CN113637671A - LncRNA related to epidermal stem cell differentiation and application thereof - Google Patents
LncRNA related to epidermal stem cell differentiation and application thereof Download PDFInfo
- Publication number
- CN113637671A CN113637671A CN202110907003.3A CN202110907003A CN113637671A CN 113637671 A CN113637671 A CN 113637671A CN 202110907003 A CN202110907003 A CN 202110907003A CN 113637671 A CN113637671 A CN 113637671A
- Authority
- CN
- China
- Prior art keywords
- lncrna
- differentiation
- epidermal stem
- expression
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to LncRNA related to epidermal stem cell differentiation, wherein the LncRNA is RP4-784A16.2, and the LncRNA sequence is SEQ ID No. 1. And the application of LncRNA in preparing the medicine for promoting the healing of skin wounds. And a method for identifying LncRNA, comprising the steps of: separating and culturing human epidermal stem cells and inducing differentiation; detecting a cell microarray chip before and after differentiation; real-time fluorescent quantitative PCR detection proves that RP4-784A16.2 is up-regulated in the differentiation of epidermal stem cells. The RP4-784A16.2 can promote cell differentiation, provides a detailed basis and a new idea for the subsequent research of regulating and controlling epidermal stem cell differentiation, and can be applied to the technical field of preparing medicines for promoting healing of skin wounds.
Description
Technical Field
The invention relates to the technical field of biology, in particular to identification and application of LncRNA RP4-784A16.2 differentiated from epidermal stem cells.
Background
The skin, the largest organ of the human body, covers the surface of the human body, and has important effects in resisting radiation, preventing infection, excreting metabolites, regulating body temperature balance, and sensing external stimuli due to external direct contact. The epidermis is the surface structure of the skin and consists of two parts, namely a cuticle and a hair growth layer. The horny layer has no life activity, water can not pass easily, and the functions of better abrasion resistance, infection resistance and the like are achieved.
Epidermal Stem Cells (ESCs) are important for maintaining the skin constantly refreshed, existing in the skin, derived from the embryonic ectoderm, with a strong potential for self-renewal and bidirectional differentiation: can migrate downwards to be differentiated into an epidermal basal layer or migrate upwards to be differentiated into various epidermal cells, and is the basis for ensuring the homeostasis of skin tissues. At present, for severe injuries of organs and tissues, a main treatment method is organ transplantation, and autotransplantation is mostly adopted for skin. However, autologous skin is limited in number, insufficient in skin source, and may affect beauty, and skin wound repair is difficult. The research on the differentiation of the epidermal stem cells provides a new idea for the treatment of skin wounds.
The length of long non-coding RNA (LncRNA) is more than 200nt, usually has no function of translating into protein, the expression abundance is generally low, and the long non-coding RNA has a conserved secondary structure. Although LncRNA was initially considered as "noise" of the transcription process, and ignored as mRNA-like transcripts that do not encode proteins, increasing research has shown that LncRNA has a specific function. With the intensive research on LncRNA, it was found to be involved in many important biological functions: regulate transcription, participate in translation, stabilize proteins, and the like. In the field of epidermal stem cell differentiation, the regulatory role of LncRNA is not clear.
Disclosure of Invention
The purpose of the invention is as follows:
the invention aims to provide LncRNA related to epidermal stem cell differentiation and application thereof, wherein the LncRNA related to epidermal stem cell differentiation is called RP4-784A 16.2; the expression change of LncRNA in the differentiation process of the epidermal stem cells is analyzed in a high-throughput manner, so that the regulation mechanism of LncRNA RP4-784A16.2 is obtained, and the LncRNA RP4-784A16.2 differentiated by the epidermal stem cells is disclosed, so that the LncRNA RP4-784A16.2 is applied to the preparation of related medicines for treating skin wounds and promoting healing, and has wide application prospects in the field of promoting healing of the wounds.
The technical scheme is as follows:
an LncRNA related to epidermal stem cell differentiation, wherein the LncRNA is RP4-784A16.2, and the LncRNA sequence is SEQ ID No. 1.
Application of LncRNA in preparing a medicament for promoting healing of skin wounds.
A method for identifying LncRNA comprises the following steps:
step one, carrying out isolated culture and induced differentiation on human epidermal stem cells;
step two, detecting a cell microarray chip before and after differentiation;
and step three, real-time fluorescent quantitative PCR detection proves that the expression of RP4-784A16.2 is up-regulated in epidermal stem cell differentiation.
The product capable of detecting the expression and/or transcription of the LncRNA in the third step is a primer sequence capable of amplifying the full-length sequence or partial sequence of the LncRNA, and the primer sequence is as follows:
RP4-784A16.2 specific forward primer:
5’-ATGGAGGAGCTGGCTGACTG-3’；
RP4-784A16.2 specific reverse primer:
5’-CACGCCCACTTGTGACTTTC-3’。
the primer sequences for detecting the GAPDH internal reference in step three are as follows:
internal control GAPDH specific forward primer: 5'-GGGAAACTGTGGCGTGAT-3', respectively;
internal control GAPDH specific reverse primer: 5'-GAGTGGGTGTCGCTGTTGA-3' are provided.
Has the advantages that:
the invention identifies the transcriptomes of mRNA and LncRNA before and after differentiation of the epidermal stem cells, analyzes and integrates the results, and further analyzes the differential expression genes of the mRNA and LncRNA. An active factor RP4-784A16.2 for epithelial differentiation is screened, and researches show that RP4-784A16.2 can promote cell differentiation, provide detailed foundation and new ideas for the subsequent research of regulating and controlling epidermal stem cell differentiation, and can be applied to the technical field of preparing medicines for promoting healing of skin wounds.
Drawings
FIG. 1 shows the isolation and culture of epidermal stem cells and the differentiation of cells induced by high calcium (A is epidermal stem cells and B is 1.5mM CaCl)2 Induced differentiated cells);
FIG. 2 is a heat map of differential expressed mRNA and LncRNA before and after differentiation analyzed by the microarray chip (A is mRNA, B is LncRNA);
FIG. 3 is a graph of GO enrichment results for differentially expressed mRNA (A is expression upregulation and B is expression downregulation);
FIG. 4 shows the relationship between the differentially expressed LncRNA and the adjacent coding gene (A is up-regulation of expression and B is down-regulation of expression);
FIG. 5 is a graph showing the detection of the expression level of RP4-784A16.2 in differentiated cells by a real-time fluorescent quantitative PCR method;
FIG. 6 siRNA of RP4-784A16.2 is capable of silencing expression of RP4-784A16.2 in epidermal stem cells;
FIG. 7 at 1.5mM CaCl2 Under induction, the knock-down of RP4-784A16.2 can reduce the expression of differentiation markers CK10 and Involucrin.
Detailed Description
The invention is described in more detail below with reference to the accompanying drawings.
The invention starts from the separation and purification of human epidermal stem cells, and 1.5mM CaCl is added2Inducing the differentiation of the epidermal stem cells, carrying out differential analysis on the cells before and after differentiation by using an LncRNA expression chip, wherein the differential standard is that the fold change is more than 2 and the P is less than 0.05, and screening LncRNA RP4-784A16.2 with expression change in the differentiation process. The invention relates to deep research of stem cells, regenerative medicine and non-coding RNA in the field of basic medicine and clinical transformation application, and is mainly applied to basic research of LncRNA in promoting epidermal differentiation and development of wound healing technology.
The method for identifying LncRNA comprises the following steps:
example 1
Isolated culture and induced differentiation of human epidermal stem cells
The material and the method are as follows:
neutral proteolytic enzyme (Roch)
Pancreatin (trypsin) BI
Epilife medium (Thermo)
FBS（Hyclone）
Washing discarded skin pieces after the skin wrapping operation by PBS to remove blood cells, removing subcutaneous fat cells, cutting into tissue blocks with the size of 2 x 1cm to 1 x 1cm, digesting with 2mg/mL neutral proteolytic enzyme at 4 ℃ for 10-12h, continuing to digest at 37 ℃ for 3h, and separating to obtain the epidermis. Cutting epidermis, digesting with 0.25% pancreatin-EDTA at 37 deg.C for 20min, adding DMEM/F12 culture solution containing 10% FBS to stop digestion, beating into single cell suspension, grinding with 200 mesh sieve, and filtering. Cells were harvested by centrifugation at 1000rpm for 10 min. Supernatants were removed, washed 1 time with PBS and cells were suspended in Epilife. At 3X 106The density of (A) was inoculated in NUNC plastic culture flasks and incubated at 37 ℃ for 20 min. And (4) replacing the liquid to remove suspended cells and impurities, wherein the cells adhered to the bottom surface of the culture bottle are epidermal stem cells. 5% CO at 37 ℃2And (5) continuing incubation culture in the incubator. The cells grow to 70% -80%, and are subjected to digestion and subculture by pancreatin-EDTA. Second or third generation cells grown to 20% -30% and replaced with 1.5mM CaCl2The Epilife broth was cultured for a further 72h, and as shown in fig. 1, the cells became stratified and grew, indicating that the cells had differentiated. After 72h, cells were washed 3 times with PBS and total cellular RNA was extracted using Trizol.
Cell microarray chip detection before and after differentiation:
shanghai Kangcheng bioengineering Co., Ltd is entrusted to use "Arraystar Human LncRNA Microarray" to perform Microarray chip analysis, mRNA and LncRNA which are differentially expressed before and after differentiation are screened out, and co-expression and correlation research between the two are carried out.
(1) The total RNA before and after the differentiation was examined using an LncRNA chip product (Human LncRNA Microarray) from ArrayStar, Inc., of Shanghai Kangcheng bioengineering, Inc., and the differential expression of mRNA and LncRNA was analyzed by Microarray chip as shown in FIG. 2, where each row of the heat map represents a gene, each column represents a sample, red represents an increase in expression level, and green represents a decrease. The brightness of the color represents the degree of increase or decrease.
(2) Differential analysis of mRNA and LncRNA expression before and after differentiation: and (3) comparing the expression results obtained by statistics in the step (1), carrying out differential analysis, screening mRNA and LncRNA with obvious differential expression according to the difference standard that the fold change is more than 2 and the P is less than 0.05, and recording the differential expression result. And functionally enriching the mRNA and LncRNA which are differentially expressed.
As a result:
comparing mRNA expression profiles of the differentiated group and the undifferentiated group, 6665 differentially expressed genes are detected, wherein 3129 genes are differentially expressed up and 3536 genes are differentially expressed down, the differential gene GO enrichment result is shown in figure 3, and the expression of a large number of genes in the differential genes is related to epidermal differentiation, epidermal development and epidermal keratinization.
Comparing the expression profiles of LncRNA in the differentiated group and the undifferentiated group, 6750 differentially expressed LncRNA were detected, wherein 3217 of the differentially expressed LncRNA were up-regulated and 3533 of the differentially expressed LncRNA were down-regulated, and the classification of these LncRNA is shown in fig. 4, in which panel a shows the LncRNA whose expression is up-regulated after differentiation, and in which panel B shows the LncRNA whose expression is down-regulated after differentiation. LncRNA with upregulated expression is classified as follows, bidirectional 8%, exon-overlapping 11%, intergenic 46%, intron sensors-overlapping 3%, intron intensities 17%, and native intensities 15%. LncRNA with downregulated expression was classified as follows, bidirectional 7%, exon-overlapping 13%, intergenic 44%, intron present-overlapping 5%, intron antisense 14%, and native antisense 17%.
From the analysis of FIG. 3, it was found that the expression of Wnt pathway gene is generally down-regulated after differentiation, which suggests that the Wnt pathway is an important differentiation regulation network, and from LncRNA co-expression and association studies, RP4-784A16.2 is located in Chromosome 1: 53,288,024-53,289,706, which is native antigen sequence of Wnt pathway gene LRP8 (low-specificity protein receptor-related protein 8), therefore we speculate that it can structurally bind to mRNA of LRP8 and play an important role in regulating differentiation of epidermal stem cells.
Example 2
Real-time fluorescent quantitative PCR detection proves that RP4-784A16.2 is up-regulated in the differentiation of epidermal stem cells.
The material and the method are as follows:
2× PCR master mix（Superarray）
the primer sequence is as follows:
RP4-784A16.2 specific forward primer: 5'-ATGGAGGAGCTGGCTGACTG-3' (SEQ ID NO. 2)
RP4-784A16.2 specific reverse primer: 5'-CACGCCCACTTGTGACTTTC-3' (SEQ ID NO. 3)
For convenient detection, GAPDH can be used as an internal reference, and the primers for detecting the GAPDH internal reference have the following sequences:
internal control GAPDH specific forward primer: 5'-GGGAAACTGTGGCGTGAT-3' (SEQ ID NO. 8)
Internal control GAPDH specific reverse primer: 5'-GAGTGGGTGTCGCTGTTGA-3' (SEQ ID NO. 9)
All cDNA samples were prepared in a Realtime PCR reaction system. The system is configured as follows:
2× Master Mix 5 µL
10uM PCR specific primer F0.5 mu L
PCR specific primer R0.5 mu L of 10uM
cDNA 2 µL
Adding water to the total volume of 10 mu L
Mixing the solution at the bottom of the flick tube, centrifuging briefly at 5000 rpm, setting PCR reaction: at 95 ℃ for 10 min; 40 PCR cycles (95 ℃, 10 sec; 60 ℃, 60 sec). In order to establish the melting curve of the PCR product, after the amplification reaction is finished, the temperature is controlled according to the formula (95 ℃, 10 seconds, 60 ℃, 60 seconds, 95 ℃, 15 seconds); and slowly heated from 60 c to 99 c. After the reaction was completed, the CT value of the gene and the reference Gene (GAPDH) were normalized, and the P value was calculated by T test.
As a result:
as shown in FIG. 5, real-time fluorescent quantitative PCR detection showed that RP4-784A16.2 expression was up-regulated in the differentiated cells, with expression levels 3.156-fold (P < 0.05) before differentiation, consistent with expectations.
Example 3
Effect of RP4-784A16.2 on epidermal differentiation
The material and the method are as follows:
1 day before transfection, cells were plated at 4X 105Perwell was seeded in 6-well plates. Transfection solutions were prepared using the following amounts per well:
A. 75pmol siRNA (SEQ ID NO. 10) was diluted in 125. mu.L of Opti-MEM medium and gently mixed;
RP4-784A16.2 siRNA sequence 5'-GGCGUGGCAGGGCUUUGUUTT-3' (SEQ ID NO. 10)
B. Dilute 7.5uL Lipofectamine 3000 with 125 uL of Opti-MEM medium;
C. the DNA diluted in the first two steps was mixed with Lipofectamine 3000 (to make the total volume 250. mu.L), gently mixed, and left at room temperature for 15 minutes;
add 250. mu.L of transformation medium to each well of cells and mix gently. After incubation at 37 ℃ for 24 hours, 1.5mM CaCl was added2Gene expression was measured after 48 hours.
2× PCR master mix（Superarray）
The primer sequence is as follows:
CK 10-specific forward primer: 5'-AGGAGGAGTGTCATCCCTAAG-3' (SEQ ID NO. 4)
CK 10-specific reverse primer: 5'-AAGCTGCCTCCATAACTCCC-3' (SEQ ID NO. 5)
Involucrin specific forward primer: 5'-TCCTCCAGTCAATACCCATCAG-3' (SEQ ID NO. 6)
Involucrin specific reverse primer: 5'-CAGCAGTCATGTGCTTTTCCT-3' (SEQ ID NO. 7)
Internal control GAPDH specific forward primer: 5'-GGGAAACTGTGGCGTGAT-3' (SEQ ID NO. 8)
Internal control GAPDH specific reverse primer: 5'-GAGTGGGTGTCGCTGTTGA-3' (SEQ ID NO. 9)
All cDNA samples were prepared in a Realtime PCR reaction system. The system is configured as follows:
2× Master Mix 5 µL
10uM PCR specific primer F0.5 mu L
PCR specific primer R0.5 mu L of 10uM
cDNA 2 µL
Adding water to the total volume of 10 mu L
Mixing the solution at the bottom of the flick tube, centrifuging briefly at 5000 rpm, setting PCR reaction: at 95 ℃ for 10 min; 40 PCR cycles (95 ℃, 10 sec; 60 ℃, 60 sec). In order to establish the melting curve of the PCR product, after the amplification reaction is finished, the temperature is controlled according to the formula (95 ℃, 10 seconds, 60 ℃, 60 seconds, 95 ℃, 15 seconds); and slowly heated from 60 c to 99 c. After the reaction was completed, the CT value of the gene and the reference Gene (GAPDH) were normalized, and the P value was calculated by T test.
As a result:
as shown in FIG. 6, real-time fluorescent quantitative PCR detection shows that the siRNA of RP4-784A16.2 can significantly reduce the expression of RP4-784A16.2 in epidermal stem cells, and the expression level is 56.2% of that of a control (P < 0.05).
As shown in FIG. 7, at 1.5mM CaCl2Under induction, the knock-down of RP4-784A16.2 can significantly reduce the expression of differentiation markers CK10 and Involucrin, and the expression amounts are 37.9% and 45% of that of a control (P is less than 0.05), respectively. This indicates that RP4-784A16.2, acting as a differentiation promoting factor, if silenced during differentiation, will inhibit epidermal differentiation.
While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Liaoning Aminoao Stem cell and regenerative medicine research institute Co., Ltd
<120> LncRNA related to epidermal stem cell differentiation and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 675
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aggtagcaag gcagccccct ctgataacca cgatgaggct cctctccctt ccctttctgc 60
ccccattgtc ggagtatctg ctggagtgag gtacggatgg aggagctggc tgactgcagt 120
gagggaaaag ctccctgtgg ccacaaaccc tccccaagca cacactccag gctgcacacg 180
actctgcctc ttgttagctg ggcagccttg ggcaaggggg agaactgact ccagcctcag 240
tttctttctc tctacatggg gatcctcctc cttccttagg ttgccaagag gatgaaagtc 300
acaagtgggc gtggcagggc tttgttcctt ctgaagctca ggacaaagca ctgggcttcc 360
ctcctgcctg ggagccttca ggacggcatg gagtttggcc acgtggtggg agcgccctga 420
gtgcgtggca cagaatgttc aactggttac ctctcctgag cctcacagcc tctcaaggaa 480
gatctgagga ggaaactgag gcccaccccc acccagactg aggggcagga ctcactgcaa 540
gtggcctcgg actcatcgga gccatcagga cactcctcct cgccgtcaca cttccaccgt 600
tcgtggatgc agtggccgtt gtcacaggtg aagtcactgt ctgcacaggt cttcttggct 660
gcagggcaag gaaga 675
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cacgcccact tgtgactttc 20
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aggaggagtg tcatccctaa g 21
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aagctgcctc cataactccc 20
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcctccagtc aatacccatc ag 22
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cagcagtcat gtgcttttcc t 21
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gggaaactgt ggcgtgat 18
<210> 9
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gagtgggtgt cgctgttga 19
<210> 10
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggcguggcag ggcuuuguut t 21
Claims (5)
1. An LncRNA associated with epidermal stem cell differentiation, characterized in that: the LncRNA is RP4-784A16.2, and the sequence of the LncRNA is SEQ ID No. 1.
2. The use of LncRNA of claim 1, wherein: the LncRNA is applied to preparing a medicine for promoting healing of skin wounds.
3. A method for identifying LncRNA according to claim 1, wherein:
the method comprises the following steps:
step one, carrying out isolated culture and induced differentiation on human epidermal stem cells;
step two, detecting a cell microarray chip before and after differentiation;
and step three, real-time fluorescent quantitative PCR detection proves that the expression of RP4-784A16.2 is up-regulated in epidermal stem cell differentiation.
4. The method for identifying LncRNA of claim 3, wherein: the product capable of detecting the expression and/or transcription of the LncRNA in the third step is a primer sequence capable of amplifying the full-length sequence or partial sequence of the LncRNA, and the primer sequence is as follows:
RP4-784A16.2 specific forward primer: 5'-ATGGAGGAGCTGGCTGACTG-3', respectively;
RP4-784A16.2 specific reverse primer: 5'-CACGCCCACTTGTGACTTTC-3' are provided.
5. The method for identifying LncRNA of claim 3, wherein: the primer sequences for detecting the GAPDH internal reference in step three are as follows:
internal control GAPDH specific forward primer: 5'-GGGAAACTGTGGCGTGAT-3', respectively;
internal control GAPDH specific reverse primer: 5'-GAGTGGGTGTCGCTGTTGA-3' are provided.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110907003.3A CN113637671B (en) | 2021-08-09 | 2021-08-09 | LncRNA related to epidermal stem cell differentiation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110907003.3A CN113637671B (en) | 2021-08-09 | 2021-08-09 | LncRNA related to epidermal stem cell differentiation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113637671A true CN113637671A (en) | 2021-11-12 |
| CN113637671B CN113637671B (en) | 2023-07-14 |
Family
ID=78420146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110907003.3A Active CN113637671B (en) | 2021-08-09 | 2021-08-09 | LncRNA related to epidermal stem cell differentiation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113637671B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
-
2021
- 2021-08-09 CN CN202110907003.3A patent/CN113637671B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
Non-Patent Citations (4)
| Title |
|---|
| BLUM，H. 等: "GenBank：AL110400.1", 《NCBI》 * |
| NONCODE: "NONHSAT003307.2", 《HTTP://WWW.NONCODE.ORG/SHOW_RNA.PHP?ID=NONHSAT003307&VERSION=2&UTD=1#》 * |
| SHANSHAN HU 等: "LncRNAs in Stem Cells", 《STEM CELLS INTERNATIONAL》 * |
| 张涛: "lncRNA调控皮肤角质形成细胞分化", 《第十届全国创伤修复(愈合)与组织再生学术会议、第三届中欧创面修复学术会议、中国医师协会创伤外科医师分会创面修复医师专业委员会会议》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113637671B (en) | 2023-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101040042A (en) | Isolation of stem/progenitor cells from amniotic membrane of umbilical cord | |
| CN101914495A (en) | Culture method for largely amplifying hair follicle stem cells in vitro | |
| CN111154763B (en) | Application of long-chain non-coding RNA lncMGPF in regulation and control of pig muscle development function | |
| CN108504625B (en) | Mouse fibroblast and application thereof | |
| CN109971710B (en) | Jian carp brain cell line and establishing method and application thereof | |
| CN101821383A (en) | A kind of primary mesenchymal stem cells and application thereof that is used for substratum, method and the acquisition of the external mass-producing cultivation of human adult primary mesenchymal stem cells | |
| CN106636210A (en) | Method for inducing transdifferentiation of fibroblast into similar testicular interstitial cells by combination of transcription factors | |
| CN111454953A (en) | Bone marrow mesenchymal stem cell adipogenic transformation promoter | |
| CN109628404A (en) | The construction method and purposes of Preadipocyte immortalized cell line under pigskin | |
| CN110777120B (en) | Application of TGFBI as marker for regulating and controlling mesenchymal stem cell adipogenic differentiation | |
| CN112143708A (en) | Umbilical cord mesenchymal stem cells, stem cell essence factor and application of umbilical cord mesenchymal stem cells and stem cell essence factor in aspect of resisting skin aging | |
| CN113144221B (en) | Exosome preparation and preparation method and application thereof | |
| CN111228501B (en) | Application of DCUN1D1 gene in preventing and treating vitiligo | |
| CN113637671B (en) | LncRNA related to epidermal stem cell differentiation and application thereof | |
| CN101074428B (en) | Method for improving embryonic stem cell to differentiate to cardiac muscle cell | |
| CN115607689A (en) | Application of KLF7 gene in preparation of drug for reversing cell senescence | |
| CN113789333A (en) | Application of Chi3l1 in regulating and controlling hUC-MSCs to inhibit Th17 differentiation-mediated immunoregulation effect | |
| CN113528576A (en) | Recurrent grapevine patient specific induced pluripotent stem cell line containing NLRP7 pure and mutant and construction method thereof | |
| CN113106059A (en) | High-migration mesenchymal stem cell and preparation method and application thereof | |
| CN112980940A (en) | Application of epidermal growth factor Betacellulin in preparation of peripheral nerve regeneration regulation and control medicine | |
| CN113801843A (en) | Method for enhancing dryness of human urinary stem cells | |
| CN111549033B (en) | Lentiviral-infected human epidermal keratinocyte strain and construction method and application thereof | |
| CN112941183B (en) | Application of non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment | |
| CN113350368B (en) | Application of gene inhibitor in preparation of epidermal stem cell migration pharmaceutical preparation | |
| CN113549593B (en) | Pharmaceutical preparation for promoting proliferation of epidermal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |