CN111500578B - 调控ADSCs成骨分化及组织再生Circ RNA-FTO及其应用 - Google Patents
调控ADSCs成骨分化及组织再生Circ RNA-FTO及其应用 Download PDFInfo
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Abstract
本发明的调控ADSCs成骨分化及组织再生Circ RNA‑FTO及其应用,属于骨组织再生技术领域,该Circ RNA‑FTO circbase ID为has‑circ‑0005941,cDNA序列如SEQ ID NO:1所示,是由如该序列所示的序列首尾连接成的环状结构。通过Circ RNA‑FTO调控ADSCs成骨分化和组织再生的体内外实验,证实了Circ RNA‑FTO在脂肪干细胞成骨分化和/组织再生过程中均起了重要的调控作用;Circ RNA‑FTO可作为组织再生治疗的潜在靶点,推动开发出新的关于Circ RNA‑FTO相关药物或因子,并最终将其运用到再生医学领域,达到临床上真正的精准医疗。
Description
技术领域:
本发明属于骨组织再生技术领域,具体涉及调控ADSCs成骨分化及组织再生CircRNA-FTO及其应用。
背景技术:
创伤,肿瘤和感染引起的骨缺损是临床的常见病,自体骨移植一直是骨缺损的首选治疗方法,但由于其来源有限、可塑性差,且可导致供体部位损伤而受到极大的限制。同种异体骨移植存在免疫排斥反应。如果为了降低异体骨的免疫原性而进行去除细胞成分的处理则会促使其丧失成骨活性。其来源有限、感染率高。异种骨移植的免疫排斥反应更严重,可能传染人畜共患疾病。它们造成了沉重的社会和经济负担,严重影响了患者的生活质量。脂肪干细胞(adipose Derived Stem Cells,ADSCs)最近几年引起了广泛的关注。脂肪干细胞来源广泛、含量较多、获取方式简单、患者承受的痛苦也较少。在适当的环境下,脂肪干细胞可以分化为脂肪细胞、成骨细胞、成软骨细胞以及肌细胞等。ADSCs具有多向分化能力和较明确的成骨潜能,因此成为骨组织工程中重要的种子细胞。
环状RNA(circRNA)是非编码RNA中的一种,是继微小RNA(microRNA,miRNA)及长链非编码RNA(long noncoding RNA,lncRNA)后的RNA家族又一研究新热点。circRNA由无5'帽或3'聚腺苷酸尾巴的共价闭环结构组成。与线性RNA相比,circRNA具有以下特征:高度稳定性;高度进化保守性;耐受核糖核酸外切酶的消化,不易降解;大多数circ RNA定位于细胞质,少数circ RNA则在细胞核内发挥作用;某种组织细胞或细胞的某个时期可特异性的表达特定的circ RNA。环状RNA表现出发育阶段或组织特异性表达的能力使其成为疾病诊断和靶向治疗的理想生物标志物。多项研究探索了circRNA在细胞活性,胚胎发育,神经发育以及各种人类疾病的发展中的重要作用。
但是,Circ RNA-FTO在脂肪干细胞中的作用以及与脂肪干细胞成骨分化及组织再生相关作用研究,目前未见报道。
发明内容:
针对上述现有技术,发明人课题率先提出利用脂肪干细胞进行骨组织再生的新理念,并进行了系列相关体内、外实验,如在裸鼠异位成骨及兔下颌骨缺损动物模型上成功再生骨组织;将自体异体脂肪干细胞附合生物支架用于组织再生,取得了良好的效果。进一步通过研究Circ RNA-FTO调控ADSCs成骨分化和组织再生的体内外实验,进行分子水平研究和体内组织再生研究,结果显示,Circ RNA-FTO在脂肪干细胞成骨分化和组织再生过程中起了重要的调控作用,通过反向论证,当Circ RNA-FTO下调,ADSCs成骨向分化和骨组织再生能力受到抑制,通过本发明阐明脂肪干细胞介导组织再生中Circ RNA-FTO的调控功能,为调控脂肪干细胞促进骨组织再生提供重要的依据。
为实现上述目的,本发明采用以下技术方案:
调控ADSCs成骨分化及组织再生Circ RNA,具体为Circ RNA-FTO,其circbase ID为hsa-circ-0005941。
所述的Circ RNA-FTO的cDNA序列如SEQ ID NO:1所示,是由如该序列所示的序列首尾连接成的环状结构。
所述的circRNA-FTO在ADSCs成骨分化及组织再生调控中的应用,所述的调控包括正调控和负调控,正调控是指促进脂肪干细胞成骨分化和组织再生,负调控是指抑制脂肪干细胞成骨分化和组织再生。
所述的circRNA-FTO在ADSCs成骨分化及组织再生调控中的应用,Circ RNA-FTO表达提高,促进脂肪干细胞成骨分化和组织再生;Circ RNA-FTO表达下降,ADSCs成骨向分化和骨组织再生能力受到抑制。
Circ RNA-FTO促进剂在制备促进脂肪干细胞成骨分化和组织再生药物中的用途。
所述的Circ RNA-FTO促进剂包括任何可以提高Circ RNA-FTO的活性、增加CircRNA-FTO表达量的物质,例如增加Circ RNA-FTO表达量的Circ RNA-FTO的过表达载体。
Circ RNA-FTO抑制剂在制备抑制脂肪干细胞成骨分化和组织再生药物中的用途。
所述的Circ RNA-FTO抑制剂包括任何可以降低Circ RNA-FTO的活性、减少CircRNA-FTO表达量的物质均可用于本发明,具体包括拮抗剂、下调剂、阻滞剂、阻断剂等。
所述的Circ RNA-FTO抑制剂为抑制Circ RNA-FTO表达的小干扰RNA序列。
所述的Circ RNA-FTO的si RNA靶点序列为:
正义链:GGAGGGUGUGAUGAUCUCAUU
反义链:UGAGAUCAUCACACCCUCCAA。
一种促进脂肪干细胞成骨分化和骨组织再生的方法,所述方法包括提高CircRNA-FTO的活性和/或增加Circ RNA-FTO的表达。
所述的促进脂肪干细胞成骨分化和骨组织再生的方法,还包括将提高Circ RNA-FTO活性和/增加Circ RNA-FTO表达后的脂肪干细胞,与人工骨支架材料形成脂肪干细胞复合物制备成骨材料的步骤。
一种抑制脂肪干细胞成骨分化和骨组织再生的方法,所述方法包括抑制CircRNA-FTO的表达。
所述的Circ RNA-FTO作为非疾病诊断和治疗用途的靶点在调控脂肪干细胞成骨分化和组织再生中的应用。
脂肪干细胞具有多种分化潜能,在体外通过适当的诱导能够形成骨、软骨、神经、脂肪、血管等多种组织,(即便在定向诱导过程中)脂肪干细胞在诱导时也会伴随多种分化细胞的产生(成骨细胞+脂肪细胞、成骨细胞+血管细胞等),通过部分抑制成骨分化,可以有效的诱导干细胞向其他特定组织方向分化。
一种调控脂肪干细胞的成骨分化和组织再生的药物组合物,所述药物组合物含有有效量的上述Circ RNA-FTO促进剂或Circ RNA-FTO抑制剂。
所述的调控脂肪干细胞的成骨分化和组织再生的药物组合物中还包含:药学上可接受的载体。
所述的调控脂肪干细胞的成骨分化和组织再生的药物组合物中“药学上可接受的载体”为常规的给药载体,包括各种赋形剂或稀释剂等。
本申请的Circ RNA-FTO来源于chr16:53907697-53922863,基因长度15166碱基,由FTO第5,6,7个外显子环化形成,成环长度为334核苷酸。FTO是一个非常大的基因,其9个外显子在16号染色体上的跨度超过400kb。据报道,脂肪量和肥胖相关的FTO是调节体重和脂肪量的关键因素之一,它参与调控了常见的肥胖和体重指数。FTO主要通过在脂肪形成过程中调节脂肪形成而参与脂肪积累。据报道,抑制FTO或FTO突变导致体重减少和脂肪堆积减少。相反,FTO的过度表达导致体重和脂肪量增加。
本发明的有益效果:
本发明通过Circ RNA-FTO调控ADSCs成骨分化和组织再生的体内外实验,证实了Circ RNA-FTO在脂肪干细胞成骨分化和/组织再生过程中均起了重要的调控作用;CircRNA-FTO可作为组织再生治疗的潜在靶点,推动开发出新的关于Circ RNA-FTO相关药物或因子,并最终将其运用到再生医学领域,达到临床上真正的精准医疗。
附图说明:
图1为实施例1的hADSCs细胞表面标志物的流式检测图;
图2为实施例1的hADSCs的多向分化图,由左向右依次为:培养至第3代的hADSCs;培养后的hADSCs的成脂分化;hADSCs的成骨分化;hADSCs的成软骨分化;
图3为实施例1的脂肪干细胞成骨向诱导后的茜素红染色结果图;由左向右依次为:脂肪干细胞成骨诱导7天、14天和21天进行茜素红染色结果;
图4为实施例2的RT-q PCR检测脂肪干细胞成骨向诱导后0天、3天、5天、7天CircRNA-FTO的表达变化情况图;
图5为实施例2的小干扰RNA转染nc对照组和Circ RNA-FTO实验组的沉默效率的检测荧光显微镜图;
图6为实施例2的RT-PCR检测nc对照组和实验组的Circ RNA-FTO的表达情况图;
图7为实施例2的Western blot检测分别经siRNA-NC组和siRNA-FTO组转染的hADSCs成骨向诱导后成骨分化相关蛋白相对表达的检测;
图8为实施例2的分别经siRNA-NC组和siRNA-FTO组转染的hADSCs成骨向诱导后的碱性磷酸酶染色及茜素红染色检测成骨分化钙结节的数量和程度图,其中,上为碱性磷酸酶染色,下为茜素红染色;
图9为实施例2的经siRNA circFTO和siRNA NC转染hADSCs+Bio-Oss组分别经HE染色和Masson染色后,体内组织再生能力的变化图。
具体实施方式:
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作和/或它们的组合。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
调控ADSCs成骨分化及组织再生Circ RNA,具体为Circ RNA-FTO,其circbase ID为hsa-circ-0005941。
所述的Circ RNA-FTO的cDNA序列如SEQ ID NO:1所示,是由如该序列所示的序列首尾连接成的环状结构。
所述的circRNA-FTO在ADSCs成骨分化及组织再生调控中的应用,所述的调控包括正调控和负调控,正调控是指促进脂肪干细胞成骨分化和组织再生,负调控是指抑制脂肪干细胞成骨分化和组织再生。
所述的circRNA-FTO在ADSCs成骨分化及组织再生调控中的应用,Circ RNA-FTO表达提高,促进脂肪干细胞成骨分化和组织再生;Circ RNA-FTO表达下降,ADSCs成骨向分化和骨组织再生能力受到抑制。
Circ RNA-FTO促进剂在制备促进脂肪干细胞成骨分化和组织再生药物中的用途。
所述的Circ RNA-FTO促进剂包括任何可以提高Circ RNA-FTO的活性、增加CircRNA-FTO表达量的物质,例如增加Circ RNA-FTO表达量的Circ RNA-FTO的过表达载体。
Circ RNA-FTO抑制剂在制备抑制脂肪干细胞成骨分化和组织再生药物中的用途。
所述的Circ RNA-FTO抑制剂包括任何可以降低Circ RNA-FTO的活性、减少CircRNA-FTO表达量的物质均可用于本发明,具体包括拮抗剂、下调剂、阻滞剂、阻断剂等。
所述的Circ RNA-FTO抑制剂为抑制Circ RNA-FTO表达的小干扰RNA序列。
所述的Circ RNA-FTO的si RNA靶点序列为:
正义链:5'-ggagggugugaugaucucauu-3'
反义链:5'-ugagaucaucacacccuccaa-3'
一种促进脂肪干细胞成骨分化和骨组织再生的方法,所述方法包括提高CircRNA-FTO的活性和/或增加Circ RNA-FTO的表达。
所述的促进脂肪干细胞成骨分化和骨组织再生的方法,还包括将提高Circ RNA-FTO活性和/增加Circ RNA-FTO表达后的脂肪干细胞,与人工骨支架材料形成脂肪干细胞复合物制备成骨材料的步骤。
一种抑制脂肪干细胞成骨分化和骨组织再生的方法,所述方法包括抑制CircRNA-FTO的表达。
所述的Circ RNA-FTO作为非疾病诊断和治疗用途的靶点在调控脂肪干细胞成骨分化和组织再生中的应用。
脂肪干细胞具有多种分化潜能,在体外通过适当的诱导能够形成骨、软骨、神经、脂肪、血管等多种组织,(即便在定向诱导过程中)脂肪干细胞在诱导时也会伴随多种分化细胞的产生(成骨细胞+脂肪细胞、成骨细胞+血管细胞等),通过部分抑制成骨分化,可以有效的诱导干细胞向其他特定组织方向分化。
一种调控脂肪干细胞的成骨分化和组织再生的药物组合物,所述药物组合物含有有效量的上述Circ RNA-FTO促进剂或Circ RNA-FTO抑制剂。
所述的调控脂肪干细胞的成骨分化和组织再生的药物组合物中还包含:药学上可接受的载体。
所述的调控脂肪干细胞的成骨分化和组织再生的药物组合物中“药学上可接受的载体”为常规的给药载体,包括各种赋形剂或稀释剂等。
本申请的Circ RNA-FTO来源于chr16:53907697-53922863,基因长度15166碱基,由FTO第5,6,7个外显子环化形成,成环长度为334核苷酸。FTO是一个非常大的基因,其9个外显子在16号染色体上的跨度超过400kb。据报道,脂肪量和肥胖相关的FTO是调节体重和脂肪量的关键因素之一,它参与调控了常见的肥胖和体重指数。FTO主要通过在脂肪形成过程中调节脂肪形成而参与脂肪积累。据报道,抑制FTO或FTO突变导致体重减少和脂肪堆积减少。相反,FTO的过度表达导致体重和脂肪量增加。
实施例1脂肪干细胞生物学特性
1、人脂肪干细胞的分离和培养
吸脂手术中取腹部脂肪组织储存于20mL无菌注射器中备用,并冰袋保存。脂肪组织以1000rpm/5min在50mL离心管中离心,去除下层液体。脂肪组织以1:1的比例与0.2%的I型胶原酶在37℃环境下摇床消化40分钟,静置后可见其分成三层。移出上层油脂层,进行充分震荡和过滤(200目滤网)。随后1000rmp/5min三次进行离心移出上层液体,可见其中的细胞沉淀。用细胞培养液将细胞吹打均匀。随后将细胞接种于培养皿中,密度为5×105/mL。随后将细胞放入37℃、5%CO2、95%湿度的培养箱中。传代培养至第三代,获得第三代脂肪干细胞hADSCs。(第三代细胞完成后续实验)
2、脂肪干细胞表面标志物的表达情况
应用流式细胞术和免疫荧光术,观察脂肪干细胞FITC-CD73,CD44,CD105,CD45等间充质干细胞标志物的表达情况。根据中国医药生物技术协会关于《脂肪组织来源的干细胞提取、制备及储存质量管理专家共识》,本实验细胞符合间充质干细胞的表面标记特点。
3、多向分化能力
对步骤1的第三代人脂肪干细胞分别在成骨向诱导培养基、脂肪向诱导培养基和成软骨向诱导培养基培养3周,然后分别通过茜素红染色、油红O染色及甲苯胺蓝染色检测其多向分化潜能。其中,hADSCs细胞表面标志物的流式检测图如图1所示,hADSCs的多向分化图如图2所示,由左向右依次为:培养至第3代的hADSCs;培养后的hADSCs的成脂分化;hADSCs的成骨分化;hADSCs的成软骨分化,本发明分离培养的hADSCs通过鉴定和诱导,可分化为成骨细胞、脂肪细胞和成软骨细胞,具备干细胞的多向分化潜能。脂肪干细胞成骨向诱导后的茜素红染色结果图如图3所示;由左向右依次为:脂肪干细胞成骨诱导7天、14天和21天进行茜素红染色结果,用成骨诱导培养基诱导hADSCs 7天,14天,21天。7天组钙结节数量少,体积小,着色浅,分布零散。14天组钙结节数量比7天明显增多,体积增大,可见数个钙结节聚集成团。21天的hADSCs的茜素红染色明显强于14天诱导的hADSCs,视野内充满钙结节。
实施例2筛选出Circ RNA-FTO,并对其进行细胞定位及在成骨向诱导中的表达变化
1、将脂肪干细胞分别成骨向诱导0d、3d、5d、7d,提取RNA,通过RT-q PCR检测CircRNA-FTO不同时间的表达变化趋势。
实验结果如图4所示,Circ RNA-FTO的表达在成骨分化过程中随着时间上升。
2、小干扰RNA序列的设计及转染
Circ RNA-FTO序列(circbase ID:hsa-circ-0005941)设计引物,引物由上海吉玛生物科技有限公司合成并纯化,设计小干扰RNA序列抑制Circ RNA-FTO的表达(因FTO序列过大,极难成功构建过表达载体,因此本发明通过抑制Circ RNA-FTO表达进行反向研究论证)。
根据人Circ RNA-FTO序列起止点为其拼接点,于拼接点附近设计基于si RNA的热稳定性,末尾的碱基选择,GC含量设计出该circ RNA的si RNA最佳靶点,挑选最佳的1条相应的靶点序列如下:
正义链:5'-ggagggugugaugaucucauu-3'
反义链:5'-ugagaucaucacacccuccaa-3'
阴性对照NC(Negative Control)序列如下:
正义链:5'-uucuccgaacgugucacguuu-3'
反义链:5'-acgugacacguucggagaauu-3'
将细胞进行分组:siRNA-NC组、siRNA-FTO组,利用脂质体转染法将siRNA及阴性对照(Negative control,NC)分别转入hADSCs。本实验设置实验组与阴性对照组。实验组为转染siRNA后的细胞;阴性对照组为NC对照的细胞。12h后换回正常培养基,孵育24h后在荧光显微镜下观察,出现绿色荧光说明转染成功。提取总RNA,完成后续RT-q PCR的相关验证。
转染及沉默效率的检测结果显示,siRNA转染hADSC细胞可明显抑制Circ RNA-FTO的表达,小干扰RNA转染nc对照组和Circ RNA-FTO实验组的沉默效率的检测荧光显微镜图如图5所示,RT-PCR检测nc对照组和实验组的Circ RNA-FTO的表达情况图如图6所示;
3、沉默Circ RNA-FTO检测成骨相关蛋白及成骨相关染色
用无血清培养基清洗细胞,将siRNA-NC组、siRNA-FTO组,按照转染步骤说明书加入到细胞中。12h后换用正常的培养基,孵育72h,待转染稳定后给予成骨向诱导培养基培养。完成后续的Circ RNA-FTO对脂肪干细胞成骨分化的功能研究。
Western blot检测分别经siRNA-NC组和siRNA-FTO组转染的ADSCs成骨向诱导后成骨分化相关蛋白相对表达的检测实验结果如图7所示,siRNA-FTO组成骨能力相关因子(ALP、Runx2、OCN)的表达均较siRNA-NC组的表达降低,证明Circ RNA-FTO具备成骨向细胞分化的能力,分别经siRNA-NC组和siRNA-FTO组转染的ADSCs成骨向诱导后的碱性磷酸酶染色及茜素红染色检测成骨分化钙结节的数量和程度图如图8所示,其中,上为碱性磷酸酶染色,下为茜素红染色;结果表明,ALP染色和茜素红染色显示hADSCs成骨向诱导后siRNA-NC组与siRNA-FTO组有明显差异。
实施例3Circ RNA-FTO调控脂肪干细胞体内组织再生的研究
将人脂肪干细胞复合物植入到裸鼠背部皮下,观察其组织形成能力。
1、利用脂质体转染法将siRNA及阴性对照(Negative control,NC)分别转入hADSCs
采用组织块法和酶消化法培养人的脂肪干细胞,将生长旺盛的第三代脂肪干细胞接种在60mm培养皿,培养成分为α-MEM培养基(含10%胎牛血清,2mmol/L谷氨酰胺,100U/ml青霉素,100μg/ml链霉素)。利用脂质体转染法将siRNA及阴性对照(Negative control,NC)分别转入脂肪干细胞,实验分为2组:(1)siRNA-NC转染脂肪干细胞组(2)siRNA-FTO转染脂肪干细胞组。
2、生物支架材料的制备
将经过高压灭菌的Bio-Oss骨胶原生物支架材料与体外培养的脂肪干细胞进行共培养,比例50mg:1*107个细胞。随机分为2组:(1)siRNA-NC转染脂肪干细胞+Bio-Oss组(2)siRNA-FTO转染脂肪干细胞+Bio-Oss组。
3、支架材料/干细胞复合体体内回植
裸鼠随机分为2组:(1)siRNA NC转染hADSCs+Bio-Oss组;(2)siRNA circFTO转染hADSCs+Bio-Oss组;回植8w后,处死裸鼠获取标本,固定,脱钙,进行HE和Masson染色,观察生物支架组织再生情况。
实验结果显示如图9所示,Circ RNA-FTO后体内组织再生能力的变化下降明显(H&E染色和Masson染色显示体内8w后)siRNA-FTO转染脂肪干细胞+Bio-Oss组的骨组织含量极少,证实Circ RNA-FTO对组织再生能力有促进作用)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 辽宁省肿瘤医院
<120> 调控ADSCs成骨分化及组织再生Circ RNA-FTO及其应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 30
<211> 1518
<212> DNA
<213> hsa-circ-0005941基因的cDNA序列(Unknown)
<400> 30
atgaagcgaa ccccaaccgc cgaggaacga gagcgcgaag ctaagaaact gaggcttctt 60
gaagagctgg aagacacttg gcttccttat ctgaccccca aagatgatga attctatcag 120
cagtggcagc tgaaataccc taagctaatt ctccgagaag caggcagcgt ccctgaggga 180
ctccacaaag aggttcaaga agccttcctc gcactgcaca agcatggctg cttatttcgg 240
gacctggtca ggatccaagg caaagatttg ctcacgccag tatctcgcct cctcattggt 300
aaccccggct gcacctacaa gtacctgaac accaggctct tcacggtccc ctggccagtg 360
aagggctctg atgcaaagta caatgaggcc gagataggcg ccgcctgcca gaccttcctc 420
aagctcaacg actacctgca gattgagacc atccaggcgc tggaggaact cgctgccaag 480
gagaaagcca atatcgacac cgtgccggtg tgtataggtc cagatttccc cagggtcggc 540
atggggtcat cctttgacgg gcatgacgag gtggacagga agagcagagc cgcctacaac 600
ctaactttgt tgaacttcat ggatccccag aaaatgccgt acctgaaaga ggagccctac 660
tttggcatgg ggaagatggc tgtgagctgg catcacgatg aaaatctggt ggacaggtca 720
gcggtggcag tgtacaatta tagctgtgaa ggccctgaag aggaaagcga ggatgatccc 780
cagctcgaag gcagagatcc cgatgtgtgg catgttggct ttaagatctc atgggacata 840
gagacccctg gtttggcgat accccttcac caaggagact gctactttat gctggatgat 900
ctcaatgcca cccaccaaca ctgtgttttg gctggtttac caccccggtt tagttccacc 960
caccgagtgg ccgagtgctc gacgggaacc ttggattaca tcttacagcg ctgccagttg 1020
gccctgcaga atgtccgtga tgaggcggac agtggtgaag tctctttgaa atccttggag 1080
cctgcggttt tgaaacaagg agaagaaatc cacaacgagg tcgagtttga gtggctgaga 1140
cagttttggt ttcaaggcaa tcgatacaaa aagtgcaccg attggtggtg tcaacccatg 1200
actcagctgg aagagctttg gaagaagatg gaaggtgcga cccatgctgt gcttcgtgaa 1260
gttaggagag agggggcccc tgtggaacag agcagtgaca tcctgactgc catcctagcc 1320
gtgctcacca ctcgccagaa cctgaggagg gagtggcatg ccaggtgcca gtcccgaatt 1380
gcccgaactc tgcctgtgga ccagaagcca gaatgccggc cgtattggga aaaggatgat 1440
ccctccatgc ctctgccgtt tgatctcaca gacactgtgg ctgaactcag aggtctgctt 1500
ctggaagcca aaccctag 1518
<210> 29
<211> 21
<212> RNA
<213> hsa-circ-0005941的小干扰RNA序列正向引物(Unknown)
<400> 29
ggagggugug augaucucau u 21
<210> 30
<211> 21
<212> RNA
<213> hsa-circ-0005941的小干扰RNA序列反向引物(Unknown)
<400> 30
ugagaucauc acacccucca a 21
<210> 29
<211> 21
<212> RNA
<213> 阴性对照NC序列正向引物(Unknown)
<400> 29
uucuccgaac gugucacguu u 21
<210> 30
<211> 21
<212> RNA
<213> 阴性对照NC序列反向引物(Unknown)
<400> 30
acgugacacg uucggagaau u 21
Claims (1)
1. Circ RNA-FTO的抑制剂的应用,其特征在于,所述的Circ RNA-FTO的cDNA序列如SEQ ID NO:1所示,是由如该序列所示的序列首尾连接成的环状结构;用于制备抑制脂肪干细胞成骨分化和组织再生药物,所述的Circ RNA-FTO抑制剂包括可以降低Circ RNA-FTO的活性、减少Circ RNA-FTO表达量的物质;
所述的Circ RNA-FTO抑制剂为抑制Circ RNA-FTO表达的si RNA序列,所述的CircRNA-FTO的si RNA靶点序列为:
正义链:5'-ggagggugugaugaucucauu-3'
反义链:5'-ugagaucaucacacccuccaa-3'。
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