能涉及间充质干细胞牙本质发生分化的关键miRNAs的调控。但是，需要更多的研究进一步审查这个假说。
本发明使用芯片分析确定了牙源性间充质干细胞和非牙源性间充质干细胞之间的miRNAs差异表达序列。使用生物信息分析确定了潜在的调控机制和关键靶基因。结果还指出了两个miRNAs(miR-615-3p和miR-196b)是间充质干细胞牙本质发生分化所需要的。本发明为进一步理解这些miRNAs和mRNAs控制间充质干细胞牙本质发生分化的详细作用和机制提供了基础，同时为间充质干细胞生物学的研究提供了重要的资源。另外，本发明运用差异表达在间充质干细胞牙本质发生分化获得的miRNAs可以作为口腔组织工程学的潜在方向。
本发明研究不同miRNAs的表达情况，以及牙源性组织来源的间充质干细胞包括SCAPs、DPSCs、PDLSCs和非口腔组织包括WJCMSCs、BMSCs、ADSCs的功能网络分析通过运用生物芯片检测和生物信息分析。含碱磷酸酶活动检测、茜素红染色、牙本质发生和骨发生相关基因表达用来分析直系分化潜能。
所有统计计算使用SPSS11统计软件。统计分析包括比较T检验、Fisher精确检验、卡方检验和皮尔逊相关系数，选择适当方法。P值小于0.05时认为有统计学意义。
缩写：
MicroRNAs：miRNA；MSCs：间充质干细胞；ALP：碱性磷酸酶；DPSCs：牙髓干细胞；SCAPs：根尖牙乳头干细胞；WJCMSCs：脐带干细胞；BMSCs：骨髓间充质干细胞；ADSCs：脂肪间充质干细胞；MAPK：丝裂原活化蛋白激酶；ZNF367：锌指蛋白367；NR4A3：核受体4亚家族A组3号；SATB2：SATB同源异形盒2；PAX9：配对盒9；PDGFRA：血小板生长因子受体α；TFAP2C：转录因子AP-2γ；SEMA6D：导向蛋白6D；KEGG：京都基因与基因组百科全书。
本发明提供的miRNA的应用中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1细胞培养和成骨分化
所有涉及人类干细胞的研究均遵守ISSCR“人类胚胎干细胞研究的行为指导方针”。牙齿组织的获取均得到了患者的同意，并严格遵守首都医科大学附属北京口腔医院的指导规定。牙齿首先用75％酒精消毒，然后用磷酸缓冲液清洗。牙周膜干细胞、牙髓干细胞和根尖乳头干细胞按照之前描述进行分离、培养和辨认。简要说，根尖乳头干细胞分离自根尖牙乳头，牙周膜干细胞分离自根中三分之一的牙周韧带，牙髓干细胞分离自冠髓。接着，将这些干细胞放入37℃的3mg/ml的胶原蛋白酶I(Worthington Biochemical Corp.,Lakewood,NJ)和4mg/ml的分散酶(Roche Diagnostics Corp.,Indianapolis,IN)溶液中1h。通过70-um的过滤器(BD Biosciences,San Jose,CA,)和细胞传代得到单一细胞的悬浮液。人骨髓干细胞、脂肪干细胞和脐带干细胞从ScienCell Research Laboratories(Carlsbad,CA)获得。干细胞生长在5％CO2和37℃的加湿保温箱中，使用杜尔伯克改良伊格尔培养基(DMEM)(Invitrogen,Carlsbad,CA)，加15％胎牛血清、2mmol/l谷氨酰胺、100U/ml青霉素和100mg/ml链霉素。培养基每3天更换一次。
所有干细胞在传代3-5代用于后续实验。对于成骨/成牙本质分化，干细胞被种植在使用常规培养基的密度为2.0X105细胞/槽的6槽盒中。当细胞达到80-90％的融合，更换培养基，然后细胞将被培养在STEMPRO成骨作用分化试剂盒的促矿化培养基中。成骨/成牙本质分化培养基每3天更换。
实施例2总RNA分离和微点阵杂交
来源于不同个体的牙周膜干细胞、牙髓干细胞、根尖乳头干细胞、脐带间充质干细胞、骨髓间充质干细胞和脂肪间充质干细胞的三组总RNA样本通过使用抽提试剂和RNA抽提试剂盒(Qiagen,德国)被分别提出。总RNA通过分光光度仪ND-2100(赛默飞世尔)定量，使用安捷伦2100(安捷伦科技)评估RNA完整性。每组总RNA样本都使用RNA抽提试剂盒和RNase-Free DNase Set(QIAGEN,GmBH,Germany)进一步提纯。微点阵分析使用人MiRNAs 4.0点阵(Affymetrix)。样本标记、微点阵杂交和洗涤基于生产者标准执行。简要说，总RNA被聚腺嘌呤示踪，接着用生物素标记。之后，标记的RNA在微点阵上被杂交。(Compass biotechnologyLtd,Beijing,China)。点阵通过使用
扫描仪3000(Affymetrix,CA,US)扫描。微点阵原始数据通过Affymetrix表达控制软件使用MAS5统计算法标准化。
实施例3生物信息分析
生物信息分析完成(Compass biotechnology Ltd,Beijing,China)。Affymetrix基因芯片命令控制台软件(版本4.0，Affymetrix)用于分析芯片图像来得到原始数据，然后提供受体调节分析标准化。接着，R软件用于继续下面的数据分析。通过折叠变化辨认差异表达miRNAs。差异表达miRNAs的靶基因就是通过5个数据库(miRanda,miRDB,miRTarbase,TargetScan,and miRecords)预测的交点。KEGG分析被用于确定这些靶基因的作用。分级群集被用于显示样本中可区分的miRNAs表达模式。
实施例4逆转录(RT)PCR和实时逆转录PCR
总RNA通过抽提试剂(英杰)从间充质干细胞中分离。对于mRNA检测，依据生产厂家的标准(英杰)，通过使用随机六边结构和逆转录合成RNA的等量样本。使用荧光PCR(Qiagen)和IcycleriQ多色实时荧光定量PCR技术检测系统进行实时PCR反应。引物设计为使用在线引物3程序，并列在表5。GAPDH作为内部调控。相对mRNA水平通过使用2-ΔΔCt方法计算。对于miRNA检测，成熟miRNAs的稳定状态水平通过使用miR-特异TaqManTM微RNA检测盒(Applied Biosystems,Foster City,CA)。TaqMan探针是hsa-miR-196a-5p(Assay ID:241070_mat,Applied Biosystems,Foster City,CA)；hsa-miR-196b-5p(Assay ID:002215,Applied Biosystems,Foster City,CA),hsa-miR-10b-5p(Assay ID:002218,Applied Biosystems,Foster City,CA),hsa-miR-10a-5p(Assay ID:000387,AppliedBiosystems,Foster City,CA),hsa-miR-615(Assay ID:001960,Applied Biosystems,Foster City,CA),and U6(Assay ID:001973,Applied Biosystems,Foster City,CA)作为内部控制。相对表达使用2-ΔΔCt方法计算。
实施例5质粒构建和病毒感染
质粒根据标准方法构建，并且所有结构通过适当限定酶解分析和/或序列测定核实。靶miRNAs的补偿序列miRNAs抑制因子和绿色荧光蛋白(GFP)(Genepharma Company,Suzhou,China)被克隆进入LV3慢病毒载体。慢病毒包装由GenePharma Inc.(Shanghai,China)制作。间充质干细胞培养过夜，接着在凝聚胺(6μg/mL,Sigma-Aldrich,St.Louis,MO,USA)参与下转染慢病毒6h。48h后，用1μg/mL嘌呤霉素选择感染细胞7天。miRNA的靶序列是：LV3shRNA(Consh),5’-TTCTCCGAACGTGTCACGTTTC-3’；hsa-miR-615-3p抑制序列,5’-AAGAGGGAGACCCAGGCTCGGA-3’；hsa-miR-196b抑制序列,5’-CCCAACAACAGGAAACTACCTA-3’。
实施例6碱性磷酸酶活性测定和茜素红染色
间充质干细胞生长在使用StemPro成骨分化试剂盒(英杰)中进行促矿化培养。培养7天，碱性磷酸酶(ALP)活性测定通过生产厂家标准(Sigma-Aldrich,St.Louis,MO,USA)ALP盒进行，同时基于蛋白量来标准化。使用Bio-Rad蛋白检测溶液(Bio-Rad LaboratoriesHercules,CA,USA)定量蛋白聚集。对于检测矿化，细胞促矿化三周后，混合70％酒精，并用2％茜素红(Sigma-Aldrich)染色。
实施例7非牙源性组织来源的间充质干细胞和牙源性组织来源的间充质干细胞表达的不同miRNAs的序列
为了辨别非牙源性组织来源的间充质干细胞和牙源性组织来源的间充质干细胞表达的不同miRNAs，我们首先应用t检验过滤出被差异表达的miRNAs，接着根据p值选择出有两倍变化的差异表达基因。在非牙源性组织来源的间充质干细胞(包括脐带间充质干细胞、骨髓间充质干细胞和脂肪间充质干细胞)和牙源性组织来源的间充质干细胞(包括牙周膜干细胞、牙髓干细胞和根尖乳头干细胞)中总共有15个miRNAs被差异表达。其中，相较于牙周膜干细胞、牙髓干细胞和根尖乳头干细胞，脐带干细胞、骨髓干细胞和脂肪干细胞中有9个miRNAs高表达，6个miRNAs低表达。(表1)
为了证明微点阵数据的可靠性，本发明随机选择了5个差异表达的miRNAs(包括hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p和hsa-miR-10a-5p)，通过实时反转录PCR来分析它们的表达情况。实时反转录PCR结果显示：对比牙周膜干细胞、牙髓干细胞和根尖乳头干细胞，在脐带干细胞、骨髓干细胞和脂肪干细胞中hsa-miR-196a-5p、hsa-miR-10b-5p、hsa-miR-615-3p、hsa-miR-196b-5p和hsa-miR-10a-5p高度表达，证实了随机挑选的5个miRNAs的表达水平与微点阵结果一致。(图1)
实施例8抑制hsa-miR-615-3p或hsa-miR-196b-5p可以促进脐带干细胞的成牙本质和成骨分化潜能
选择2个在非牙源性组织来源的间充质干细胞中高度表达的miRNAs——hsa-miR-615-3p和hsa-miR-196b-5p作比较，来评价它们对脐带干细胞的成牙本质和成骨分化的影响。我们将hsa-miR-615-3p的抑制序列插入慢病毒载体，然后通过慢病毒感染转导进脐带干细胞(图2A)。矿化诱导一周后，结果显示hsa-miR-615-3p的抑制提高了脐带干细胞中ALP的活性(图2B)。矿化诱导三周后，通过茜素红染色结果显示：与对照组相比，抑制hsa-miR-615-3p促进了脐带干细胞的体外矿化(图2C)。同时，与对照组相比，hsa-miR-615-3p受抑制脐带干细胞的实时反转录PCR分析显示了成牙本质标志物的高度表达(包括矿化诱导后0、7、14天的DSPP和7、14天的DMP1)，还有成骨标志物(包括矿化诱导后0、7、14天的BSP)(图2D-F)。
通过慢病毒感染将hsa-miR-196b-5p转导入脐带干细胞。通过实时反转录PCR确认脐带干细胞的hsa-miR-196b-5p受抑制(图3A)。矿化诱导一周后，结果显示hsa-miR-615-3p的抑制提高了脐带干细胞中ALP的活性(图3B)。矿化诱导三周后，茜素红染色结果显示：与对照组相比，抑制hsa-miR-615-3p促进了脐带干细胞的体外矿化(图3C)。同时，与对照组相比，实时反转录PCR结果显示hsa-miR-196b-5p受抑制提高了DSPP和DMP1(矿化诱导后14天)、BSP(矿化诱导后0、7、14天)的表达(图3D-F)。
实施例9非牙源性组织来源的间充质干细胞和牙源性组织来源的间充质干细胞的微点阵数据的生物学分析
实时RT-PCR检测这些基因在非牙源性间充质干细胞和牙源性组织间充质干细胞中的表达。实时定量RT-PCR结果表明锌指蛋白367(ZNF367)、核受体亚家族4组A成员3(NR4A3)、SATB同源盒2(SATB2)、配对盒9(PAX9)、血小板生长因子受体α(PDGFRA)、转录因子AP-2γ(TFAP2C)和脑信号蛋白6D(SEMA6D)mRNA的表达在牙周膜干细胞、牙髓干细胞和根尖乳头干细胞(与脐带间充质干细胞、骨髓间充质干细胞和脂肪间充质干细胞相比)中高很多(图4)。同时，其他13个基因的表达在非牙源性组织来源的间充质干细胞和牙源性组织来源的间充质干细胞之间没有明显差异。
表3用于实时反转录PCR的引物序列
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 首都医科大学附属北京口腔医院
<120> miRNA的应用
<130> MP1719605
<160> 49
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> LV3 shRNA (Consh)
<400> 1
ttctccgaac gtgtcacgtt tc 22
<210> 2
<211> 22
<212> DNA
<213> hsa-miR-615-3p inhibition sequence
<400> 2
aagagggaga cccaggctcg ga 22
<210> 3
<211> 22
<212> DNA
<213> hsa-miR-196b inhibition sequence
<400> 3
cccaacaaca ggaaactacc ta 22
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
agggtgccat cttgacaact 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
ggggctccat catctgaaca 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 6
acaaaagcgg aagatgctgc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
cttcctcgca aacatgcacc 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 8
aagaccaaca acggcactca 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 9
cttcgtgcgt caggagaact 20
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 10
agaagccact ccgtgcatc 19
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 11
ggggagttat cagctcgctc 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 12
gttatcggcg tcctcctcag 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 13
taggcttgga tgggcaacag 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 14
gggcccaatt acccatcctt 20
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 15
gaaggtgggg tgagcatcat 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 16
gggcagaaaa gattccggga 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 17
aaggcactga agtcgatgca 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 18
aatgtgtggg aggcatcagg 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 19
ggaacacggt ttgcaagcat 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 20
agtacaatgt gccctccgtg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 21
agatgtggtt gtagggcagc 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 22
gagtgaagtg agctggcagt 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 23
gctcacttcc aagaccgtca 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 24
ccttcgtgtg cctcatcctt 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 25
ctgctcccct tctctcctct 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 26
tgtcttcgaa ctggacaggc 20
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 27
ccttctggct ccttcacctg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 28
accacgggaa gctgaaagag 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 29
cctcctcagc ctccactttg 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 30
ggtagagtga ccccagggat 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 31
accatggcag tcccctagat 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 32
accggtggca agcaaatcta 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 33
actatttccc caagggcagc 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 34
ctgccccttg ttctctggta 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 35
aggccaggat gacgatgtag 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 36
ttgctgcacg atcagacagt 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 37
tcagtggggt tcattacggc 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 38
ttacctcccc acgaacttgc 20
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 39
tgtcctggct cggtagatga 20
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 40
actgtccaag tgggaggcta 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 41
tctcggaagt caatggtgcc 20
<210> 42
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 42
tgagaggccc tatctgtgtg a 21
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 43
ctgctcaggc agccattttc 20
<210> 44
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 44
caggccacga tattatcttt aca 23
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 45
ctcctcttct tcctcctcct c 21
<210> 46
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 46
cgacataggt cacaatgagg atgtcg 26
<210> 47
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 47
ttgcttccag ctacttgagg tc 22
<210> 48
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 48
cgtggacaaa gaagatagca actccacg 28
<210> 49
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 49
ttccggctct ctatctcaat gttt 24
Claims (2)
1.抑制和/或消除hsa-miR-615-3p和/或hsa-miR-196b-5p的制剂在制备脐带干细胞中牙本质发生标记物的表达上调的促进剂中的应用；所述牙本质发生标记物包括DMP 1和/或DSPP。
2.抑制和/或消除hsa-miR-196b-5p的试剂在制备促进脐带干细胞体外矿化的制剂中的应用。
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| CN103820438A (zh) * | 2014-02-13 | 2014-05-28 | 中国人民解放军军事医学科学院基础医学研究所 | 改善间充质干细胞成骨能力的rna分子及其作用靶点和应用 |
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| CN104667294A (zh) * | 2013-11-29 | 2015-06-03 | 中国医学科学院基础医学研究所 | miR-216在制备促进成骨分化的药物中的用途 |
| CN103820438A (zh) * | 2014-02-13 | 2014-05-28 | 中国人民解放军军事医学科学院基础医学研究所 | 改善间充质干细胞成骨能力的rna分子及其作用靶点和应用 |
| CN106687602A (zh) * | 2014-06-13 | 2017-05-17 | 维也纳自然资源与生命科学大学 | 用于诊断和治疗骨折和骨病的组合物及方法 |
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