CN115054612B - 一种可用于促进骨形成的增强子及其应用 - Google Patents
一种可用于促进骨形成的增强子及其应用 Download PDFInfo
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Abstract
本发明属于生物领域,具体涉及一种可用于促进骨形成的增强子及其应用。本发明提供了了一个与骨形成过程有着密切关系的增强子,该增强子可以调控成骨前体细胞的增殖和分化为成骨细胞,从而影响骨的形成;本发明通过敲除成骨前体细胞中特定增强子片段,提供了所述增强子调控成骨前体细胞增殖和向成骨细胞分化的方法,从而为研究预防和治疗骨质疏松症的相关药物提供了技术思路。
Description
技术领域
本发明涉及生物领域,具体涉及一种可用于促进骨形成的增强子及其应用。
背景技术
增强子是基因组上的一段可以与转录因子结合,促进基因转录的一类长约300bp的DNA非编码序列。增强子可以与启动子、沉默子等顺式作用元件结合,也可以与转录因子等反式作用元件相互协作,共同调控基因的表达。大量全基因组关联分析显示,与疾病相关联的变异富集在非编码调控区,尤其是细胞特异性的增强子区。增强子序列的缺失或扩增、表观遗传修饰变化等因素都可能导致增强子活性异常,从而引起疾病的发生发展,进而影响人类健康。
骨形成过程包括了骨间充质干细胞分化成为成骨细胞前体细胞;成骨细胞前体细胞分化为成骨细胞;成骨细胞进一步分化分泌胞外基质并矿化,成为骨细胞。骨形成过程中,越来越多的基因被鉴定出参与骨发生过程中,例如ALP、RUNX2和OCN。增强子可以与转录因子结合,调控骨形成中的关键基因,从而在骨质疏松的发生发展中发挥重要作用。但是增强子与其调控靶基因的距离和方向不确定,并且一个增强子可以调控多个基因。因此,在骨形成过程中,大量增强子尚未被鉴定,它们的调控机制也未被解析。
骨质疏松症是一种全身性的骨代谢疾病,表现为骨量减少、骨质量下降和骨折风险增加。随着中国社会老龄化问题的加重,骨质疏松症所引发的中老年健康问题将会逐年凸显。不仅如此,长期执行太空任务的宇航员,由于外太空的特殊环境,例如失重及辐射等,也会诱发宇航员患有骨质疏松症。目前临床上用于治疗骨质疏松的药物,例如雌激素、降钙素或合成类固醇等药物,均存在效果不理想且副作用大,长期服用甚至增加患癌风险。目前,与骨质疏松相关的增强子研究知之甚少,对骨形成过程中增强子的研究和鉴定,将为骨质疏松的预防和治疗提供新的策略,将成为攻克增龄性骨丢失或空间骨丢失新的突破口。
发明内容
本发明为了解决以上提出的问题,经过发明人的深入研究和分析,发现在chr11(mm10):86624000-86624800处有一个增强子;该增强子在骨形成过程中处于激活状态并参与成骨前体细胞的增殖和向成骨细胞分化,是促进骨形成的潜在增强子;该增强子敲除后,成骨前体细胞的增殖能力和分化为成骨细胞的能力均显著降低。
本发明目的之一在于提供一种成骨前体细胞中的增强子,所述增强子的核苷酸序列为SEQ ID NO.1所示。
SEQ ID NO.1:
GATGGCTCATCTGTTAAGAATACTGGCTGCTCTCCCCCTGGCAGAAGGTCTAGGCTCAATTTTCAGCACCCACTTAGTAGCTCACAACCATCTATGATTCGAGTTCAGGAGTTCCAACAACTTCTTCTGAGCTCTTCACAAATACGGTACACATACACACATGCAGGCAAATTCACACACTTAAAATAAAAATAAAGATGTTAAGCAACAAAATCCTGAATGTCACTGGCTGTGTGTGGTTGGTGGGATCTGAAAGGAGATCTACATGACAAGACCTCGAATGAGCCTAGAATGAACATTTCTAATACTTAATGCAAATAAGCAAAAGATCCTGAGAGGAGGAAGCTTTTCCCAGGAACTGACACAACACAAACCATATCGTTGCTGCAAAGCTTAATGCAAACCCTCTAGAGTCCACGTTGACAAATTCTTCAACAAGCCCTGTGGTTACAGGGAAGCTGGTTTCACTCTGACTCACGCCTGCTCAAACAGTTTACTGGAAATGAAGAGGAGGGGGAATTTTCAGAGAAGTCATGTGATGGCCAGACAAACCACAACCAGCAAAGGGTTCAAAAGAGTCAAAAACAGGTCCCAGCAACAAATGACATTAAAAACATATGAAAATGACCCCTGTTTAGTTTCAGATCATCTATACTATACCATGAGCTCTTTCATAAAATCTTTGTATGAAAAAGAACAGATACTTCATAGGGGGAAAACCCAGCAGGCACTGACTGACCTGGCATATCCAACTGCTAACACTCACAGGAAAATGTGCTCAACAAACAGAACATAAATGGA
本发明目的之二在于提供一种促进或抑制成骨前体细胞增殖和分化为成骨细胞的方法,具体技术方案如下:
一种体外抑制成骨前体细胞增殖和分化为成骨细胞的方法,敲除上述细胞中的增强子。
具体步骤如下:
(1)设计sgRNA,敲除所述增强子序列;
(2)利用CRISPR/Cas9切割所述增强子序列片段。
优选的:敲除所述增强子序列的sgRNA序列分别如SEQ ID NO.2、SEQ ID NO.3所示:
SEQ ID NO.2:CTAGACAGTTTAAGTATTAC(上游);GCTGCTTGGCAGGCTGAGTC(下游)
SEQ ID NO.3:CTGTTGGCTCTCCCTGGAAC(上游);GATAAGTAGTTTACCTAGTC(下游)
本发明目的之三在于提供一种体外抑制成骨前体细胞增殖和分化为成骨细胞的方法,具体为,通过敲除所述增强子从而抑制所述增强子活性。
优选的:成骨前体细胞为MC3T3-E1细胞。
总之,本发明所述的增强子在骨形成过程中发挥了重要作用,该增强子可以促进MC3T3-E1细胞的增殖和分化为成骨细胞。
本发明的有益之处在于:本发明找到了一个增强子,并通过设计sgRNA敲除所述增强子序列,提供了多方位的抑制成骨前体细胞增殖和分化为成骨细胞的方法,表明该增强子在骨形成过程中的积极作用,从而为研究预防和治疗骨质疏松的相关药物提供了技术思路和方法。
附图说明
图1为通过设计两对sgRNA,CRISPR/Cas9敲除所述增强子区域筛选出两株双拷贝敲除的细胞株(#1和#2),经分子克隆后进行测序分析以鉴定敲除结果。
图2为实施例2中CCK8检测增强子敲除的两组细胞株(#1和#2)相比对照细胞(WT)的增殖能力图。(*P<0.05;***P<0.001)
图3为实施例2中EdU检测增强子敲除的两组细胞株(#1和#2)相比对照细胞(WT)的增殖能力图。(*P<0.05;**P<0.01)
图4为实施例2中ALP染色检测增强子敲除的两组细胞株(#1和#2)相比对照细胞(WT)的碱性磷酸酶活性图。
图5为实施例2中茜素红染色检测增强子敲除的两组细胞株(#1和#2)相比对照细胞(WT)的钙结节图。
具体实施方式
接下来将会通过实施例进一步解释和说明本发明细则,需要说明的是,以下的实施例描述仅是对本发明的解释,并不用于限定本发明。
实施例1 MC3T3-E1细胞培养
细胞培养基配置:含10%FBS(胎牛血清)、1%P/S(双抗)的MEM-α培养基,4℃冰箱保存.
实验前准备:细胞间和无菌超净台预先紫外消毒30min,水浴锅恒温至37℃,细胞培养基孵育至37℃放入超净台备用。
细胞复苏和培养:从液氮罐或-80℃冰箱取出相应的细胞冻存管(需带手套防止冻伤),置于37℃水浴锅并轻微震荡,待细胞液完全融化后取出置于超净台,移液器吹打几次悬液后转入离心管,1000rpm离心3-5min;弃掉上清,加入适量细胞培养夜重悬细胞,转移到培养皿中并补足培养基,轻轻摇匀,置于37℃,5%CO2培养箱中培养。24h首次换液,以后每2-3天换液一次,定期观察细胞状态。
实施例2通过CRISPR-Cas9技术敲除增强子片段,获得敲除增强子的单细胞克隆,并验证该增强子敲除后对细胞增殖和分化能力的影响
1)CRISPR-Cas9技术敲除增强子片段
根据该增强子的位置,在该增强子上游200bp和下游200bp范围内选定敲除区域,首先设计sgRNA,sgRNA序列分别如SEQ ID NO.2、SEQ ID NO.3所示;接下来将设计好的sgRNA分别克隆至带有mCherry和GFP荧光信号的质粒载体(Addgene#64324和#48138)中,通过Lipofectamine 3000(Thermo Fisher Scientific)将包含sgRNA和带有荧光信号的质粒转染至MC3T3-E1细胞中;转染24小时后,通过流式分选技术筛选出同时含有mCherry和GFP的单细胞,并在96孔板中进行细胞扩增,等待扩增至足够数量的细胞即可进行后续验证试验。
通过Sanger测序验证敲除效果,最终测序结果显示成功获得两株该增强子双拷贝敲除的细胞株#1和#2(图1)。
2)CCK8和EdU实验检测细胞增殖能力
接下来,通过CCK8和EdU验证该增强子敲除后是否影响MC3T3-E1细胞增殖能力。对照组细胞(WT)和两株所述增强子双拷贝敲除的细胞株(#1和#2)分别以4000细胞/孔的密度均匀铺在96孔板中,置于37℃,5%CO2培养箱中继续培养。48小时后按照CCK-8CellCounting kit(Vazyme)说明书处理细胞并使用酶标仪检测450nm处的吸光度;72小时后按照Click-iT EdU成像试剂盒(百瑞极)说明书处理细胞,荧光显微镜下拍照并计算EdU阳性细胞率。
CCK8和EdU实验结果均显示,相较对照组细胞(WT),两株该增强子双拷贝敲除的细胞株(#1和#2)增殖能力受到显著抑制(图2和图3)。以上结果说明该增强子可以调控MC3T3-E1细胞的增殖。
3)碱性磷酸酶(ALP)染色和茜素红染色检测细胞向成骨细胞分化能力
ALP是成熟成骨细胞的早期标志性酶,同时成骨细胞形成的钙结节也是成骨细胞的标志物。通过ALP染色,碱性磷酸酶活性部位可以呈现蓝色;茜素红染色即茜素红与钙发生显色反应,产生一种深红色的带色化合物,成骨诱导的细胞外面沉积的钙结节可以被染成深红色。因此,ALP染色和茜素红染色可以验证MC3T3-E1细胞中敲除所述增强子对细胞向成骨细胞分化的影响。
按照实施例2的细胞培养方法将MC3T3-E1细胞和两株该增强子双拷贝敲除的细胞株(#1和#2)铺在六孔板,置于37℃,5%CO2培养箱中继续培养三天,待细胞汇合度达到80%以上,更换为诱导培养基,诱导培养基组分包括10%FBS(胎牛血清)、1%P/S(双抗)的MEM-α培养基,并添加终浓度为50μg/ml的L-抗坏血酸,10mM的β-甘油磷酸二钠盐,100nM的地塞米松。每两天换液一次,在诱导14天后通过碱性磷酸酶染色液(Solarbio)检测碱性磷酸酶活性,诱导21天后通过茜素红染色液(Solarbio)检测钙结节。
实验结果显示,相较对照组细胞(WT),两株该增强子双拷贝敲除的细胞株(#1和#2)的碱性磷酸酶活性显著降低(图4),茜素红染色颜色较浅,无明显钙结节(图5)。以上结果说明该增强子可以调控MC3T3-E1细胞向成骨细胞的分化,影响骨发生过程。
最后需要说明的是,对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
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Claims (4)
1.一种增强子在制备治疗骨质疏松症的药物中的用途,其特征在于,所述增强子的核苷酸序列为SEQ ID NO.1所示。
2.如权利要求1所述的用途,其特征在于,所述增强子促进成骨前体细胞增殖。
3.如权利要求1所述的用途,其特征在于,所述增强子促进成骨前体细胞向成骨细胞分化。
4.一种体外抑制成骨前体细胞增殖和分化的方法,其特征在于,抑制成骨前体细胞中的权利要求1中所述的增强子的活性,具体步骤包括:(1)通过设计sgRNA,敲除所述增强子序列,(2)利用CRISPR/Cas9切割所述增强子序列片段;设计的sgRNA的序列分别如SEQ IDNO.2、SEQ ID NO.3所示;成骨前体细胞为小鼠MC3T3-E1细胞。
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