CN108441549A - A kind of identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time - Google Patents
A kind of identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time Download PDFInfo
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Abstract
The invention discloses a kind of, and the identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time, includes the qPCR primers for measuring hsa_circ_0042254, hsa_circ_0033037 and hsa_circ_0057635 expression；The identification mesenchymal stem cell freezes the time and refers to identification and distinguish to freeze and mesenchymal stem cell and freeze mesenchymal stem cell in 7~December 1~June.The present invention can quickly determine that the mesenchymal stem cell to be measured that freezes freezes for 1~June or freezes the mesenchymal stem cell in 7~December.
Description
Technical field
The invention belongs to genetic test fields, are related to a kind of gene detection reagent of identification mesenchymal stem cell cryopreserving time
Box.
Background technology
It is most popular in mescenchymal stem cell, most foreground to surely belong to mesenchymal stem cell and umbilical cord mesenchyma dry thin
Born of the same parents.
Liquid nitrogen frozen preserves mesenchymal stem cell in recent years or umbilical cord mesenchymal stem cells technology is used widely.
However the shortcomings that liquid nitrogen cryopreservation cell is its highly volatile, Long-term Cryopreservation can cause liquid nitrogen to consume, it is necessary to be replenished in time.Therefore,
All -80 DEG C of the uses of many research institutions carry out freezing protection to mesenchymal stem cell or umbilical cord mesenchymal stem cells (not to surpass
Spend 12 months).
But -80 DEG C freeze the time to mesenchymal stem cell or umbilical cord mesenchymal stem cells biological characteristics and increasing
The influence for growing vigor does not have unified come to a conclusion also.In general, according to document and the experience that studies for a long period of time, -80 DEG C freeze 1~6 month
Substantially the biological characteristics and proliferation activity of mesenchymal stem cell or umbilical cord mesenchymal stem cells will not be significantly affected, and-
The nucleus and cytoplasm stability meeting of 80 DEG C of mesenchymal stem cells for freezing 7~12 months or umbilical cord mesenchymal stem cells
It is deteriorated, proliferative capacity and differentiation capability can be a greater impact, and cause the poor (bibliography of experimental repeatability：Cryopreservation is to people
The influence of umbilical cord mesenchymal stem cells biological characteristics, Acad J PLA Postgrad Med Sch, o. 11th of volume 33 in 2012；It is filled between marrow
Matter stem cell freezing under -80 DEG C of cryogenic conditions, Chinese Tissue Engineering Study, the 10th phase of volume 20 in 2016).Therefore, it grinds
The person of studying carefully usually selects -80 DEG C of mesenchymal stem cells frozen in 1~6 month or umbilical cord mesenchymal stem cells careful as kind
Born of the same parents study.
But there is following deficiency in the prior art：
First, laboratory freezes in -80 DEG C of refrigerators to be filled as between the mesenchymal stem cell or people's umbilical cord of seed cell
Matter stem cell can not usually determine freezing for the mesenchymal stem cell or human umbilical cord mesenchymal stem cells because of tag damage
Time；
Second, although after mesenchymal stem cell or human umbilical cord mesenchymal stem cells can be recovered, by measuring it
Proliferation activity and phenotype probably judge the time that freezes of the cell, but this method first by the mesenchymal stem cell frozen or
Human umbilical cord mesenchymal stem cells are recovered, then in vitro culture, its proliferation activity of re-test and phenotype, time and effort consuming.
The present invention is intended to provide a kind of solving above-mentioned insufficient technical solution, to identify mesenchymal stem cell or people's navel
The band mesenchymal stem cell cryopreserving time, while recovery need not be carried out to the cell frozen and can be operated.
Invention content
When being frozen present invention aims at a kind of identification mesenchymal stem cell of offer or human umbilical cord mesenchymal stem cells
Between gene detecting kit, with quickly determine the mesenchymal stem cell or umbilical cord mesenchymal stem cells to be measured of freezing be to freeze
Still the mesenchymal stem cell or umbilical cord mesenchymal stem cells in 7~December are frozen 1~June.
Above-mentioned purpose is achieved through the following technical solutions：
Mesenchymal stem cell-long-chain non-coding RNA
A kind of identification mesenchymal stem cell based on long-chain non-coding RNA freezes the gene detecting kit of time,
Include the qPCR primers for measuring LncRNAAK046999, LncRNA CDKN2B and LncRNAAK043773 expressions；Institute
It states identification mesenchymal stem cell and freezes the time and refer to identification and distinguish to freeze and mesenchymal stem cell and freeze 7~12 1~June
Month mesenchymal stem cell.
Preferably, measurement LncRNAAK046999, LncRNACDKN2B and LncRNAAK043773 expression refers to
Measure the relative expression levels of LncRNAAK046999, LncRNACDKN2B and LncRNAAK043773 relative to internal reference U6.
The qPCR sense primers of LncRNAAK046999 are 5'-TCTAAGAGTTTCGGCGAGCACCT-3'；
The qPCR downstream primers of LncRNAAK046999 are 5'-TACACCATGCTACGACTGGTTTG-3'.
The qPCR sense primers of LncRNACDKN2B are 5'-GTATCCTACATCAGATGAGTCTG-3'；
The qPCR downstream primers of LncRNACDKN2B are 5'-GGCATTCGTATACAATAGTCGTA-3'.
The qPCR sense primers of LncRNAAK043773 are 5'-GCAATCTTGTACGATACGAGGCTG-3'；
The qPCR downstream primers of LncRNAAK043773 are 5'-GATTGACCAGTATGTAACCTAGGC-3'.
The qPCR sense primers of internal reference U6 are 5'-TTTAGGCTTGCCTCTGTAGTA-3'；
The qPCR downstream primers of internal reference U6 are 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, further include reverse transcription reagents and qPCR reagents etc..
Mesenchymal stem cell-Microrna
A kind of identification mesenchymal stem cell based on Microrna freezes the gene detecting kit of time, including with
In the qPCR primers for measuring hsa-miR-505-5p, hsa-miR-197-5p and hsa-miR-635 expression；Between identification marrow
Mesenchymal stem cells freeze the time and refer to identification and distinguish to freeze and mesenchymal stem cell and freeze medulla mesenchyma in 7~December 1~June
Stem cell.
Preferably, measurement hsa-miR-505-5p, hsa-miR-197-5p and hsa-miR-635 expression refers to survey
Determine the relative expression levels of hsa-miR-505-5p, hsa-miR-197-5p and hsa-miR-635 relative to internal reference U6.
The qPCR sense primers of hsa-miR-505-5p are 5'-TGTCCTACGCCATGCGCATATAAAG-3'；
The qPCR downstream primers of hsa-miR-505-5p are 5'-AGTAACTAAGCGGCAGATGAATGG-3'.
The qPCR sense primers of hsa-miR-197-5p are 5'-TGTTTGACTTCACTGTGCCTCTGTTG-3'；
The qPCR downstream primers of hsa-miR-197-5p are 5'-CACAATGGTTGTGGCTCGAACTGTG-3'.
The qPCR sense primers of hsa-miR-635 are 5'-CTTCAAGACTAGAAAGTGACACCT-3'；
The qPCR downstream primers of hsa-miR-635 are 5'-TGCGCTAGGGGCGAGCAGTAGTG-3'.
The qPCR sense primers of internal reference U6 are 5'-TTTAGGCTTGCCTCTGTAGTA-3'；
The qPCR downstream primers of internal reference U6 are 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, further include reverse transcription reagents and qPCR reagents etc..
Mesenchymal stem cell-circular rna
A kind of identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time, including with
In the qPCR primers for measuring hsa_circ_0042254, hsa_circ_0033037 and hsa_circ_0057635 expression；
The identification mesenchymal stem cell freeze the time refer to identification distinguish freeze mesenchymal stem cell in 1~June and freeze 7~
December mesenchymal stem cell.
Preferably, hsa_circ_0042254, hsa_circ_0033037 and hsa_circ_0057635 are measured and expresses water
Flat refer to measures hsa_circ_0042254, hsa_circ_0033037 and hsa_circ_0057635 relative to the opposite of internal reference U6
Expression.
The qPCR sense primers of hsa_circ_0042254 are 5'-GCAACTGCTTTATGAACACAAGGA-3'；
The qPCR downstream primers of hsa_circ_0042254 are 5'-GACTCCACCACGTGTCATGGGCAA-3'.
The qPCR sense primers of hsa_circ_0033037 are 5'-ACCATGCGAATCTGAGGTCTACAAGT-3'；
The qPCR downstream primers of hsa_circ_0033037 are 5'-CCTAGCATTAGCATGTATCCAACGTG-3'.
The qPCR sense primers of hsa_circ_0057635 are 5'-GACTTACTCGTGGTAATGACATGA-3'；
The qPCR downstream primers of hsa_circ_0057635 are 5'-AACTCACTCAGTCGATCCATGCTA-3'.
The qPCR sense primers of internal reference U6 are 5'-TTTAGGCTTGCCTCTGTAGTA-3'；
The qPCR downstream primers of internal reference U6 are 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, further include reverse transcription reagents and qPCR reagents etc..
Umbilical cord mesenchymal stem cells-long-chain non-coding RNA
A kind of identification umbilical cord mesenchymal stem cells based on long-chain non-coding RNA freeze the gene detecting kit of time,
Include the qPCR for measuring LncRNA LINC01001, LncRNA LINC00265 and LncRNA LINC00152 expressions
Primer；The identification umbilical cord mesenchymal stem cells freeze the time refer to identification distinguish freeze umbilical cord mesenchymal stem cells in 1~June and jelly
Deposit umbilical cord mesenchymal stem cells in 7~December.
Preferably, described measurement LncRNALINC01001, LncRNALINC00265 and LncRNALINC00152 express water
It is flat to refer to the opposite table for measuring LncRNALINC01001, LncRNALINC00265 and LncRNALINC00152 relative to internal reference U6
Up to level.
Preferably, the qPCR sense primers of LncRNALINC01001 are 5'-TAGTGAGATTCGCTCTCGTGG-3',
QPCR downstream primers are 5'-TGCAGATCTCGTGCTTGCTTT-3'.
The qPCR sense primers of LncRNALINC00265 are 5'-TGAGTCAATGCAAGGCTGATAGG-3', the downstreams qPCR
Primer is 5'-CGTCTGACTCATGACGTAGGAAC-3'.
The qPCR sense primers of LncRNALINC00152 are 5'-TCTAATGCAGAGATAATGCTGGC-3', the downstreams qPCR
Primer is 5'-ACAGGACACCAACAGACATGG-3'.
Preferably, the qPCR sense primers of internal reference U6 are 5'-TTTAGGCTTGCCTCTGTAGTA-3', qPCR downstream primers
For 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, further include reverse transcription reagents and qPCR reagents etc..
Umbilical cord mesenchymal stem cells-Microrna
A kind of identification umbilical cord mesenchymal stem cells based on Microrna freeze the gene detecting kit of time, including with
In the qPCR primers for measuring hsa-miR-615-3p, hsa-miR-455-5p and hsa-miR-219b-3p expression；The mirror
Determine umbilical cord mesenchymal stem cells and freeze the time to refer to identification and distinguish to freeze and umbilical cord mesenchymal stem cells and freeze navel in 7~December 1~June
Band mescenchymal stem cell.
Preferably, measurement hsa-miR-615-3p, hsa-miR-455-5p and hsa-miR-219b-3p expression
Refer to the relative expression's water for measuring hsa-miR-615-3p, hsa-miR-455-5p and hsa-miR-219b-3p relative to internal reference U6
It is flat.
Preferably, the qPCR sense primers of hsa-miR-615-3p are 5'-CACGTTGCTACATGGTACGAAC-3',
QPCR downstream primers are 5'-AAGAACAGAGTGTGGTACACG-3'.
Preferably, the qPCR sense primers of hsa-miR-455-5p are 5'-GTTGATAGAGAGGTGTGACGTG-3',
QPCR downstream primers are 5'-CGAGCCAGAACGATAATAGTCT-3'.
Preferably, the qPCR sense primers of hsa-miR-219b-3p are 5'-GATCCTTCTGGTAGTGGACGG-3',
QPCR downstream primers are 5'-TGCCGGTCTGACTTGTCTTCG-3'.
Preferably, the qPCR sense primers of internal reference U6 are 5'-TTTAGGCTTGCCTCTGTAGTA-3', qPCR downstream primers
For 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, further include reverse transcription reagents and qPCR reagents etc..
Umbilical cord mesenchymal stem cells-circular rna
A kind of identification umbilical cord mesenchymal stem cells based on circular rna freeze the gene detecting kit of time, including with
In the qPCR primers for measuring hsa_circ_0012332, hsa_circ_0083703 and hsa_circ_0023538 expression；
The identification umbilical cord mesenchymal stem cells freeze the time refer to identification distinguish freeze umbilical cord mesenchymal stem cells in 1~June and freeze 7~
December umbilical cord mesenchymal stem cells.
Preferably, hsa_circ_0012332, hsa_circ_0083703 and hsa_circ_0023538 are measured and expresses water
Flat refer to measures hsa_circ_0012332, hsa_circ_0083703 and hsa_circ_0023538 relative to the opposite of internal reference U6
Expression.
The qPCR sense primers of hsa_circ_0012332 are 5'-GATGCTGTTCCCAAGAAACCTGAA-3', under qPCR
Trip primer is 5'-CTTAAATGCTGACTGGTCAGTAACT-3'.
The qPCR sense primers of hsa_circ_0083703 are 5'-CGTGACCGATTGACCATACATTGAG-3', qPCR
Downstream primer is 5'-GTGAACTCCAGCTTAACAGGTCTA-3'.
The qPCR sense primers of hsa_circ_0023538 are 5'-TACACTGAAGGTGTGCCAATTACA-3', under qPCR
Trip primer is 5'-ACATCTAGGAGCACTGTCAGGAGT-3'.
Preferably, the qPCR sense primers of internal reference U6 are 5'-TTTAGGCTTGCCTCTGTAGTA-3', qPCR downstream primers
For 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, further include reverse transcription reagents and qPCR reagents etc..
Gene detecting kit provided by the invention has the following advantages that：
First, laboratory freezes in -80 DEG C of refrigerators to be filled as between the mesenchymal stem cell or people's umbilical cord of seed cell
Matter stem cell can not usually determine freezing for the mesenchymal stem cell or human umbilical cord mesenchymal stem cells because of tag damage
Time, kit can measure the human marrow mesenchymal stem cell through the invention or human umbilical cord mesenchymal stem cells belong to and freeze
Still the mesenchymal stem cell or human umbilical cord mesenchymal stem cells in 7~December are frozen 1~June；
Second, the mescenchymal stem cell that can directly freeze carries out analysis detection, simple, efficient without recovery of thawing.
Description of the drawings
Fig. 1 is LncRNAAK046999, LncRNA CDKN2B, LncRNAAK043773 joints are identified to distinguish and freeze 1~6
ROC curve moon mesenchymal stem cell and freeze mesenchymal stem cell in 7~December；
Fig. 2 is that hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 joint identification differentiation freeze for 1~June
Mesenchymal stem cell and the ROC curve for freezing mesenchymal stem cell in 7~December；
Fig. 3 is that jelly is distinguished in hsa_circ_0042254, hsa_circ_0033037, hsa_circ_0057635 joint identification
It deposits mesenchymal stem cell in 1~June and freezes the ROC curve of mesenchymal stem cell in 7~December；
Fig. 4 is LncRNALINC01001, LncRNALINC00265, LncRNA LINC00152 joint identification are distinguished and frozen
The ROC curve 1~June umbilical cord mesenchymal stem cells and freeze umbilical cord mesenchymal stem cells in 7~December；
Fig. 5 be hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p joint identification distinguish freeze 1~
ROC curve June umbilical cord mesenchymal stem cells and freeze umbilical cord mesenchymal stem cells in 7~December；
Fig. 6 is that jelly is distinguished in hsa_circ_0012332, hsa_circ_0083703, hsa_circ_0023538 joint identification
It deposits umbilical cord mesenchymal stem cells in 1~June and freezes the ROC curve of umbilical cord mesenchymal stem cells in 7~December.
Specific implementation mode
Technical scheme of the present invention is introduced with reference to specific embodiment.
Embodiment 1：Identification mesenchymal stem cell freezes the gene detecting kit of time
One, experiment material
L-DMEM culture mediums and fetal calf serum are purchased from Gibco companies of the U.S..RNA extracts kits TRIzol is purchased from
Invitrogen companies, Reverse Transcriptase kit are purchased from TaKaRa companies, and qPCR reagents are purchased from Vazyme companies.
Two, experimental method
1, the in-vitro separation of mesenchymal stem cell, culture, pass on and freeze
The bone marrow fluid 5ml and test tube of hepari of aseptic aspiration healthy volunteer, the L- with equivalent volumes, containing 10% fetal calf serum
DMEM culture solution mixings are placed in ficoll separating liquids (proportion 1.077), and 2000r/min centrifuges 20min, visible mixed after centrifugation
It closes liquid and is divided into four layers from top to bottom：Flaxen top layer is serum；The second layer is the tunica albuginea layer, that is, mononuclearcell belittled；The
Three layers of lymphocyte separation medium for water white transparency；The bottom is red blood cell layer.The mononuclearcell in tunica albuginea layer is collected, to contain
The L-DMEM culture solutions of 10% fetal calf serum are resuspended cell and are counted, with 1 × 106The density of/ml is inoculated in the training of 50ml
It supports in bottle, is placed in 37 DEG C, the CO of volume fraction 5%2Incubator is incubated.It is non-adherent to discard to replace culture medium for full dose after for 24 hours
Hereafter cell carries out half amount and changes liquid, as primary (P0) mesenchymal stem cell every other day.
When primary cultured cell pastes culture bottle floor space up to 60%~70% or so, exhausts culture medium and wash 1 with PBS
It is secondary, 0.25% trypsase and each 0.5ml mixture slakings cells of 0.02%EDTA of 37 DEG C of pre-temperatures is added, it can under mirror after about 3min
See that cellular contraction shortens, gap increases, the L-DMEM containing 10% fetal calf serum is added and terminates digestion.Gently blown and beaten with suction pipe until
Attached cell suspends, and draws cell suspension, and 1000r/min centrifuges 5min, washs 2 times.Finally with the L- of 10% fetal calf serum
Cell is resuspended in DMEM culture solutions, is inoculated in respectively in the culture bottle of two bottles of 50ml, cell at this time is known as P1 for cell.With method into
Respectively for the secondary culture of cell until reaching P3 generations after row.It is thin that P3 is sub-packed in for mesenchymal stem cell to 1.5ml respectively
- 80 DEG C of born of the same parents' cryopreservation tube freezes for 1~December, if monthly main pipe, label is spare.
2, Total RNAs extraction and target RNA relative expression levels measure
Cryopreservation tube is taken out from liquid nitrogen, the method RNA extracts kits of RNA are directly extracted according to freeze-stored cell
TRIzol extracts total serum IgE, and NanoDrop ND-1000 measure 260nm/280nm absorbance ratios range 1.8~2.0.
Using U6 as internal reference, each sample is detected using qPCR methods according to Reverse Transcriptase kit, qPCR reagent operation instructions
As a result the relative expression levels of middle target RNA use 2-ΔΔCtMethod carries out relative quantitative assay.
The qPCR primers of target RNA and internal reference U6 are as shown in the table.
3, data analysis
It is for statistical analysis using 19.0 softwares of SPSS.The measurement data of normal distribution indicates with means standard deviation, two
Comparison among groups use two independent samples t tests；Non-Gaussian Distribution measurement data indicates that two comparison among groups use Mann- with M (QR)
Whitney U are examined；Enumeration data compares using Fisher's exact propability.
LncRNA AK046999, LncRNA CDKN2B, LncRNA are calculated using binary Logistic regression analyses
The united Logistic regression equations of AK043773；Draw LncRNAAK046999, LncRNACDKN2B, LncRNA
AK043773 and its joint identification, which are distinguished, freezes mesenchymal stem cell in 1~June and to freeze 7~December medulla mesenchyma dry thin
Receiver operating curve's (ROC curve) of born of the same parents calculates area (AUC) under ROC curve.There is statistics with P ＜ 0.05 for difference
Learn meaning.
Hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 are calculated using binary Logistic regression analyses
United Logistic regression equations；Draw hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 and its joint
The subject work spy for freezing mesenchymal stem cell in 1~June and freezing mesenchymal stem cell in 7~December is distinguished in identification
Curve (ROC curve) is levied, area (AUC) under ROC curve is calculated.It is that difference is statistically significant with P ＜ 0.05.
Hsa_circ_0042254, hsa_circ_0033037, hsa_ are calculated using binary Logistic regression analyses
The united Logistic regression equations of circ_0057635；Draw hsa_circ_0042254, hsa_circ_0033037, hsa_
Circ_0057635 and its joint identification, which are distinguished, to be frozen mesenchymal stem cell in 1~June and freezes medulla mesenchyma in 7~December
Receiver operating curve's (ROC curve) of stem cell calculates area (AUC) under ROC curve.Have for difference with P ＜ 0.05
Statistical significance.
Three, experimental result
1, LncRNAAK046999, LncRNACDKN2B, LncRNAAK043773 and its joint identification are distinguished and freeze 1~6
Value moon mesenchymal stem cell and freeze mesenchymal stem cell in 7~December
It randomly selects and freezes mesenchymal stem cell in 1~June and monthly each 20 pipe measurement result and freeze marrow in 7~December
Mescenchymal stem cell monthly each 20 pipe measurement result as training set；Another randomly select freezes mesenchymal stem cell in 1~June
Monthly monthly each 20 pipe measurement result collects each 20 pipe measurement result as verification with mesenchymal stem cell in 7~December is frozen.
In training set, LncRNAAK046999, LncRNACDKN2B, LncRNAAK043773 are freezing marrow in 7~December
Relative expression quantity in mescenchymal stem cell is shown respectively than the relative expression quantity in freezing mesenchymal stem cell in 1~June
Write 3~4 times of up-regulation.LncRNAAK046999, LncRNA CDKN2B, LncRNAAK043773 identifications are distinguished and freeze 1~6 lunar
Bone marrow-drived mesenchymal stem and the AUC for freezing mesenchymal stem cell in 7~December are respectively 0.628,0.685,0.677.With bone
Bone marrow-drived mesenchymal stem algebraically is dependent variable (assignment：1~June=0 is frozen, 7~December=1 is frozen), LncRNA
AK046999, LncRNACDKN2B, LncRNAAK043773 are independent variable (continuous variable), carry out binary Logistic recurrence
Analysis, the results show that Logit (P)=- 2.172+4.833 × LncRNA AK046999+5.074 × LncRNA CDKN2B+
4.565 × LncRNAAK043773, identification, which is distinguished, to be frozen mesenchymal stem cell in 1~June and freezes between marrow in 7~December
The AUC of mesenchymal stem cells is 0.921, and cutoff value is 2.886 (ROC curve is shown in Fig. 1).LncRNAAK046999、LncRNA
CDKN2B, LncRNAAK043773 joint identification differentiation freeze mesenchymal stem cell in 1~June and freeze marrow in 7~December
The AUC of mescenchymal stem cell is noticeably greater than LncRNAAK046999, LncRNA CDKN2B, LncRNA AK043773 and individually identifies
Distinguish the AUC for freezing mesenchymal stem cell in 1~June and freezing mesenchymal stem cell in 7~December.
Verification is concentrated, respectively that each sample LncRNAAK046999, LncRNA CDKN2B, LncRNAAK043773 is opposite
Expression substitutes into binary Logistic regression equations, and the medulla mesenchyma in 7~December that is predicted as freezing higher than cutoff value is done carefully
Born of the same parents are predicted as freezing mesenchymal stem cell in 1~June less than cutoff value, and compared with practical algebraically, as a result this method is pre-
It is 92.08% to survey accuracy rate.
2, hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 and its joint identification are distinguished and freeze for 1~June
Mesenchymal stem cell and the value for freezing mesenchymal stem cell in 7~December
It randomly selects and freezes mesenchymal stem cell in 1~June monthly each 20 pipe and to freeze 7~December medulla mesenchyma dry
Cell monthly each 20 pipe as training set；Another randomly select freezes mesenchymal stem cell in 1~June and monthly each 20 pipe and freezes
7~December mesenchymal stem cell monthly each 20 pipe collect as verification.
In training set, hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 are between freezing marrow in 7~December
Relative expression quantity in mesenchymal stem cells is more notable than the relative expression quantity difference in freezing mesenchymal stem cell in 1~June
3~4 times of up-regulation.Hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 identification differentiation freeze between marrow in 1~June
Mesenchymal stem cells and the AUC for freezing mesenchymal stem cell in 7~December are respectively 0.646,0.659,0.653.With between marrow
Mesenchymal stem cells algebraically is dependent variable (assignment：1~June=0 is frozen, 7~December=1 is frozen), hsa-miR-505-5p, hsa-
MiR-197-5p, hsa-miR-635 are independent variable (continuous variable), carry out binary Logistic regression analyses, the results show that
Logit (P)=- 3.144+5.138 × hsa-miR-505-5p+5.569 × hsa-miR-197-5p+4.147 × hsa-miR-
635, identifying that differentiation freezes mesenchymal stem cell in 1~June and freezes the AUC of mesenchymal stem cell in 7~December is
0.918, cutoff value is 3.073 (ROC curve is shown in Fig. 2).Hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 join
Close identification distinguish freeze 1~June mesenchymal stem cell and freeze mesenchymal stem cell in 7~December AUC it is significantly big
Individually identify that differentiation freezes medulla mesenchyma in 1~June and does in hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635
Cell and the AUC for freezing mesenchymal stem cell in 7~December.
Verification is concentrated, respectively by each sample hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635 with respect to table
Binary Logistic regression equations are substituted into up to level, are predicted as freezing mesenchymal stem cell in 7~December higher than cutoff value,
It is predicted as freezing mesenchymal stem cell in 1~June less than cutoff value, and compared with practical algebraically, as a result this method is predicted
Accuracy rate is 90.83%.
3, hsa_circ_0042254, hsa_circ_0033037, hsa_circ_0057635 and its joint identification are distinguished
It freezes mesenchymal stem cell in 1~June and freezes the value of mesenchymal stem cell in 7~December
It randomly selects and freezes mesenchymal stem cell in 1~June monthly each 20 pipe and to freeze 7~December medulla mesenchyma dry
Cell monthly each 20 pipe as training set；Another randomly select freezes mesenchymal stem cell in 1~June and monthly each 20 pipe and freezes
7~December mesenchymal stem cell monthly each 20 pipe collect as verification.
In training set, hsa_circ_0042254, hsa_circ_0033037, hsa_circ_0057635 freeze 7~
Relative expression quantity in December mesenchymal stem cell is than the relative expression in freezing mesenchymal stem cell in 1~June
Amount significantly 3.2~4.2 times of up-regulation respectively.Hsa_circ_0042254, hsa_circ_0033037, hsa_circ_0057635 reflect
It is fixed distinguish to freeze mesenchymal stem cell in 1~June and freeze the AUC of mesenchymal stem cell in 7~December be respectively
0.702、0.671、0.686.Using mesenchymal stem cell algebraically as dependent variable (assignment：1~June=0 is frozen, freezes 7~12
Month=1), hsa_circ_0042254, hsa_circ_0033037, hsa_circ_0057635 are that (continuous type becomes independent variable
Amount), binary Logistic regression analyses are carried out, the results show that Logit (P)=- 2.759+5.812 × hsa_circ_
0042254+6.303 × hsa_circ_0033037+4.909 × hsa_circ_0057635, identification are distinguished and freeze for 1~June
Mesenchymal stem cell and the AUC for freezing mesenchymal stem cell in 7~December are 0.929, and cutoff value is 3.295 (ROC songs
Line is shown in Fig. 3).Hsa_circ_0042254, hsa_circ_0033037, hsa_circ_0057635 joint identification, which are distinguished, freezes 1
It mesenchymal stem cell and freezes the AUC of mesenchymal stem cell in 7~December and is noticeably greater than hsa_circ_~June
0042254, hsa_circ_0033037, hsa_circ_0057635 individually identify that differentiation freezes medulla mesenchyma in 1~June and does carefully
Born of the same parents and the AUC for freezing mesenchymal stem cell in 7~December.
Verification is concentrated, respectively by each sample hsa_circ_0042254, hsa_circ_0033037, hsa_circ_
0057635 relative expression levels substitute into binary Logistic regression equations, are predicted as freezing marrow in 7~December higher than cutoff value
Mescenchymal stem cell is predicted as freezing mesenchymal stem cell in 1~June less than cutoff value, and compared with practical algebraically, knot
Fruit this method predictablity rate is 92.50%.
4, identification differentiation freezes mesenchymal stem cell in 1~June and freezes the examination of mesenchymal stem cell in 7~December
Agent box
A kind of identification differentiation freezes mesenchymal stem cell in 1~June and freezes mesenchymal stem cell in 7~December
Kit, the qPCR primers containing LncRNAAK046999, LncRNACDKN2B, LncRNAAK043773, internal reference U6, also contains
Reverse transcription reagents, qPCR reagents.LncRNAAK046999, LncRNACDKN2B, LncRNAAK043773, internal reference U6 qPCR draw
Object is as shown in the table.
A kind of identification differentiation freezes mesenchymal stem cell in 1~June and freezes mesenchymal stem cell in 7~December
Kit, the qPCR primers containing hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635, internal reference U6, also contains
Reverse transcription reagents, qPCR reagents.Hsa-miR-505-5p, hsa-miR-197-5p, hsa-miR-635, internal reference U6 qPCR draw
Object is as shown in the table.
A kind of identification differentiation freezes mesenchymal stem cell in 1~June and freezes mesenchymal stem cell in 7~December
Kit, the qPCR containing hsa_circ_0042254, hsa_circ_0033037, hsa_circ_0057635, internal reference U6 draw
Object also contains reverse transcription reagents, qPCR reagents.hsa_circ_0042254、hsa_circ_0033037、hsa_circ_
0057635, the qPCR primers of internal reference U6 are as shown in the table.
Embodiment 2：Identification umbilical cord mesenchymal stem cells freeze the gene detecting kit of time
One, experiment material
DMEM/F12 culture mediums and fetal calf serum are purchased from Gibco companies of the U.S..RNA extracts kits TRIzol is purchased from
Invitrogen companies, Reverse Transcriptase kit are purchased from TaKaRa companies, and qPCR reagents are purchased from Vazyme companies.
Two, experimental method
1, the in-vitro separation of umbilical cord mesenchymal stem cells, culture and continuous passage
1. digesting：8cm or so umbilical cord tissues (newborn from health full term Cesarean esction) are taken, are washed with sterile PBS
Except residual blood, about 1mm × 1mm × 1mm sizes are cut into, through 0.15% clostridiopetidase A II, 37 DEG C incubate digestion 4h, and 200 mesh screens filter,
Collect cell and with trypan blue staining living cell counting number.2. adherent separation：Cell is suspended in containing 10% tire ox of volume fraction
The DMEM/F12 complete culture solutions of serum, are inoculated into T25 culture bottles, control inoculating cell a concentration of 2.4 × 104/cm2, set 37
DEG C, volume fraction 5%CO2In incubator, full dose changes liquid after saturated humidity culture 3d, continues to cultivate and observe cell growth feelings
Condition (this cell is primary umbilical cord mesenchymal stem cells P0).3. passing on：When culture cell reaches 90% fusion, digestive juice is used
(0.25% pancreatin -0.01%EDTA solution) processing about 3min, it centrifuges, abandon supernatant, the resuspension of sedimentation cell complete culture solution simultaneously
It is counted, is inoculated in new T25 culture bottles with trypan blue, control inoculating cell a concentration of 2.4 × 104/cm2, set CO2Incubator
In continue to cultivate and observe cell growth status, this cell is P1 for cell.It is respectively trained for the passage of cell after being carried out with method
It supports.P3 is sub-packed in -80 DEG C of 1.5ml cell cryopreservation tubes for umbilical cord mesenchymal stem cells respectively and freezes for 1~December, it is monthly several
Pipe, label are spare.
2, Total RNAs extraction and target RNA relative quantifications measure
Cryopreservation tube is taken out from liquid nitrogen, the method RNA extracts kits of RNA are directly extracted according to freeze-stored cell
TRIzol extracts total serum IgE, and NanoDrop ND-1000 measure 260nm/280nm absorbance ratios range 1.8~2.0.
Using U6 as internal reference, each sample is detected using qPCR methods according to Reverse Transcriptase kit, qPCR reagent operation instructions
As a result the relative expression levels of middle target RNA use 2-ΔΔCtMethod carries out relative quantitative assay.
The qPCR primers of target RNA and internal reference U6 are as shown in the table.
3, data analysis
It is for statistical analysis using 19.0 softwares of SPSS.The measurement data of normal distribution indicates with means standard deviation, two
Comparison among groups use two independent samples t tests；Non-Gaussian Distribution measurement data indicates that two comparison among groups use Mann- with M (QR)
Whitney U are examined；Enumeration data compares using Fisher's exact propability.
LncRNA LINC01001, LncRNA LINC00265, LncRNA are calculated using binary Logistic regression analyses
The united Logistic regression equations of LINC00152；Draw LncRNALINC01001, LncRNALINC00265, LncRNA
LINC00152 and its joint identification are distinguished to freeze umbilical cord mesenchymal stem cells in 1~June and freeze umbilical cord mesenchyma in 7~December and be done
Receiver operating curve's (ROC curve) of cell calculates area (AUC) under ROC curve.There is system with P ＜ 0.05 for difference
Meter learns meaning.
Hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR- are calculated using binary Logistic regression analyses
The united Logistic regression equations of 219b-3p；Draw hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-
3p and its joint identification distinguish freeze 1~June umbilical cord mesenchymal stem cells and freeze umbilical cord mesenchymal stem cells in 7~December by
Examination person's performance curve (ROC curve) calculates area (AUC) under ROC curve.It is that difference is statistically significant with P ＜ 0.05.
Hsa_circ_0012332, hsa_circ_0083703, hsa_ are calculated using binary Logistic regression analyses
The united Logistic regression equations of circ_0023538；Draw hsa_circ_0012332, hsa_circ_0083703, hsa_
Circ_0023538 and its joint identification, which are distinguished, to be frozen umbilical cord mesenchymal stem cells in 1~June and freezes umbilical cord mesenchyma in 7~December
Receiver operating curve's (ROC curve) of stem cell calculates area (AUC) under ROC curve.Have for difference with P ＜ 0.05
Statistical significance.
Three, experimental result
1, LncRNA LINC01001, LncRNA LINC00265, LncRNA LINC00152 and its joint identification are distinguished
It freezes umbilical cord mesenchymal stem cells in 1~June and freezes the value of umbilical cord mesenchymal stem cells in 7~December
It randomly selects and freezes umbilical cord mesenchymal stem cells in 1~June monthly each 20 pipe and to freeze 7~December umbilical cord mesenchyma dry
Cell monthly each 20 pipe as training set；Another randomly select freezes umbilical cord mesenchymal stem cells in 1~June and monthly each 20 pipe and freezes
7~December umbilical cord mesenchymal stem cells monthly each 20 pipe collect as verification.
In training set, LncRNALINC01001, LncRNALINC00265, LncRNALINC00152 are freezing for 7~December
Relative expression quantity in umbilical cord mesenchymal stem cells is than the relative expression quantity in freezing umbilical cord mesenchymal stem cells in 1~June point
3~4 times are not raised not significantly.LncRNALINC01001, LncRNA LINC00265, LncRNALINC00152 identifications are distinguished and are frozen
1~June umbilical cord mesenchymal stem cells and freeze the AUC of umbilical cord mesenchymal stem cells in 7~December be respectively 0.680,0.693,
0.678.Using umbilical cord mesenchymal stem cells algebraically as dependent variable (assignment：1~June=0 is frozen, 7~December=1 is frozen),
LncRNA LINC01001, LncRNA LINC00265, LncRNALINC00152 are independent variable (continuous variable), carry out two
First Logistic regression analyses, the results show that Logit (P)=- 3.410+4.055 × LncRNALINC01001+4.172 ×
LncRNA LINC00265+3.992 × LncRNA LINC00152, identification are distinguished and freeze umbilical cord mesenchymal stem cells in 1~June
It is 0.909 with the AUC of umbilical cord mesenchymal stem cells in 7~December is frozen, cutoff value is 2.718 (ROC curve is shown in Fig. 4).LncRNA
LINC01001, LncRNA LINC00265, LncRNALINC00152 joint identification differentiations freeze umbilical cord mesenchyma in 1~June and do
Cell and the AUC for freezing umbilical cord mesenchymal stem cells in 7~December are noticeably greater than LncRNA LINC01001, LncRNA
LINC00265, LncRNALINC00152 individually identify that differentiation freezes umbilical cord mesenchymal stem cells in 1~June and freezes for 7~December
The AUC of umbilical cord mesenchymal stem cells.
Verification is concentrated, respectively by each sample LncRNALINC01001, LncRNALINC00265, LncRNALINC00152
Relative expression levels substitute into binary Logistic regression equations, are predicted as freezing umbilical cord mesenchyma in 7~December higher than cutoff value
Stem cell is predicted as freezing umbilical cord mesenchymal stem cells in 1~June less than cutoff value, and compared with practical algebraically, as a result the party
Method predictablity rate is 90.42%.
2, hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p and its joint identification are distinguished and freeze 1
The value~June umbilical cord mesenchymal stem cells and freeze umbilical cord mesenchymal stem cells in 7~December
It randomly selects and freezes umbilical cord mesenchymal stem cells in 1~June monthly each 20 pipe and to freeze 7~December umbilical cord mesenchyma dry
Cell monthly each 20 pipe as training set；Another randomly select freezes umbilical cord mesenchymal stem cells in 1~June and monthly each 20 pipe and freezes
7~December umbilical cord mesenchymal stem cells monthly each 20 pipe collect as verification.
In training set, hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p are freezing navel in 7~December
With the relative expression quantity in mescenchymal stem cell than the relative expression quantity difference in freezing umbilical cord mesenchymal stem cells in 1~June
Significantly 3~4 times of up-regulation.Hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p identification, which are distinguished, freezes 1~6
Month umbilical cord mesenchymal stem cells and to freeze the AUC of umbilical cord mesenchymal stem cells in 7~December be respectively 0.686,0.713,0.695.
Using umbilical cord mesenchymal stem cells algebraically as dependent variable (assignment：1~June=0 is frozen, 7~December=1 is frozen), hsa-miR-
615-3p, hsa-miR-455-5p, hsa-miR-219b-3p are independent variable (continuous variable), carry out binary Logistic and return
Return analysis, the results show that Logit (P)=- 4.144+5.975 × hsa-miR-615-3p+6.344 × hsa-miR-455-5p+
5.324 × hsa-miR-219b-3p, identification, which is distinguished, to be frozen umbilical cord mesenchymal stem cells in 1~June and freezes umbilical cord in 7~December
The AUC of mescenchymal stem cell is 0.915, and cutoff value is 2.803 (ROC curve is shown in Fig. 5).hsa-miR-615-3p、hsa-miR-
455-5p, hsa-miR-219b-3p joint identification differentiation freeze umbilical cord mesenchymal stem cells in 1~June and freeze umbilical cord in 7~December
The AUC of mescenchymal stem cell is noticeably greater than hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p and individually reflects
It is fixed to distinguish the AUC for freezing umbilical cord mesenchymal stem cells in 1~June and freezing umbilical cord mesenchymal stem cells in 7~December.
Verification is concentrated, respectively by each sample hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p phase
Binary Logistic regression equations are substituted into expression, the umbilical cord mesenchyma in 7~December that is predicted as freezing higher than cutoff value is done
Cell is predicted as freezing umbilical cord mesenchymal stem cells in 1~June less than cutoff value, and compared with practical algebraically, as a result this method
Predictablity rate is 91.25%.
3, hsa_circ_0012332, hsa_circ_0083703, hsa_circ_0023538 and its joint identification are distinguished
It freezes umbilical cord mesenchymal stem cells in 1~June and freezes the value of umbilical cord mesenchymal stem cells in 7~December
It randomly selects and freezes umbilical cord mesenchymal stem cells in 1~June monthly each 20 pipe and to freeze 7~December umbilical cord mesenchyma dry
Cell monthly each 20 pipe as training set；Another randomly select freezes umbilical cord mesenchymal stem cells in 1~June and monthly each 20 pipe and freezes
7~December umbilical cord mesenchymal stem cells monthly each 20 pipe collect as verification.
In training set, hsa_circ_0012332, hsa_circ_0083703, hsa_circ_0023538 freeze 7~
Relative expression quantity in December umbilical cord mesenchymal stem cells is than the relative expression in freezing umbilical cord mesenchymal stem cells in 1~June
Amount significantly 3.5~4.5 times of up-regulation respectively.Hsa_circ_0012332, hsa_circ_0083703, hsa_circ_0023538 reflect
It is fixed distinguish to freeze umbilical cord mesenchymal stem cells in 1~June and freeze the AUC of umbilical cord mesenchymal stem cells in 7~December be respectively
0.725、0.694、0.708.Using umbilical cord mesenchymal stem cells algebraically as dependent variable (assignment：1~June=0 is frozen, freezes 7~12
Month=1), hsa_circ_0012332, hsa_circ_0083703, hsa_circ_0023538 are that (continuous type becomes independent variable
Amount), binary Logistic regression analyses are carried out, the results show that Logit (P)=- 3.797+6.181 × hsa_circ_
0012332+7.144 × hsa_circ_0083703+6.291 × hsa_circ_0023538, identification are distinguished and freeze for 1~June
Umbilical cord mesenchymal stem cells and the AUC for freezing umbilical cord mesenchymal stem cells in 7~December are 0.930, and cutoff value is 3.045 (ROC songs
Line is shown in Fig. 6).Hsa_circ_0012332, hsa_circ_0083703, hsa_circ_0023538 joint identification, which are distinguished, freezes 1
It umbilical cord mesenchymal stem cells and freezes the AUC of umbilical cord mesenchymal stem cells in 7~December and is noticeably greater than hsa_circ_~June
0012332, hsa_circ_0083703, hsa_circ_0023538 individually identify that differentiation freezes umbilical cord mesenchyma in 1~June and does carefully
Born of the same parents and the AUC for freezing umbilical cord mesenchymal stem cells in 7~December.
Verification is concentrated, respectively by each sample hsa_circ_0012332, hsa_circ_0083703, hsa_circ_
0023538 relative expression levels substitute into binary Logistic regression equations, are predicted as freezing umbilical cord in 7~December higher than cutoff value
Mescenchymal stem cell is predicted as freezing umbilical cord mesenchymal stem cells in 1~June less than cutoff value, and compared with practical algebraically, knot
Fruit this method predictablity rate is 93.75%.
4, identification differentiation freezes umbilical cord mesenchymal stem cells in 1~June and freezes the examination of umbilical cord mesenchymal stem cells in 7~December
Agent box
A kind of identification differentiation freezes umbilical cord mesenchymal stem cells in 1~June and freezes umbilical cord mesenchymal stem cells in 7~December
Kit, the qPCR containing LncRNA LINC01001, LncRNA LINC00265, LncRNA LINC00152, internal reference U6 draw
Object also contains reverse transcription reagents, qPCR reagents.LncRNA LINC01001、LncRNA LINC00265、LncRNA
LINC00152, the qPCR primers of internal reference U6 are as shown in the table.
A kind of identification differentiation freezes umbilical cord mesenchymal stem cells in 1~June and freezes umbilical cord mesenchymal stem cells in 7~December
Kit, the qPCR primers containing hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p, internal reference U6, also
Contain reverse transcription reagents, qPCR reagents.Hsa-miR-615-3p, hsa-miR-455-5p, hsa-miR-219b-3p, internal reference U6
QPCR primers it is as shown in the table.
A kind of identification differentiation freezes umbilical cord mesenchymal stem cells in 1~June and freezes umbilical cord mesenchymal stem cells in 7~December
Kit, the qPCR containing hsa_circ_0012332, hsa_circ_0083703, hsa_circ_0023538, internal reference U6 draw
Object also contains reverse transcription reagents, qPCR reagents.hsa_circ_0012332、hsa_circ_0083703、hsa_circ_
0023538, the qPCR primers of internal reference U6 are as shown in the table.
Above-mentioned specific embodiment is only used for explaining technical scheme of the present invention, it will be appreciated by those skilled in the art that, this hair
Bright protection domain is not limited to above-mentioned specific embodiment.
Claims (7)
1. a kind of identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time, feature exists
In：Include for measuring hsa_circ_0042254, hsa_circ_0033037 and hsa_circ_0057635 expression
QPCR primers；The identification mesenchymal stem cell freeze the time refer to identification distinguish freeze mesenchymal stem cell in 1~June
With freeze mesenchymal stem cell in 7~December.
2. gene detecting kit according to claim 1, it is characterised in that：The measurement hsa_circ_0042254,
Hsa_circ_0033037 and hsa_circ_0057635 expressions, which refer to, measures hsa_circ_0042254, hsa_circ_
Relative expression levels of the 0033037 and hsa_circ_0057635 relative to internal reference U6.
3. gene detecting kit according to claim 2, it is characterised in that：
The qPCR sense primers of hsa_circ_0042254 are 5'-GCAACTGCTTTATGAACACAAGGA-3'；
The qPCR downstream primers of hsa_circ_0042254 are 5'-GACTCCACCACGTGTCATGGGCAA-3'.
4. gene detecting kit according to claim 2, it is characterised in that：
The qPCR sense primers of hsa_circ_0033037 are 5'-ACCATGCGAATCTGAGGTCTACAAGT-3'；
The qPCR downstream primers of hsa_circ_0033037 are 5'-CCTAGCATTAGCATGTATCCAACGTG-3'.
5. gene detecting kit according to claim 2, it is characterised in that：
The qPCR sense primers of hsa_circ_0057635 are 5'-GACTTACTCGTGGTAATGACATGA-3'；
The qPCR downstream primers of hsa_circ_0057635 are 5'-AACTCACTCAGTCGATCCATGCTA-3'.
6. gene detecting kit according to claim 2, it is characterised in that：
The qPCR sense primers of internal reference U6 are 5'-TTTAGGCTTGCCTCTGTAGTA-3'；
The qPCR downstream primers of internal reference U6 are 5'-GAACCCTGCCAGGTGATTTTG-3'.
7. according to any gene detecting kits of claim 1-6, it is characterised in that：Further include reverse transcription reagents and
QPCR reagents etc..
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| CN201810280397.2A CN108441549A (en) | 2018-04-02 | 2018-04-02 | A kind of identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time |
| PCT/CN2018/117346 WO2019192194A1 (en) | 2018-04-02 | 2018-11-24 | Gene detection kit for identifying cryopreservation time of mesenchymal stem cells |
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| CN201810280397.2A CN108441549A (en) | 2018-04-02 | 2018-04-02 | A kind of identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019192194A1 (en) * | 2018-04-02 | 2019-10-10 | 田红青 | Gene detection kit for identifying cryopreservation time of mesenchymal stem cells |
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| CN107072188A (en) * | 2014-08-08 | 2017-08-18 | Cefo有限公司 | A kind of plant source recombination human serum albumin and the composition of plant peptide for including as active component preserved for cell |
| CN107771782A (en) * | 2017-10-12 | 2018-03-09 | 北京臻惠康生物科技有限公司 | A kind of mesenchyme stem cell protection solution and application thereof |
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| WO2019192194A1 (en) * | 2018-04-02 | 2019-10-10 | 田红青 | Gene detection kit for identifying cryopreservation time of mesenchymal stem cells |
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