CN105925706B - The application of long-chain non-coding RNA LOC100506530 - Google Patents
The application of long-chain non-coding RNA LOC100506530 Download PDFInfo
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- CN105925706B CN105925706B CN201610435511.5A CN201610435511A CN105925706B CN 105925706 B CN105925706 B CN 105925706B CN 201610435511 A CN201610435511 A CN 201610435511A CN 105925706 B CN105925706 B CN 105925706B
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Abstract
The invention discloses a kind of long-chain non-coding RNA (long no-coding RNA, LncRNA) the application of LOC100506530, the mescenchymal stem cell in prediction people's umbilical cord source is used to prepare to the detection reagent of osteoblast differentiation, especially prepares the mescenchymal stem cell in Real time PCR method prediction people's umbilical cord source to the kit of osteoblast differentiation.LncRNA LOC100506530 is confirmed after osteogenic induction culture processing by research, therefore the expression of LOC100506530, is used for prediction of the mescenchymal stem cell to osteoblast differentiation, has far-reaching clinical meaning and generalization by LOC100506530 high expression.
Description
Technical field
The present invention relates to stem cell molecular biology fields, and in particular to long-chain non-coding RNA LOC100506530 is making
Application of the mescenchymal stem cell in standby prediction people's umbilical cord source into osteoblast differentiation preparation.
Background technique
Mescenchymal stem cell (Mesenchymal stem cells, MSCs) is a kind of with the of self-replication capacity and multidirectional
The adult stem cell of differentiation potential belongs to non-terminally differentiated cells, in vitro under specific inductive condition, can be divided into fat,
The Various Tissues cell such as cartilage, bone, muscle, tendon, nerve, liver, cardiac muscle, beta Cell of islet and endothelium, continuous passage culture and cold
Freezing still has multi-lineage potential after saving, be a kind of ideal Seeding Cells in Bone Tissue Engineering.Current most widely used bone
The mescenchymal stem cell in the sources such as marrow, fat, umbilical cord and Cord blood, they all have multi-lineage potential；And it is easily obtained
And amplification, still there is multi-lineage potential after continuous passage culture and freezen protective, can be mentioned for the reparation and regeneration of histoorgan
For required a large amount of cells；Immunogenicity is low simultaneously, whether self or allogenic mescenchymal stem cell, generally not
It can cause the immune response of host, also there is unique immunoloregulation function.
Long-chain non-coding RNA (Long non-coding RNA, lncRNAs) is a kind of not encode egg greater than 200bp
White RNA.With universal and to LncRNAs the understanding of high throughput sequencing technologies, it has been found that LncRNAs is in gene expression
Very important effect is played in regulation.Equally during mescenchymal stem cell directed differentiation, LncRNA is also by regulation
The expression of related gene affects the direction of differentiation.
Summary of the invention
The object of the present invention is to provide the applications of LncRNA LOC100506530 a kind of.LncRNA LOC100506530
(Gene ID:100506530), is positioned at No. 15 chromosomes, and expression can be used as whether judge umbilical cord mesenchymal stem cells
It is induced to differentiate into the foundation of osteoblast, therefore the mescenchymal stem cell that can be used for preparing prediction people's umbilical cord source is thin to skeletonization
The preparation of born of the same parents' differentiation is further capable of providing the kit that a kind of cost performance is high, and the skeletonization efficiency of application easy to spread is evaluated.
The application of long-chain non-coding RNA LOC100506530 is used to prepare the mescenchymal stem cell in prediction people's umbilical cord source
To osteoblast differentiation preparation, the transcript sequence of long-chain non-coding RNA LOC100506530 is as shown in SEQ NO:1.
The mescenchymal stem cell in prediction people's umbilical cord source includes detecting the non-volume of long-chain to osteoblast differentiation preparation
The real-time fluorescence quantitative PCR detection reagent of code RNA LOC100506530 expression quantity.
The real-time fluorescence quantitative PCR detection reagent includes the specific primer of real-time fluorescence quantitative PCR:
LncRNA LOC100506530 specific forward primer: 5'-ATGCAATGTTTCGATCATGG-3'
LncRNA LOC100506530 specific reverse primers: 5'-ACCATCCCCAGGGTCTTAGT-3'.
To the kit of osteoblast differentiation, which includes: for a kind of mescenchymal stem cell that predicting people's umbilical cord source
LncRNA LOC100506530 specific forward primer: 5'-ATGCAATGTTTCGATCATGG-3'
LncRNA LOC100506530 specific reverse primers: 5'-ACCATCCCCAGGGTCTTAGT-3'.
The kit further include:
The specific primer of internal reference β-actin:
Forward primer: 5'-CCTATCGAGCATGGAGTGGT-3'
Reverse primer: 5'-CTGAGGCATAGAGGGACAGC-3'
The specific primer of internal reference α-Tubulin:
Forward primer: 5'-CCTGATGTATGCCAAACGTG-3'
Reverse primer: 5'-TCCCTGTAAAAGCAGCACCT-3'.
The kit full set reagent includes:
(1) from the mescenchymal stem cell extracted total RNA agents useful for same of induction, including Trizol reagent, chloroform, isopropyl
Alcohol, no enzyme water；(2) it is cDNA agents useful for same by lncRNA LOC100506530 reverse transcription by template of total serum IgE, including reverses
Record buffer, dNTP, RNase inhibitor, MMLV reverse transcriptase and random primer；(3) it will be tried used in cDNA real-time quantitative PCR
Agent, specific primer, real time fluorescent quantitative SYBR dyestuff including lncRNA LOC100506530, without enzyme water.
The present invention induces the mescenchymal stem cell in people's umbilical cord source to mention after processing 1,2,3 weeks by Osteogenic Induction Medium
Cell total rna is taken, the expression of real-time fluorescence quantitative PCR analysis LncRNA LOC100506530 is carried out after reverse transcription, discovery: is lured
Leading LncRNA LOC100506530 expression in 14 days is more than 4000 times not induced, and expression is increased significant after induction, other classics
Osteoblast molecule marker such as osteocalcin and osteopontin also increase, but multiple is not so good as LncRNA
The expression of LOC100506530 is increased significantly.Accordingly, applicant proposes to utilize LncRNA LOC100506530 preparation prediction people's navel
Mescenchymal stem cell with source is to osteoblast differentiation reagent.LncRNA LOC100506530 is used to predict that people's umbilical cord to come
The mescenchymal stem cell in source whether to osteoblast differentiation method: (1) collect after osteogenic induction or people's umbilical cord for not inducing come
The mescenchymal stem cell in source, extracted total RNA；It (2) is cDNA by LncRNA LOC100506530 reverse transcription by template of total serum IgE；
(3) real-time fluorescence quantitative PCR amplification is carried out with LncRNA LOC100506530 specific primer and internal control primer obtain opposite table
Up to amount, induction and the expression difference not induced are obvious, can be used as whether the prediction reagent of Osteoblast Differentiation.
It can detecte people umbilical cord source of the LncRNA LOC100506530 after osteogenic induction using reagent of the invention
The expression of mescenchymal stem cell provides to judge whether Osteoblast Differentiation for human umbilical cord mesenchymal stem cells Osteoblast Differentiation
The molecular marker of lncRNA has far-reaching practice significance and generalization.
The present invention is further illustrated below in conjunction with Detailed description of the invention and specific embodiment, is not intended to limit the present invention.
Detailed description of the invention
Fig. 1 is that real-time fluorescence quantitative PCR is analyzed after LncRNA LOC100506530 osteogenic induction or people's umbilical cord for not inducing
The differential expression of the mescenchymal stem cell in source；Although the expression of induction 21 days will be lower than induction 14 days, it may be possible to due to
Caused by other genes or the factor regulate and control jointly, no matter but the length of induction time will be than induction after capable of still finding out induction
Preceding expression obviously increases；The lncRNA LOC100506530 can be used as umbilical cord mesenchymal stem cells to osteoblast point
Change marker；
Fig. 2 is the correlation analysis of skeletonization marker molecules OPN expression；
QC1205con representative ordinary culture medium culture group；
QC1205diff representative Osteogenic Induction Medium culture group.
Specific embodiment
1 Osteogenic Induction Medium of embodiment induce 1,2,3 week and the human umbilical cord mesenchymal stem cells that do not induce in LncRNA
The detection kit of LOC100506530
1. isopropanol 100ml
2.Trizol reagent 100ml
3. chloroform 50ml
4. 1 μM of random 50 μ l of reverse transcriptase primer
5. without enzyme water 2ml
6. 10mM dNTP 100μl
7. 50 μ l of 200U/ μ l RNA reverse transcriptase
8. 5 × RT Buffer 1ml
9. 500 μ l of 40U/ μ l RNA inhibitor
10.Premix Ex Taq 50μl
11. 10 μM of 50 μ l of lncRNA LOC100506530 specific primer
The specific primer of quantitative fluorescent PCR:
LncRNA LOC100506530 forward primer: 5'-ATGCAATGTTTCGATCATGG-3'
LncRNA LOC100506530 reverse primer: 5'-ACCATCCCCAGGGTCTTAGT-3'
12. 10 μM of internal references compare each 50 μ l of primer
The specific primer of internal reference β-actin:
5'-CCTATCGAGCATGGAGTGGT-3'
5'-CTGAGGCATAGAGGGACAGC-3'
The specific primer of internal reference α-Tubulin:
5'-CCTGATGTATGCCAAACGTG-3'
5'-TCCCTGTAAAAGCAGCACCT-3'
After 2 human umbilical cord mesenchymal stem cells osteogenic induction of embodiment LncRNA LOC100506530 detection (1) collect at
The mescenchymal stem cell in people's umbilical cord source that 1,2,3 Zhou Houhe of self-bone grafting is not induced, extracted total RNA: the culture after going most induction
1ml Trizol is added in culture medium in plate, is horizontally arranged 5min at room temperature, so that lysate is uniformly distributed in cell surface, so
Make cell detachment with liquid-transfering gun piping and druming cell afterwards；Cell pyrolysis liquid is transferred in centrifuge tube, 200 μ l chloroforms, lid is added
Tight centrifuge tube pipe lid, oscillation 15 seconds, are stored at room temperature 3 minutes, 12,000g, 4 DEG C are centrifuged 15 minutes up and down.Take out centrifuge tube, sample
It is divided into three layers: lower layer's organic phase of the supernatant water phase of light color, intermediate white layer and pink.It is careful to draw light color supernatant water phase
Another centrifuge tube is moved to, isometric isopropanol is added, mixes gently, is stored at room temperature 10 minutes, then 12,000g, 4 DEG C of centrifugations
10 minutes, in the visible RNA precipitate of tube bottom.Careful removal supernatant, is slowly added 1mL75% ethyl alcohol along tube wall, mixes gently.12,
000g, 4 DEG C are centrifuged 10 minutes, carefully exhaust supernatant.Drying at room temperature precipitate 2~5 minutes, be added 30~50 μ L without RNase water
RNA precipitate is dissolved, spectrophotometer detects the concentration and quality of RNA, and OD260/280 ratio is between 1.8-2.0, -70 DEG C of guarantors
It deposits.
It (2) is cDNA by LncRNA LOC100506530 reverse transcription by template of total serum IgE；
Oligo(dT)18primer(100μM) 1μl
Total RNA 1μg
Without 12 μ l of enzyme water Total
Reverse transcription first step condition: 62 DEG C 5 minutes
5 × RT Buffer, 4 μ l
dNTP(10mM) 2μl
1 μ l of RNase inhibitor (40U/ μ l)
1 μ l of MMLV reverse transcriptase (200U/ μ l)
12 μ l of first step product
Total 20μl
Reverse transcription second step program, 42 DEG C 60 minutes, 72 DEG C 5 minutes.
(3) real-time fluorescence quantitative PCR is carried out with LncRNA LOC100506530 specific primer and internal control primer:
LOC100506530 specific primer DNA sequence dna is synthesized by Invitrogen company.
Reverse transcription product is first diluted 5 times, is mixed, 20 μ l reaction systems are as follows:
SYBR Premix 2× 10μl
1 μ l of LOC100506530 or internal control primer
0.5 μ l of cDNA product
Without 20 μ l of enzyme water Total
Real-time fluorescence quantitative PCR react 95 DEG C 30 seconds, 40 circulation 95 DEG C 5 seconds, 65 DEG C 30 seconds, 72 DEG C 30 seconds, 65~
95 DEG C, increase by 0.5 DEG C within every 0.05 second.
LncRNA LOC100506530 forward primer: 5'-ATGCAATGTTTCGATCATGG-3'
LncRNA LOC100506530 reverse primer: 5'-ACCATCCCCAGGGTCTTAGT-3'
The specific primer of internal reference β-actin:
Forward primer: 5'-CCTATCGAGCATGGAGTGGT-3'
Reverse primer: 5'-CTGAGGCATAGAGGGACAGC-3'
The specific primer of internal reference α-Tubulin:
Forward primer: 5'-CCTGATGTATGCCAAACGTG-3'
Reverse primer: 5'-TCCCTGTAAAAGCAGCACCT-3'.
(4) measurement of osteogenic induction: this experimental data uses the analysis method of relative quantification, β-actin and α-Tubulin
As reference gene, data are analyzed using GraphPad Prism.Success inducing umbilical cord mesenchymal stem is thin to skeletonization
LncRNA LOC100506530 is significantly raised after born of the same parents' differentiation, and difference has conspicuousness (p < 0.05).
Above studies have shown that lncRNA LOC100506530 can be used as umbilical cord mesenchymal stem cells to osteoblast differentiation
Marker.
Claims (3)
1. the application of long-chain non-coding RNA LOC100506530, which is characterized in that be used to prepare between prediction people's umbilical cord source
Preparation of the mesenchymal stem cells to osteoblast differentiation, the transcript sequence such as SEQ of long-chain non-coding RNA LOC100506530
Shown in NO:1.
2. application according to claim 1, which is characterized in that the mescenchymal stem cell in prediction people's umbilical cord source is thin to skeletonization
The preparation of born of the same parents' differentiation includes the real-time fluorescence quantitative PCR detection examination for detecting long-chain non-coding RNA LOC100506530 expression quantity
Agent.
3. application according to claim 2, which is characterized in that the real-time fluorescence quantitative PCR detection reagent includes real
When quantitative fluorescent PCR specific primer:
LncRNA LOC100506530 specific forward primer: 5'-ATGCAATGTTTCGATCATGG -3'
LncRNA LOC100506530 specific reverse primers: 5'-ACCATCCCCAGGGTCTTAGT-3'.
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| CN108486256A (en) * | 2018-04-02 | 2018-09-04 | 南京千年健干细胞基因工程有限公司 | A kind of identification umbilical cord mesenchymal stem cells based on long-chain non-coding RNA freeze the gene detecting kit of time |
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Non-Patent Citations (4)
| Title |
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| "LINC02349:5";anonymous;《Lncipedia》;20120731;1-2页 |
| "MiR-29c regulates the expression of miR-34c and miR-449a by targeting DNA methyltransferase 3a and 3b in nasopharyngeal carcinoma";Man Niu等;《BMC cancer》;20160315;第16卷;第3页左栏第1段 |
| "人脐带和骨髓间充质干细胞体外分离培养及生物学特性比较";马锡慧等;《中华细胞与干细胞杂志（电子版）》;20150501;第5卷(第2期);摘要，第79页结果部分至第81页讨论部分 |
| "人骨髓间充质干细胞成骨及成软骨分化中长链非编码RNA的初步研究";王亮;《中国博士学位论文全文数据库 医药卫生科技辑》;20151115(第11期);摘要，第一部分 人骨髓间充质干细胞成骨分化中lncRNAs的研究 |
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