CN105950754B - 长链非编码rnaenst00000511346的应用 - Google Patents
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Abstract
本发明公开了一种长链非编码RNA(long no‑coding RNA,LncRNA)ENST00000511346的应用,即用于制备预测人脐带来源的间充质干细胞向成骨细胞分化的检测试剂,特别是制备出实时荧光定量分析法预测人脐带来源的间充质干细胞向成骨细胞分化的试剂盒。通过研究证实LncRNA ENST00000511346在成骨诱导培养处理后,ENST00000511346高表达,因此,将ENST00000511346的表达用于间充质干细胞向成骨细胞分化的预测,具有深远的临床意义和推广性。
Description
技术领域
本发明涉及干细胞分子生物学领域,具体涉及长链非编码RNA ENST00000511346在制备预测人脐带来源的间充质干细胞向成骨细胞分化制剂中的应用。
背景技术
间充质干细胞(Mesenchymal stem cells,MSCs)是一种具有自我复制能力和多向分化潜能的成体干细胞,属于非终末分化细胞,在体外特定的诱导条件下,可分化为脂肪、软骨、骨、肌肉、肌腱、神经、肝、心肌、胰岛β细胞和内皮等多种组织细胞,连续传代培养和冷冻保存后仍具有多向分化潜能,是一种理想的骨组织工程种子细胞。目前应用最广泛的骨髓、脂肪、脐带和脐带血等来源的间充质干细胞,它们均具有多向分化潜能;而且易于获得及扩增,连续传代培养和冷冻保存后仍具有多向分化潜能,可为组织器官的修复和再生提供所需的大量细胞;同时免疫原性低,不论是自体还是同种异源的间充质干细胞,一般都不会引起宿主的免疫反应,还具有独特的免疫调节功能。
长链非编码RNA(Long non-coding RNA,lncRNAs),是一类大于200bp的不编码蛋白的RNA。随着高通量测序技术的普及以及对LncRNAs的了解,人们发现LncRNAs在基因表达调控中起着举足轻重的作用。同样在间充质干细胞定向分化的过程中,LncRNA也通过调控相关基因的表达影响着分化的方向。
发明内容
本发明的目的是提供一种LncRNA ENST00000511346的应用。LncRNAENST00000511346(NONCODE ID:NONHSAT099313.2)定位于4号染色体,其表达情况能作为判断脐带间充质干细胞是否诱导分化为成骨细胞的依据,因此可以用于制备预测人脐带来源的间充质干细胞向成骨细胞分化的制剂,更进一步能够提供一种性价比高,易于推广应用的成骨效率评价的试剂盒。
长链非编码RNA ENST00000511346的应用,用于制备预测人脐带来源的间充质干细胞向成骨细胞分化制剂,该长链非编码RNA ENST00000511346的转录本序列如SEQ NO:1所示。
所述的预测人脐带来源的间充质干细胞向成骨细胞分化制剂包括检测长链非编码RNA ENST00000511346表达量的实时荧光定量PCR检测试剂。
所述的实时荧光定量PCR检测试剂包括实时荧光定量PCR的特异性引物:
lncRNA ENST00000511346特异性正向引物:
5'-CTTCAGTCTCCCAGGATCGT-3';
lncRNA ENST00000511346特异性反向引物:
5'-TCGAGAAACCGAACACTGATT-3'。
一种预测人脐带来源的间充质干细胞向成骨细胞分化的试剂盒,该试剂盒包括:
lncRNA ENST00000511346特异性正向引物:
5'-CTTCAGTCTCCCAGGATCGT-3';
lncRNA ENST00000511346特异性反向引物:
5'-TCGAGAAACCGAACACTGATT-3'。
该试剂盒还包括:
内参β-actin的特异性引物:
正向引物5'-CCTATCGAGCATGGAGTGGT-3'
反向引物5'-CTGAGGCATAGAGGGACAGC-3'
内参α-Tubulin的特异性引物:
正向引物5'-CCTGATGTATGCCAAACGTG-3'
反向引物5'-TCCCTGTAAAAGCAGCACCT-3'。
该试剂盒全套试剂包括:
(1)从诱导的间充质干细胞抽提总RNA所用试剂,包括Trizol试剂,三氯甲烷,异丙醇,无酶水;(2)以总RNA为模板将lncRNA ENST00000511346逆转录为cDNA所用试剂,包括逆转录缓冲液,dNTP,RNA酶抑制剂,MMLV逆转录酶以及随机引物;(3)将cDNA实时定量PCR所用试剂,包括lncRNA ENST00000511346的特异性引物、实时荧光定量SYBR染料、无酶水。
本发明通过成骨诱导培养基诱导人脐带来源的间充质干细胞,处理1,2,3周后,提取细胞总RNA,逆转录后进行实时荧光定量PCR分析LncRNA ENST00000511346的表达,发现:诱导14天LncRNA ENST00000511346表达是未诱导的95倍左右,诱导后表达增高显著,其它经典的成骨细胞分子marker如osteocalcin和osteopontin也增加,但倍数不如LncRNAENST00000511346的表达增高显著。据此,申请人提出利用LncRNA ENST00000511346制备预测人脐带来源的间充质干细胞向成骨细胞分化试剂。将LncRNA ENST00000511346用于预测人脐带来源的间充质干细胞是否向成骨细胞分化的方法:(1)收集成骨诱导后或未诱导的人脐带来源的间充质干细胞,抽提总RNA;(2)以总RNA为模板将LncRNA ENST00000511346逆转录为cDNA;(3)用LncRNA ENST00000511346特异性引物和内参引物进行实时荧光定量PCR扩增获得相对表达量,诱导和未诱导的表达量差异明显,可以作为是否成骨分化的预测试剂。
利用本发明的试剂可以检测LncRNA ENST00000511346在成骨诱导后的人脐带来源的间充质干细胞的表达水平从而判断是否成骨分化,为人脐带间充质干细胞成骨分化提供了lncRNA的分子标志物,具有深远的实践意义和推广性。
以下结合附图说明和具体实施方式进一步说明本发明,而非限制本发明。
附图说明
图1为实时荧光定量PCR分析LncRNA ENST00000511346成骨诱导后或未诱导的人脐带来源的间充质干细胞的表达差异;虽然诱导21天的表达水平要低于诱导14天,可能是由于其他基因或因子共同调控导致的,但是不论诱导时间的长短依旧能看出诱导后都要比诱导前的表达水平明显增高;该lncRNA ENST00000511346可作为脐带间充质干细胞向成骨细胞分化标志物;
图2为成骨标志物分子Osterix表达水平分析。
QC1205con代表用普通培养基培养组;
QC1205diff代表用成骨诱导培养基培养组。
具体实施方式
实施例1成骨诱导培养基诱导1、2、3周和未诱导的人脐带间充质干细胞中LncRNAENST00000511346的检测试剂盒
1.异丙醇100ml
2.Trizol试剂100ml
3.三氯甲烷50ml
4.1μM随机逆转录引物50μl
5.无酶水2ml
6.10mM dNTP 100μl
7.200U/μl RNA逆转录酶50μl
8.5×逆转录缓冲液1ml
9.40U/μl RNA抑制剂500μl
10.Premix Ex Taq 50μl
11.10μM lncRNA ENST00000511346特异性引物50μl
荧光定量PCR的特异性引物:
lncRNA ENST00000511346正向引物:
5'-CTTCAGTCTCCCAGGATCGT-3';
lncRNA ENST00000511346反向引物:
5'-TCGAGAAACCGAACACTGATT-3'。
12.10μM内参对照引物各50μl
内参β-actin的特异性引物:
5'-CCTATCGAGCATGGAGTGGT-3'
5'-CTGAGGCATAGAGGGACAGC-3'
内参α-Tubulin的特异性引物:
5'-CCTGATGTATGCCAAACGTG-3'
5'-TCCCTGTAAAAGCAGCACCT-3'。
实施例2人脐带间充质干细胞成骨诱导后LncRNA ENST00000511346的检测
(1)收集成骨诱导1,2,3周后和未诱导的人脐带来源的间充质干细胞,
抽提总RNA:去尽诱导后的培养板中培养基,加入1ml Trizol,在室温下水
平放置5min,使裂解液均匀分布于细胞表面,然后用移液枪吹打细胞使细胞
脱落;将细胞裂解液转移至离心管中,加入200μl三氯甲烷,盖紧离心管管
盖,上下振荡15秒,室温静置3分钟,12,000g,4℃离心15分钟。取出离心
管,样品分为三层:淡色的上清水相、中间的白色层及粉红色的下层有机相。
小心吸取淡色上清水相移至另一离心管,加入等体积的异丙醇,轻轻混匀,
室温静置10分钟,然后12,000g,4℃离心10分钟,在管底可见RNA沉淀。
小心去除上清,缓慢沿管壁加入1mL75%乙醇,轻轻混匀。12,000g,4℃离
心10分钟,小心吸尽上清。室温干燥沉淀2~5分钟,加入30~50μL的无
RNase水溶解RNA沉淀,分光光度计检测RNA的浓度和质量,OD260/280
比值在1.8-2.0之间,-70℃保存。
(2)以总RNA为模板将LncRNA ENST00000511346逆转录为cDNA;
Oligo(dT)18primer(100μM) 1μl
Total RNA 1μg
无酶水Total 12μl
逆转录第一步条件:62℃5分钟
5×逆转录缓冲液 4μl
dNTP(10mM) 2μl
RNA酶抑制剂(40U/μl) 1μl
MMLV逆转录酶(200U/μl) 1μl
第一步产物 12μl
Total 20μl
逆转录第二步程序,42℃60分钟,72℃5分钟。
(3)用LncRNA ENST00000511346特异性引物和内参引物进行实时荧光定量PCR:ENST00000511346特异性引物DNA序列由Invitrogen公司合成。
先将逆转录产物稀释5倍,混匀,20μl反应体系如下:
SYBR Premix 2×10μl
ENST00000511346或内参引物 1μl
cDNA产物 0.5μl
无酶水Total 20μl
实时荧光定量PCR反应95℃30秒,40个循环的95℃5秒,65℃30秒,72℃30秒,65~95℃,每0.05秒增加0.5℃。
lncRNA ENST00000511346特异性正向引物:5'-CTTCAGTCTCCCAGGATCGT-3'
lncRNA ENST00000511346特异性反向引物:5'-TCGAGAAACCGAACACTGATT-3'
内参β-actin的特异性引物:
正向引物5'-CCTATCGAGCATGGAGTGGT-3'
反向引物5'-CTGAGGCATAGAGGGACAGC-3'
内参α-Tubulin的特异性引物:
正向引物5'-CCTGATGTATGCCAAACGTG-3'
反向引物5'-TCCCTGTAAAAGCAGCACCT-3'
(4)成骨诱导的测定:本实验数据采用相对定量的分析方法,β-actin和α-Tubulin作为内参基因,数据利用GraphPad Prism进行分析。成功诱导脐带间充质干细胞向成骨细胞分化后lncRNA ENST00000511346显著上调,差异具有显著性(p<0.05)。
以上研究表明,lncRNA ENST00000511346可作为脐带间充质干细胞向成骨细胞分化标志物。
Claims (3)
1.长链非编码RNAENST00000511346的应用,其特征在于,用于制备预测人脐带来源的间充质干细胞向成骨细胞分化的制剂,该长链非编码RNAENST00000511346的转录本序列如SEQ NO:1所示。
2.根据权利要求1所述的应用,其特征在于,预测人脐带来源的间充质干细胞向成骨细胞分化的制剂包括检测长链非编码RNAENST00000511346表达量的实时荧光定量PCR检测试剂。
3.根据权利要求2所述的应用,其特征在于,所述的实时荧光定量PCR检测试剂包括实时荧光定量PCR的特异性引物:
lncRNA ENST00000511346特异性正向引物:
5'-CTTCAGTCTCCCAGGATCGT-3';
lncRNA ENST00000511346特异性反向引物:
5'-TCGAGAAACCGAACACTGATT-3'。
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Non-Patent Citations (5)
Title |
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ENST00000511346.1;Anonymous;《互联网文章》;20141005;第1-3页 |
MiR-29c regulates the expression of miR-34c and miR-449a by targeting DNA methyltransferase 3a and 3b in nasopharyngeal carcinoma;Man Niu et al;《BMC Cancer》;20160315;第16卷(第218期);第1-11页 |
人骨髓间充质干细胞成骨分化相关的长链非编码RNA表达谱特征;孙翔等;《广东医学》;20150531;第36卷(第9期);第1309-1313页 |
体外诱导骨髓与脐血间充质干细胞向成骨细胞分化及其成骨活性的研究;邵帅等;《中国组织工程研究》;20150604;第19卷(第23期);第3652-3657页 |
利用共培养法诱导人胎盘间充质干细胞向成骨细胞分化;张红艳等;《中华骨质疏松和骨矿盐疾病杂志》;20141231;第7卷(第4期);第325-331页 |
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