CN110305867B - 调控脂肪干细胞干性维持能力的方法及其试剂 - Google Patents
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Abstract
本发明涉及调控脂肪干细胞干性维持能力的方法及其试剂，发现LncRNA‑TCONS在脂肪干细胞干性维持过程中起了重要的调控作用，当LncRNA‑TCONS下调，脂肪的增殖、干性维持能力均受到抑制，由此确认LncRNA‑TCONS可作为多能干细胞干性维持的潜在靶点，可以用于开发出新的关于LncRNA‑TCONS相关药物。
Description
技术领域
本发明涉及生物医药领域，具体涉及调控脂肪干细胞干性维持能力的方法及其试剂。
背景技术
LncRNA是1990年由Brannan等研究人员首先在老鼠体内发现的非编码RNA，命名为H19，随后又在人类基因库中将其成功分离。在过去的二十几年中，随着新的生物信息及实验技术的发展，已经有超过50000个人类lncRNA被发现。虽然迄今为止已经发现了数以万计的lncRNA，但绝大多数还未检测其功能，在丰富多样的细胞进程中起什么具体的作用还犹未可知。目前发现的一些lncRNA能在基因内或基因间表达，存在许多转录模式，广泛参与DNA甲基化、组蛋白修饰、染色质重塑等生物学过程。由于lncRNA复杂的结构以及信息容量，使得它们在细胞进程中发挥着一系列重要的生物学作用，除了简单的信使作用外，还有如细胞分化、细胞周期调控等。LncRNA可以通过许多机制来发挥它们的调节作用。首先，它们与染色质修饰复合物紧密结合，并在转录水平调节基因表达。其次，它们对DNA损害等许多刺激都比较敏感，使外界环境与细胞应答相结合。最后，它们可以作为剪接因子调节基因表达。LncRNA有着高度的组织特异性，经常与相邻的编码基因共同表达，并能通过转录干扰、染色质重塑、组蛋白修饰、选择性剪切以及其他转录后调控来调节蛋白编码基因的表达。有研究表明，lncRNA可以在不同类型的肿瘤中调节细胞周期进程、细胞的增殖以及凋亡。LncRNA参与肿瘤细胞的增殖、侵袭、凋亡、迁移等生理过程，在肿瘤的发生、发展、复发等方面扮演着重要的角色。
干细胞(stemcells)是一类具有自我更新和多向分化潜能的细胞。自我更新是指干细胞分裂产生至少1个具有干细胞特性子细胞的过程。分化潜能是指未分化的干细胞在一定条件下可分化为各种类型的细胞。自我更新能维持干细胞具有多分化的潜能。对于组织特异性干细胞而言，自我更新是维持其终生具有分化潜能的基础。间充质干细胞(mesenchymalstemcells，MSCs)最早发现于骨髓，同时也存在于其他组织中，如脂肪组织和脐带等。MSCs增殖与分化的平衡决定其最终归宿:平衡状态下，MSCs发育成硬骨、软骨、脂肪或其他类型的细胞；失衡状态下，则可能发生骨肉瘤等骨病变。因此，明确MSCs增殖与分化的调控机制，对于骨肉瘤治疗和骨组织工程发展等都具有重要意义。
MSCs的干性维持包括两个方面:自我增殖能力和分化全能性或多能性，即在合适条件下，MSCs能一直传代下去，而且分化全能性保持不变。越来越多的研究发现:LncRNA是MSCs转录调控网络的重要组成部分，对维持MSCs多向分化潜能起着关键作用。Sheik等通过对小鼠ESCs全基因组转录调控网络分析，寻找与ESCs干性维持相关的LncRNA。他们发现LncRNAAK028326是Oct4的直接靶标，在调节反馈环中共激活Oct4。LncRNAAK141205是Nanog的直接靶标，可抑制Nanog活性。通过实验进一步证明了这些LncRNA不仅是由mESC人转录因子控制，而且LncRNA本身就可以调控mESC发育状态。Wang等报道:linc-RoR在人类胚胎干细胞(hESCs)的分化过程中表达急剧下降，linc-RoR可能作为竞争性内源RNA(ceRNA)调控Oct4、Sox2和Nanog的表达。Kretz等的研究表明:LncRNADANCR(anti-differentiationncRNA，DANCR)在细胞分化过程中表达急剧下降。Zhu等的研究表明:DANCR可通过与enhancerofzestehomolog2(EZH2)相互作用，抑制Runx2表达，从而使细胞停留在未分化状态。Jia等则发现:下调DANCR可能通过经典Wnt信号通路促进牙周膜MSCs的增殖和成骨分化。这些研究均提示DANCR在维持细胞干性方面起着重要作用，并且表明多种LncRNA在细胞干性维持方面起着重要的调控作用。
发明人在前期的CN107320726B中发现了首次发现LncRNA-TUG1在牙周膜干细胞干性维持过程中起了重要的调控作用，当LncRNA-TUG1下调，PDLSCs的增殖、自我更新、分化、迁移能力均受到抑制，由此确认LncRNA-TUG1可作为多能干细胞干性维持的潜在靶点，推动开发出新的关于LncRNA-TUG1相关药物。
然而，目前还未有关于其他的LncRNA与其他的干细胞的干性维持能力相关的更多的报道。
发明内容
针对上述现有技术，本发明的目的是提供LncRNA-TCONS在制备调控脂肪干细胞的干性维持能力的药物中的应用。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面，提供LncRNA-TCONS作为生物标志物在制备调控脂肪干细胞的干性维持能力的药物中的应用。
本发明的第二方面，提供LncRNA-TCONS促进剂在制备正性调控脂肪干细胞的干性维持能力的药物中的应用。
上述应用中，所述LncRNA-TCONS促进剂是指任何可以提高LncRNA-TCONS的活性、增加LncRNA-TCONS表达量的物质，例如增加LncRNA-TCONS表达量的LncRNA-TCONS的过表达载体。
具体的TCONS序列如SEQIDNO：1所示。
上述应用中，所述正性调控脂肪干细胞的干性维持能力包括：促进脂肪干细胞的增殖能力、促进脂肪干细胞的多向分化能力、促进脂肪干细胞的迁移能力和促进细胞干性相关因子表达的能力。
本发明的第三方面，提供LncRNA-TCONS抑制剂在制备负性调控脂肪干细胞的干性维持能力的药物中的应用。
上述应用中，所述LncRNA-TCONS抑制剂包括了拮抗剂、下调剂、阻滞剂、阻断剂等。
任何可以降低LncRNA-TCONS的活性、减少LncRNA-TCONS表达量的物质均可用于本发明。
作为优选的，所述LncRNA-TCONS抑制剂为抑制LncRNA-TCONS表达的慢病毒载体。
本发明的第四方面，提供一种调控脂肪干细胞的干性维持能力的药物组合物，
所述药物组合物含有有效量的上述LncRNA-TCONS促进剂或LncRNA-TCONS抑制剂。
进一步的，所述药物组合物中还包含：药学上可接受的载体。
所述“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。
上述技术方案具有如下有益效果：
本发明首次发现LncRNA-TCONS在脂肪干细胞干性维持过程中起了重要的调控作用，当LncRNA-TCONS下调，脂肪干细胞的增殖、干性维持能力均受到抑制，由此确认LncRNA-TCONS可作为多能干细胞干性维持的潜在靶点，可以用于开发出新的关于LncRNA-TCONS相关药物。
附图说明
图1传代的脂肪干细胞与刚分离的脂肪干细胞基因表达差异图
图2不同siRNA在抑制TCONS表达的抑制效果图，其中NC为对照
图3导入了siRNA的脂肪干细胞细胞生长曲线图
图4导入了siRNA1的脂肪干细胞中干细胞因子表达情况图
具体实施方式
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。
实施例1脂肪干细胞的制备
取皮下脂肪组织，PBS缓冲液冲洗3次，用眼科剪和眼科镊除去脂肪组织膜和血管并剪成1mm3大小的碎块，0.1％Ⅰ型胶原酶和0.25％胰酶37℃消化40min，期间搅拌5次，消化结束后加入完全培养基(含10％胎牛血清的DMEM，下同)，1500r/min离心10min，弃上清去除悬浮的脂肪细胞及脂滴，细胞沉淀重悬于完全培养基，接种于培养瓶中，于37℃，5％CO2培养箱中培养，24h后换培养液除去未贴壁细胞，之后每3天换液，细胞融合后用0.25％胰酶和1mmol/LEDTA37℃消化4min，完全培养基终止消化，1:2比例传代。经流式细胞术鉴定干细胞标志物正确，获得了所述的脂肪干细胞。
实施例2干扰载体的构建
发明人通过使用Agilent表达谱芯片按照基因表达杂交标准流程针对30代传代的脂肪干细胞与刚分离的脂肪干细胞进行表达谱分析，采用安捷伦扫描仪对完成杂交的芯片进行扫描，软件设置Dyechannel:Green,Scanresolution＝3μm,20bit.用FeatureExtraction软件10.7读取数据后采用GeneSpring软件11.0作归一化处理。挑选阈值变化>2.0或<-2.0并且Benjamini-Hochberg校正P值<0.05的差异表达基因。数据用Cluster3.0软件进行标准化和分群，并用JAVATreeview软件将数据做成树形图，发现了二个lncRNA具有较大的差异性表达。结果如图1定量所示，TCONS_00183316在多传代的脂肪干细胞中高表达，实验数据代表3次独立实验的平均值(**P<0.01)。因此，推定TCONS_00183316具有维持脂肪干细胞干性维持的功能。
根据SEQIDNO：1中的人TCONS_00183316lncRNA序列，设计3段针对该lncRNA的siRNA序列:siRNA1、siRNA2、siRNA3。各段序列见表1。
表1 lncRNA－siRNA序列
| 编号 | 序列 | 起始 |
| siRNA1 | tgctcttgggtaaggaatgc | 421 |
| siRNA2 | ggcagatcaacaagacagaa | 902 |
| siRNA3 | tagagaagcaagagcaaaca | 1408 |
根据siRNA序列设计重组质粒干扰序列的正义链和反义链。结果如表2所示。
表2重组质粒干扰序列的正义链和反义链
设计合成含有特异干扰序列的单链DNAoligo(序列见表2)，5'端引入AgeⅠ酶切位点，3'端引入EcoRⅠ酶切位点，组成特异寡核苷酸链。把上述干粉溶解于退火缓冲液中，置95℃水浴中5min，然后自然冷却至室温，退火形成黏性末端双链。将该黏性末端双链与质粒载体进行连接，连接体系如下:线性化DNA载体GV115(100ng/μl)1μl，DNAoligo(100ng/μl)1μl，10×T4噬菌体DNA连接酶缓冲液2μl，T4噬菌体DNA连接酶1μl，ddH2O补足至20μl，16℃连接过夜。连接后的重组质粒转化DH5α感受态细胞，经SOC培养基培养后，收集菌液，涂布平板，37℃过夜后每组样品挑取平板上的1个单菌落，筛选得到阳性菌落并获得相应载体。
实施例3细胞的转化
将前述实施例制备得到的干细胞，用胰蛋白酶消化对数生长期的细胞，以含10％胎牛血清的1640培养基调整细胞密度为1.5×107cells/20ml，重新接种于15cm细胞培养皿，37℃、5％CO2培养箱内培养。待细胞密度达70％～80％时(约24h)更换为无血清培养基。向一灭菌离心管中加入所制备的各DNA溶液(重组GV115载体20μg，pHelper1.0载体15μg，pHelper2.0载体10μg)，与相应体积的Opti－MEM混合均匀，调整总体积为2.5ml，在室温下温育5min。另取100μllipofectamine2000试剂在另一管中与2.4mlOpti－MEM混合，在室温下温育5min。把稀释后的DNA与lipofectamine2000进行混合，轻轻地颠倒混匀，在室温下温育20min，以便形成DNA－lipofectamine2000稀释液的转染复合物。将DNA－lipofectamine2000混合液转移至干细胞的培养液中，轻轻混匀，于37℃、5％CO2细胞培养箱中培养。培养8h后弃去培养基，PBS清洗1次，每瓶细胞中加入含10％血清的细胞培养基25ml，于37℃、5％CO2培养箱内继续培养48h。分别制备并获得相应的转基因细胞，命名为：siRNA1-干细胞，siRNA2-干细胞，siRNA3-干细胞，空白-干细胞。
实施例4基因水平检测
取2ug总RNA，按照美国Thermo公司提供的逆转录试剂盒说明书进行逆转录。获得cDNA序列。
设计引物如下：
TCONS(300bp)上游5'-accaacctctcttctcccta-3'
下游5'-tcagaggctaggattgcaac-3'
采用实时荧光定量PCR检测在转染慢病毒后脂肪干细胞中TCONS-lncRNA的表达水平，实验结果如图2所示：表明转染si-TCONS后脂肪干细胞中TCONS的表达显著降低，尤其以siRNA-1的效果最为显著。
实施例5CCK-8法检测细胞增殖
LncRNA-TCONS对脂肪干细胞增殖能力影响的分析，实验方法如下:
(1)取100微升细胞接种于96孔板中，浓度约为2000个细胞/孔，置于37摄氏度细胞培养箱中。
(2)分别在接种后24h,48h,72h向每孔中加入10微升CCK-8溶液，37摄氏度条件下再培养4小时。
(3)用酶标仪在450nm波长处检测各孔的吸光度并计算各组干细胞的增殖率。
(4)重复实验三次。结果如图3所示，沉默LncRNA，细胞的增殖能力减弱，特别是siRNA1使得细胞增殖得到显著的抑制。
实施例6干性相关因子的测定：
将导入了siRNA1的脂肪干细胞进行培养，而后采用本领域常规的方法对干性相关因子的mRNA水平进行测定，结果如图4所示。由图4可以看出，沉默LncRNA，Sox2、Nanog、Oct4这三个细胞干性相关因子表达减弱。
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> 山东如戴生物科技有限公司
<120> 调控脂肪干细胞干性维持能力的方法及其试剂
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aaagaagata catagctgaa aacacttgaa ggtatagact accttgaaag tcacgcaaga 60
actgttcttc ctgctttcca tatccaagat gcagaagtca aggcacaggg atgtgaacat 120
acgtgggtca tgtttcatag tacctggacc aagagaagag gtctctctat gttgagaaaa 180
tggcaatgcc ttcccacagc atggtacttc ttctccttct gagaatgata ctgttacata 240
aaggttttcg gcggtgcccc agaacacgga gatttccttc tgcccagctg catgaggcac 300
acatatcctg ggatggcatt aggaagatgg ccactttcac atcaacttat gtactctggc 360
accaacctct cttctcccta cccagcccaa cttcccaccc actgctattg ggatgatatg 420
tgctcttggg taaggaatgc atatccaaat gctacctcag atcagggagg taatacctgg 480
cctcaattct ctgcccttaa agaatcttgc agccaaattg ggtaaagagt caagacccat 540
cggtgtgctt tattcaggag acccatctca cgtgcaaaga cacacatggg cccaaaataa 600
agggatggag taatatttac caaggaaatg gaaagcacac acacacaaaa aaaagcagag 660
gttgcaatcc tagcctctga taaaacagac tttaaaccaa caaagatcaa aaaagacaaa 720
gaagggcatt acatactggt aaagggatca atgcaacaag aagagctaac tctcctaaat 780
atatatgcac ccaatacagg agcacccaca ttcataaagc aagttcttag agacctacaa 840
agagacttag actcccacac aataatagtg ggagactttt aacacctcac tgtcaatatt 900
aggcagatca acaagacaga aaattaacca ggatattcag gatgtgaact aagctctgga 960
ccaagaggac ctaatagaca tctgtggaac tctccacccc aaatcaatag aatgtacatt 1020
cttctcagca ccacatcgca cttattctaa aattgaccac ataattggaa ataaaacact 1080
cctcagcaaa tgcaaaagaa tggaaatcat tacagtctct cagatcacag tgcaatcaag 1140
ttagaactca ggattaagaa actcagtcag aaccggacaa cgacatggaa actgacaacc 1200
tgctcctgaa tgactactgg gtaaataaca aaattaagga agaaataaat aagttctttg 1260
aaaccaatga gaacaaagac acaatgtact agagtctctg ggacacagct aaagcagtgt 1320
ttagagggaa atttatagca ccaaatgccc acaggagaaa gcaggaaaga tctaaaatcg 1380
acaccgtaat atcacaatta aaagaactag agaagcaaga gcaaacaaat tcaaaagcta 1440
gcagaaggca agaaatgact aagatcagag cagaactgaa ggagacagag acacatggac 1500
acaggagaat gagaacacat ggacacaggg agggtaacat cacaaactgg ggcctgtcag 1560
gggtggggag tggtggggag ggatagcatt aggagaaaga tctaatgtag atgaaagttt 1620
gatgggtgca gcaaaccacc atggcatgtg catacctatg taacaaacct gcacattctg 1680
cacgtgtatc ccagaactta aagtataata taataaaaga atctcatagc catcatctgt 1740
tgtatacaac agacctcaaa ttagtctccc tctgcttctc caacagtgtg gcctcttcta 1800
tgaagctgtc tacccatggg tttccccaac atcccatccc ccagccctca ggtttctcct 1860
ccccatcatc ttcacttcag aaagagaaat gaaaaagaaa ttcaagtgac tcatcggtcc 1920
acataggcag gagtgggaga ggctgagaga gtgaatttgt gggtccagat gaatatttcc 1980
gcaggcctct tgactgcttc acacgatctg ctg 2013
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accaacctct cttctcccta 20
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Claims (2)
1.LncRNA-TCONS抑制剂在制备负性调控脂肪干细胞的干性维持能力的药物中的应用，其中LncRNA-TCONS序列如SEQ ID NO：1所示，其中抑制剂为针对LncRNA-TCONS序列的siRNA。
2.LncRNA-TCONS促进剂在制备正性调控脂肪干细胞的干性维持能力的药物中的应用，所述LncRNA-TCONS促进剂是指能增加LncRNA-TCONS表达量的物质，其中LncRNA-TCONS序列如SEQ ID NO：1所示，其中增加LncRNA-TCONS表达量的物质为LncRNA-TCONS的过表达载体。
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