CN108192960A - A kind of gene detecting kit of the identification mesenchymal stem cell passage number based on long-chain non-coding RNA - Google Patents

A kind of gene detecting kit of the identification mesenchymal stem cell passage number based on long-chain non-coding RNA Download PDF

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CN108192960A
CN108192960A CN201810279534.0A CN201810279534A CN108192960A CN 108192960 A CN108192960 A CN 108192960A CN 201810279534 A CN201810279534 A CN 201810279534A CN 108192960 A CN108192960 A CN 108192960A
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mesenchymal stem
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田红青
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Nanjing Qiannianjian Stem Cell Gene Engineering Co Ltd
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Abstract

The invention discloses a kind of gene detecting kit of the identification mesenchymal stem cell passage number based on long-chain non-coding RNA, the qPCR primers including being used to measure LncRNA CASC2, LncRNABX357664 and LncRNA LINC00520 expressions；The identification mesenchymal stem cell passage number refers to identification and distinguishes P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell.The present invention can quickly determine that the mesenchymal stem cell to be measured that freezes is 1~5 generation or 6~10 generation mesenchymal stem cells.

Description

A kind of identification mesenchymal stem cell passage number based on long-chain non-coding RNA Gene detecting kit
Technical field
The invention belongs to genetic test fields, are related to a kind of gene detection reagent for identifying mescenchymal stem cell passage number Box.
Background technology
It is most popular in mescenchymal stem cell, most prospect surely belong to mesenchymal stem cell and umbilical cord mesenchyma is done carefully Born of the same parents.
Mescenchymal stem cell content is rare in people's marrow, and with age or cell quantity of having bad physiques can gradually subtract It is few, therefore it is very necessary to carry out a large amount of amplifications, but at present about after amplification respectively for the biological characteristics of mesenchymal stem cell It is whether identical still without accurate final conclusion.Human marrow mesenchymal stem cell can grow or so 10 generations under conventional culture conditions, with reference to text Open source information and experiment experience are offered, 1~5 generation multiplication characteristic and cell viability are substantially better than 6~10 generation (bibliography：People's bone The serial passages in vitro of bone marrow-drived mesenchymal stem and identification, Colleges Of Traditional Chinese Medicine Of Guangxi's journal, the 1st phase of volume 15 in 2012；Than less The biological characteristics of simultaneous interpretation generation human marrow mesenchyme stem cell, Chinese Tissue Engineering Study and clinical rehabilitation, 2009 volume 13 49th phase).
Human umbilical cord mesenchymal stem cells are derived from embryonic development early stage mesoderm, have good versatility, induce in vitro The various kinds of cell such as fat, cartilage, nerve cell and liver cell can be divided into.Human umbilical cord mesenchymal stem cells are than filling between in people marrow Matter stem cell has preferably division passage capacity, it is however generally that, researcher can receive human umbilical cord mesenchymal in 1~20 generation Stem cell is used as the seed cell of research.With reference to document open source information and experiment experience, 1~10 generation multiplication characteristic, cell are lived Power and chromosome stability are substantially better than 11~20 generation (bibliography：The biology of long-term cultivation human umbilical cord mesenchymal stem cells is living Property and its restricted, Chinese Tissue Engineering Study and clinical rehabilitation, the 10th phase of volume 15 in 2011；Umbilical cord mesenchymal stem cells body Cell karyotypic stability after outer secondary culture, Guiyang Medical College journal, the 5th phase of volume 38 in 2013；Human umbilical cord mesenchymal is done carefully The research of born of the same parents' subculture in vitro separately hereditary capacity, modern medicine, the 9th phase of volume 43 in 2015).
In order to obtain preferable experimental result, ensure the repeatability of experiment, generally preferable 1~5 generation marrow of researcher Mescenchymal stem cell or 1~10 generation human umbilical cord mesenchymal stem cells are as research cell.But the prior art exists as follows not Foot：
First, which the mesenchymal stem cell or human umbilical cord mesenchymal stem cells bought on the market be on earth for nothing Method is confirmed, and businessman's beacon information is insincere, as seed cell poor repeatability；
Second, laboratory oneself freezes the mesenchymal stem cell or human umbilical cord mesenchymal stem cells as seed cell The algebraically of the mesenchymal stem cell or human umbilical cord mesenchymal stem cells can not be usually determined due to tag damage；
Third, although the mesenchymal stem cell or human umbilical cord mesenchymal stem cells bought or voluntarily retained can be answered Su Hou probably judges the algebraically of the cell by measuring its proliferation activity and phenotype, but this method first will be between the marrow that frozen Mesenchymal stem cells or human umbilical cord mesenchymal stem cells are recovered, then in vitro culture, its proliferation activity of re-test and phenotype take and take Power.
The present invention is intended to provide a kind of solve above-mentioned insufficient technical solution, to identify mesenchymal stem cell or people's navel Band mescenchymal stem cell passage number, while do not need to that the cell frozen recover can be operated.
Invention content
Present invention aims at provide a kind of identification mesenchymal stem cell or human umbilical cord mesenchymal stem cells passage time Several gene detecting kit, quickly to determine that the mesenchymal stem cell to be measured that freezes is 1~5 generation or 6~10 generation marrow Mescenchymal stem cell quickly determines that freezing and storing umbilical mesenchymal stem cells to be measured are filled between 1~10 generation or 11~20 generation umbilical cords Matter stem cell.
Above-mentioned purpose is achieved through the following technical solutions：
Mesenchymal stem cell-long-chain non-coding RNA
A kind of gene detecting kit of the identification mesenchymal stem cell passage number based on long-chain non-coding RNA, QPCR primers including being used to measure LncRNA CASC2, LncRNA BX357664 and LncRNA LINC00520 expressions； The identification mesenchymal stem cell passage number refers to identification and distinguishes P1~P5 for mesenchymal stem cell and P6~P10 generations Mesenchymal stem cell.
The measure LncRNA CASC2, LncRNA BX357664 and LncRNA LINC00520 expressions refer to measure LncRNA CASC2, LncRNA BX357664 and LncRNA LINC00520 relative to internal reference U6 relative expression levels.
The qPCR sense primers of LncRNA CASC2 are 5'-CGACTGGTGAGCTTGCTGATGATTC-3'；
The qPCR downstream primers of LncRNA CASC2 are 5'-AAGTGTTCCTGTTGCGCCTCTTGAT-3'.
The qPCR sense primers of LncRNA BX357664 are 5'-AGTCGTATAGCACTTGATAGAGAGGC-3'；
The qPCR downstream primers of LncRNA BX357664 are 5'-CGCAGTTCTTCACCGTCGGTAGGAAA-3'.
The qPCR sense primers of LncRNA LINC00520 are 5'-ATGCTCAGGTATATAACGATGAGTCGC-3'；
The qPCR downstream primers of LncRNA LINC00520 are 5'-CACACAAACATAGACGGGGACCATG-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, reverse transcription reagents and qPCR reagents etc. are further included.
Mesenchymal stem cell-Microrna
It is a kind of based on Microrna identification mesenchymal stem cell passage number gene detecting kit, including with In the qPCR primers for measuring hsa-miR-378a-5p, hsa-miR-325 and hsa-miR-511-3p expression；The identification Mesenchymal stem cell passage number refers to identification differentiation P1~P5 and is filled for mesenchymal stem cell and P6~P10 between marrow Matter stem cell.
Preferably, measure hsa-miR-378a-5p, hsa-miR-325 and hsa-miR-511-3p expression refers to Measure the relative expression levels of hsa-miR-378a-5p, hsa-miR-325 and hsa-miR-511-3p relative to internal reference U6.
The qPCR sense primers of hsa-miR-378a-5p are 5'-CTACACTGACGTGTGTATCACAGAC-3'；
The qPCR downstream primers of hsa-miR-378a-5p are 5'-ATGGTAGAAAACAGGCTACGACGG-3'.
The qPCR sense primers of hsa-miR-325 are 5'-TGACGGATTGATGTTAGAGGGATG-3'；
The qPCR downstream primers of hsa-miR-325 are 5'-CGGAAAGCCCTAGCAGGATAATCT-3'.
The qPCR sense primers of hsa-miR-511-3p are 5'-GTTACTTAGGCCTGCGGTTGGAAG-3'；
The qPCR downstream primers of hsa-miR-511-3p are 5'-GTCTAGTGGATCTTCGTCTGCGTC-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, reverse transcription reagents and qPCR reagents etc. are further included.
Mesenchymal stem cell-circular rna
It is a kind of based on circular rna identification mesenchymal stem cell passage number gene detecting kit, including with In the qPCR primers for measuring hsa_circ_0079888, hsa_circ_0010283 and hsa_circ_0019223 expression； Identification mesenchymal stem cell passage number refers to identification and distinguishes P1~P5 for mesenchymal stem cell and P6~P10 for marrow Mescenchymal stem cell.
Preferably, hsa_circ_0079888, hsa_circ_0010283 and hsa_circ_0019223 expression water are measured Flat refer to measures hsa_circ_0079888, hsa_circ_0010283 and hsa_circ_0019223 relative to the opposite of internal reference U6 Expression.
The qPCR sense primers of hsa_circ_0079888 are 5'-CCCACGATTGTGAGACTACCAATGA-3'；
The qPCR downstream primers of hsa_circ_0079888 are 5'-ACTCTTATGCGGATAAGTAAAGCTTC-3'.
The qPCR sense primers of hsa_circ_0010283 are 5'-CATACGTGGATCAGTTGACCCACGAT-3'；
The qPCR downstream primers of hsa_circ_0010283 are 5'-GACAGTGCCACTGTAGGACTACTTA-3'.
The qPCR sense primers of hsa_circ_0019223 are 5'-AATCATATACAAGAGGCTTGCCGTG-3'；
The qPCR downstream primers of hsa_circ_0019223 are 5'-GTACTAGCGAATTGTCCGCAGAGCA-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, reverse transcription reagents and qPCR reagents etc. are further included.
Umbilical cord mesenchymal stem cells-long-chain non-coding RNA
A kind of gene detecting kit of the identification umbilical cord mesenchymal stem cells passage number based on long-chain non-coding RNA, QPCR including being used to measure LncRNA LINC01140, LncRNA MALAT-1 and LncRNA LINC01116 expressions draws Object；The identification umbilical cord mesenchymal stem cells passage number refer to identification distinguish P1~P10 for umbilical cord mesenchymal stem cells and P11~ P20 is for umbilical cord mesenchymal stem cells.
Preferably, the measure LncRNA LINC01140, LncRNA MALAT-1 and LncRNA LINC01116 expression Level, which refers to, measures LncRNA LINC01140, LncRNA MALAT-1 and LncRNA LINC01116 relative to the opposite of internal reference U6 Expression.
The qPCR sense primers of LncRNA LINC01140 are 5'-ATTTGCTCTAGGGACACTCG-3', and qPCR draws in downstream Object is 5'-TTCATCACCATTAGGTTGCG-3'.
The qPCR sense primers of LncRNA MALAT-1 be 5'-CTTCCAGTATCGAGTGAGCA-3', qPCR downstream primers For 5'-GCTAGCATAGTAGTAATCGA-3'.
The qPCR sense primers of LncRNA LINC01116 be 5'-ATGAGTACTACATGCGAGCTGC-3', qPCR downstreams Primer is 5'-TGTGATACAGATAGACGCTACG-3'.
Preferably, the qPCR sense primers of internal reference U6 be 5'-TTTAGGCTTGCCTCTGTAGTA-3', qPCR downstream primers For 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, reverse transcription reagents and qPCR reagents etc. are further included.
Umbilical cord mesenchymal stem cells-Microrna
It is a kind of based on Microrna identification umbilical cord mesenchymal stem cells passage number gene detecting kit, including with In the qPCR primers for measuring hsa-miR-532-3p, hsa-miR-146a-5p and hsa-miR-223-5p expression；Identify navel Band mescenchymal stem cell passage number refers to identification differentiation P1~P10 and is filled for umbilical cord mesenchymal stem cells and P11~P20 between umbilical cord Matter stem cell.
Preferably, measure hsa-miR-532-3p, hsa-miR-146a-5p and hsa-miR-223-5p expression Refer to and measure the relative expression's water of hsa-miR-532-3p, hsa-miR-146a-5p and hsa-miR-223-5p relative to internal reference U6 It is flat.
The qPCR sense primers of hsa-miR-532-3p be 5'-TGTCCGCCATATGCATGATCCAA-3', qPCR downstreams Primer is 5'-AGTACAACTAGAGGCATGGAATG-3'.
The qPCR sense primers of hsa-miR-146a-5p are 5'-TGATTCGCCGTACTGTTGTTTGTCT-3', under qPCR Trip primer is 5'-CGTTGTCGTGACAATGCTGTGAAC-3'.
The qPCR sense primers of hsa-miR-223-5p are 5'-AGACTTCCACTTAGAATGAACC-3', and qPCR draws in downstream Object is 5'-TGCTGAGGGACGTAGCGTGCG-3'.
Preferably, the qPCR sense primers of internal reference U6 be 5'-TTTAGGCTTGCCTCTGTAGTA-3', qPCR downstream primers For 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, reverse transcription reagents and qPCR reagents etc. are further included.
Umbilical cord mesenchymal stem cells-circular rna
It is a kind of based on circular rna identification umbilical cord mesenchymal stem cells passage number gene detecting kit, including with In the qPCR primers for measuring hsa_circ_0052766, hsa_circ_0019389 and hsa_circ_0077559 expression； The identification umbilical cord mesenchymal stem cells passage number refers to identification and distinguishes P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 For umbilical cord mesenchymal stem cells.
Preferably, hsa_circ_0052766, hsa_circ_0019389 and hsa_circ_0077559 expression water are measured Flat refer to measures hsa_circ_0052766, hsa_circ_0019389 and hsa_circ_0077559 relative to the opposite of internal reference U6 Expression.
The qPCR sense primers of hsa_circ_0052766 be 5'-ACTGAAGCACTTTAGACAGATA-3', qPCR downstreams Primer is 5'-CTCAGACCCACGTTGGACATGA-3'.
The qPCR sense primers of hsa_circ_0019389 are 5'-TGCCCTAGATTGAGTGACCATACAG-3', qPCR Downstream primer is 5'-GCAGCTTTCCTAACCATAAGACGTG-3'.
The qPCR sense primers of hsa_circ_0077559 be 5'-TTACGATGGACTGAACGTGATA-3', qPCR downstreams Primer is 5'-CATAACTCCTCGCTGATGACCA-3'.
Preferably, the qPCR sense primers of internal reference U6 be 5'-TTTAGGCTTGCCTCTGTAGTA-3', qPCR downstream primers For 5'-GAACCCTGCCAGGTGATTTTG-3'.
Preferably, reverse transcription reagents and qPCR reagents etc. are further included.
Gene detecting kit provided by the invention has the following advantages that：
First, can be used for measuring commercialization mesenchymal stem cell be belong to proliferative capacity is high, energetic P1~ P5 generations or P6~P10 generations that proliferative capacity is weaker, vigor is weaker；It can be used for measuring commercialization human umbilical cord mesenchymal stem cells Belong to P1~P10 generations that proliferative capacity is high, energetic or P11~P20 generations that proliferative capacity is weaker, vigor is weaker；
Second, laboratory oneself freezes the mesenchymal stem cell or human umbilical cord mesenchymal stem cells as seed cell The algebraically of the mesenchymal stem cell or human umbilical cord mesenchymal stem cells can not be usually determined due to tag damage, by this hair Bright kit can measure the human marrow mesenchymal stem cell and belong to P1~P5 generations or P6~P10 generations or measure the people's umbilical cord Mescenchymal stem cell belongs to P1~P10 generations or P11~P20 generations；
Third, the mescenchymal stem cell that can directly freeze carry out analysis detection, recover without thawing, simple, efficient.
Description of the drawings
Fig. 1 is LncRNA CASC2, P1~P5 is distinguished in LncRNA BX357664, LncRNA LINC00520 joint identification For mesenchymal stem cell and P6~P10 for the ROC curve of mesenchymal stem cell；
Fig. 2 distinguishes P1~P5 for bone for hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p joint identification Bone marrow-drived mesenchymal stem and P6~P10 are for the ROC curve of mesenchymal stem cell；
Fig. 3 distinguishes P1 for hsa_circ_0079888, hsa_circ_0010283, hsa_circ_0019223 joint identification ~P5 is for mesenchymal stem cell and P6~P10 for the ROC curve of mesenchymal stem cell；
Fig. 4 for LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 joint identification distinguish P1~ P10 is for umbilical cord mesenchymal stem cells and P11~P20 for the ROC curve of umbilical cord mesenchymal stem cells；
Fig. 5 distinguishes P1~P10 for hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p joint identification For umbilical cord mesenchymal stem cells and P11~P20 for the ROC curve of umbilical cord mesenchymal stem cells；
Fig. 6 distinguishes P1 for hsa_circ_0052766, hsa_circ_0019389, hsa_circ_0077559 joint identification ~P10 is for umbilical cord mesenchymal stem cells and P11~P20 for the ROC curve of umbilical cord mesenchymal stem cells.
Specific embodiment
Technical scheme of the present invention is introduced with reference to specific embodiment.
Embodiment 1：Identify the gene detecting kit of mesenchymal stem cell passage number
First, 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.
2nd, experimental method
1st, the in-vitro separation of mesenchymal stem cell, culture and continuous passage
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 poor tunica albuginea layer, that is, mononuclearcell；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, exhaust 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 are added in, it can under mirror after about 3min See that cellular contraction shortens, gap increases, add in the L-DMEM containing 10% fetal calf serum and terminate digestion.Gently blown and beaten with suction pipe until Attached cell suspends, and draws cell suspension, and 1000r/min centrifugation 5min are washed 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 P10 generations after row.P1~P10 is sub-packed in respectively for mesenchymal stem cell - 80 DEG C of 1.5ml cell cryopreservation tubes freeze, if per generation main pipe, label are spare.
2nd, Total RNAs extraction and target RNA relative expression levels measure
Cryopreservation tube from liquid nitrogen is taken out, 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 reagents operation instructions The relative expression levels of middle target RNA, as a result using 2-ΔΔCtMethod carries out relative quantitative assay.
The qPCR primers of target RNA and internal reference U6 are as shown in the table.
3rd, data analysis
It is for statistical analysis using 19.0 softwares of SPSS.The measurement data of normal distribution represents with means standard deviation, two Comparison among groups use two independent samples t tests；Non-Gaussian Distribution measurement data represents that two comparison among groups use Mann- with M (QR) Whitney U are examined；Enumeration data compares using Fisher's exact propability.
LncRNA CASC2, LncRNA BX357664, LncRNA are calculated using binary Logistic regression analyses The united Logistic regression equations of LINC00520；Draw LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520 and its joint identification distinguish P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell Receiver operating curve's (ROC curve), calculate ROC curve under area (AUC).There is statistics for difference with P ＜ 0.05 Meaning.
Hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511- are calculated using binary Logistic regression analyses The united Logistic regression equations of 3p；Draw hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p and its connection It closes identification and distinguishes Receiver Operating Characteristics of the P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell Curve (ROC curve) calculates area (AUC) under ROC curve.It is statistically significant for difference with P ＜ 0.05.
Hsa_circ_0079888, hsa_circ_0010283, hsa_ are calculated using binary Logistic regression analyses The united Logistic regression equations of circ_0019223；Draw hsa_circ_0079888, hsa_circ_0010283, hsa_ Circ_0019223 and its joint identification are distinguished P1~P5 and are done carefully for medulla mesenchyma for mesenchymal stem cell and P6~P10 Receiver operating curve's (ROC curve) of born of the same parents calculates area (AUC) under ROC curve.There is statistics for difference with P ＜ 0.05 Learn meaning.
3rd, experimental result
1st, P1~P5 is distinguished in LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520 and its joint identification For mesenchymal stem cell and P6~P10 for the value of mesenchymal stem cell
P1~P5 is randomly selected for mesenchymal stem cell often for each 20 pipes and P6~P10 for mesenchymal stem cell Often for each 20 pipe as training set；It is another to randomly select P1~P5 for mesenchymal stem cell often for each 20 pipes and P6~P10 generations Mesenchymal stem cell often collects for each 20 pipe as verification.
In training set, LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520 are in P6~P10 for marrow Relative expression quantity in mescenchymal stem cell is more notable for the relative expression quantity difference in mesenchymal stem cell than in P1~P5 3.5~4.5 times of up-regulation.In P1~P5 generations, are distinguished in LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520 identifications Mesenchymal stem cell and P6~P10 are respectively 0.713,0.742,0.735 for the AUC of mesenchymal stem cell.With bone Bone marrow-drived mesenchymal stem algebraically is dependent variable (assignment：P1~P5 generations=0, P6~P10=1), LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520 are independent variable (continuous variable), carry out binary Logistic regression analyses, as a result show Show, Logit (P)=- 3.035+7.204 × LncRNA CASC2+4.288 × LncRNA BX357664+5.205 × LncRNA AUCs of the P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell is distinguished in LINC00520, identification It is 0.961, cutoff value is 4.382 (ROC curve is shown in Fig. 1).LncRNA CASC2、LncRNA BX357664、LncRNA AUCs of the P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell is distinguished in LINC00520 joint identifications Noticeably greater than LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520, which are individually identified, distinguishes P1~P5 for marrow Mescenchymal stem cell and P6~P10 are for the AUC of mesenchymal stem cell.
Verification is concentrated, respectively by each sample LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520 phases Binary Logistic regression equations are substituted into expression, the P6~P10 that is predicted as higher than cutoff value is done carefully for medulla mesenchyma Born of the same parents are predicted as P1~P5 for mesenchymal stem cell, and compared with practical algebraically less than cutoff value, and as a result this method is predicted Accuracy rate is 96.5%.
2nd, in P1~P5 generations, are distinguished in hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p and its joint identification Mesenchymal stem cell and P6~P10 are for the value of mesenchymal stem cell
P1~P5 is randomly selected for mesenchymal stem cell often for each 20 pipes and P6~P10 for mesenchymal stem cell Often for each 20 pipe as training set；It is another to randomly select P1~P5 for mesenchymal stem cell often for each 20 pipes and P6~P10 generations Mesenchymal stem cell often collects for each 20 pipe as verification.
In training set, hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p are in P6~P10 between marrow Relative expression quantity in mesenchymal stem cells is than on P1~P5 is notable for the relative expression quantity difference in mesenchymal stem cell Adjust 3.5~4.5 times.Hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p identification distinguish P1~P5 between marrow Mesenchymal stem cells and P6~P10 are respectively 0.702,0.709,0.748 for the AUC of mesenchymal stem cell.It is filled between marrow Matter stem cell algebraically is dependent variable (assignment：P1~P5 generations=0, P6~P10=1), hsa-miR-378a-5p, hsa-miR- 325th, hsa-miR-511-3p is independent variable (continuous variable), carries out binary Logistic regression analyses, the results show that Logit (P)=- 3.747+6.021 × hsa-miR-378a-5p+5.924 × hsa-miR-325+4.699 × hsa-miR- 511-3p identifies that distinguish P1~P5 is for the AUC of mesenchymal stem cell for mesenchymal stem cell and P6~P10 0.943, cutoff value is 3.878 (ROC curve is shown in Fig. 2).hsa-miR-378a-5p、hsa-miR-325、hsa-miR-511-3p Joint identification is distinguished P1~P5 and is noticeably greater than for mesenchymal stem cell and P6~P10 for the AUC of mesenchymal stem cell Hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p individually identify that distinguish P1~P5 does carefully for medulla mesenchyma Born of the same parents and P6~P10 are for the AUC of mesenchymal stem cell.
Verification is concentrated, respectively by each sample hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p with respect to table Binary Logistic regression equations are substituted into up to level, P6~P10 is predicted as mesenchymal stem cell higher than cutoff value, it is low P1~P5 is predicted as mesenchymal stem cell, and compared with practical algebraically, as a result this method prediction is accurate in cutoff value Rate is 93.5%.
3rd, hsa_circ_0079888, hsa_circ_0010283, hsa_circ_0019223 and its joint identification are distinguished P1~P5 is for mesenchymal stem cell and P6~P10 for the value of mesenchymal stem cell
P1~P5 is randomly selected for mesenchymal stem cell often for each 20 pipes and P6~P10 for mesenchymal stem cell Often for each 20 pipe as training set；It is another to randomly select P1~P5 for mesenchymal stem cell often for each 20 pipes and P6~P10 generations Mesenchymal stem cell often collects for each 20 pipe as verification.
In training set, hsa_circ_0079888, hsa_circ_0010283, hsa_circ_0019223 are in P6~P10 For the relative expression quantity in mesenchymal stem cell than dividing in P1~P5 for the relative expression quantity in mesenchymal stem cell 4.0~5.0 times are not raised not significantly.Hsa_circ_0079888, hsa_circ_0010283, hsa_circ_0019223 identify area Point P1~P5 for mesenchymal stem cell and P6~P10 is respectively 0.752 for the AUC of mesenchymal stem cell, 0.749, 0.757.Using mesenchymal stem cell algebraically as dependent variable (assignment：P1~P5 generations=0, P6~P10=1), hsa_circ_ 0079888th, hsa_circ_0010283, hsa_circ_0019223 are independent variable (continuous variable), carry out binary Logistic regression analyses, the results show that Logit (P)=- 4.117+5.930 × hsa_circ_0079888+5.778 × Hsa_circ_0010283+5.949 × hsa_circ_0019223, identification distinguish P1~P5 for mesenchymal stem cell and P6~P10 is 0.977 for the AUC of mesenchymal stem cell, and cutoff value is 4.005 (ROC curve is shown in Fig. 3).hsa_circ_ 0079888th, the identification of hsa_circ_0010283, hsa_circ_0019223 joint distinguishes P1~P5 for mesenchymal stem cell With P6~P10 for the AUC of mesenchymal stem cell be noticeably greater than hsa_circ_0079888, hsa_circ_0010283, Hsa_circ_0019223 individually identifies that distinguish P1~P5 does carefully for mesenchymal stem cell and P6~P10 for medulla mesenchyma The AUC of born of the same parents.
Verification is concentrated, respectively by each sample hsa_circ_0079888, hsa_circ_0010283, hsa_circ_ 0019223 relative expression levels substitute into binary Logistic regression equations, and P6~P10 is predicted as between marrow higher than cutoff value Mesenchymal stem cells are predicted as P1~P5 for mesenchymal stem cell, and compared with practical algebraically less than cutoff value, as a result should Method predictablity rate is 97.5%.
4th, kits of the P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell is distinguished in identification
It is a kind of to identify the reagent for distinguishing P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell Box, containing LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520, internal reference U6 qPCR primers, also contain Reverse transcription reagents, qPCR reagents.LncRNA CASC2, LncRNA BX357664, LncRNA LINC00520, internal reference U6 QPCR primers are as shown in the table.
It is a kind of to identify the reagent for distinguishing P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell Box, containing hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p, internal reference U6 qPCR primers, also containing reverse Record reagent, qPCR reagents.Hsa-miR-378a-5p, hsa-miR-325, hsa-miR-511-3p, internal reference U6 qPCR primers such as Shown in following table.
It is a kind of to identify the reagent for distinguishing P1~P5 for mesenchymal stem cell and P6~P10 for mesenchymal stem cell Box, containing hsa_circ_0079888, hsa_circ_0010283, hsa_circ_0019223, internal reference U6 qPCR primers, also Contain reverse transcription reagents, qPCR reagents.It is hsa_circ_0079888, hsa_circ_0010283, hsa_circ_0019223, interior The qPCR primers for joining U6 are as shown in the table.
Embodiment 2：Identify the gene detecting kit of umbilical cord mesenchymal stem cells passage number
First, 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.
2nd, experimental method
1st, the in-vitro separation of umbilical cord mesenchymal stem cells, culture and continuous passage
1. it digests：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, put 37 DEG C, volume fraction 5%CO2In incubator, full dose changes liquid after saturated humidity culture 3d, continues to cultivate and observes cell growth feelings Condition (this cell is primary umbilical cord mesenchymal stem cells P0).3. it passes 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, put CO2Incubator In continue to cultivate and observe cell growth status, this cell is P1 for cell.It is respectively trained after being carried out with method for the passage of cell It supports until reaching P20 generations.P1~P20 is sub-packed in -80 DEG C of jellies of 1.5ml cell cryopreservation tubes respectively for umbilical cord mesenchymal stem cells It deposits, if per generation main pipe, label are spare.
2nd, Total RNAs extraction and target RNA relative quantifications measure
Cryopreservation tube from liquid nitrogen is taken out, 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 reagents operation instructions The relative expression levels of middle target RNA, as a result using 2-ΔΔCtMethod carries out relative quantitative assay.
The qPCR primers of target RNA and internal reference U6 are as shown in the table.
3rd, data analysis
It is for statistical analysis using 19.0 softwares of SPSS.The measurement data of normal distribution represents with means standard deviation, two Comparison among groups use two independent samples t tests；Non-Gaussian Distribution measurement data represents that two comparison among groups use Mann- with M (QR) Whitney U are examined；Enumeration data compares using Fisher's exact propability.
LncRNA LINC01140, LncRNA MALAT-1, LncRNA are calculated using binary Logistic regression analyses The united Logistic regression equations of LINC01116；Draw LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 and its joint identification are distinguished P1~P10 and are done carefully for umbilical cord mesenchyma for umbilical cord mesenchymal stem cells and P11~P20 Receiver operating curve's (ROC curve) of born of the same parents calculates area (AUC) under ROC curve.There is statistics for difference with P ＜ 0.05 Learn meaning.
Hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR- are calculated using binary Logistic regression analyses The united Logistic regression equations of 223-5p；Draw hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223- 5p and its joint identification distinguish P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 for the tested of umbilical cord mesenchymal stem cells Person's performance curve (ROC curve) calculates area (AUC) under ROC curve.It is statistically significant for difference with P ＜ 0.05.
Hsa_circ_0052766, hsa_circ_0019389, hsa_ are calculated using binary Logistic regression analyses The united Logistic regression equations of circ_0077559；Draw hsa_circ_0052766, hsa_circ_0019389, hsa_ Circ_0077559 and its joint identification are distinguished P1~P10 and are done for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord mesenchyma Receiver operating curve's (ROC curve) of cell calculates area (AUC) under ROC curve.There is system for difference with P ＜ 0.05 Meter learns meaning.
3rd, experimental result
1st, P1 is distinguished in LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 and its joint identification ~P10 is for umbilical cord mesenchymal stem cells and P11~P20 for the value of umbilical cord mesenchymal stem cells
P1~P10 is randomly selected often to do carefully for umbilical cord mesenchyma for each 20 pipes and P11~P20 for umbilical cord mesenchymal stem cells Born of the same parents are often for each 20 pipe as training set；It is another randomly select P1~P10 for umbilical cord mesenchymal stem cells often for each 20 pipes and P11~ P20 often collects for umbilical cord mesenchymal stem cells for each 20 pipe as verification.
In training set, LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 are in P11~P20 generations Relative expression quantity in umbilical cord mesenchymal stem cells in P1~P10 for the relative expression quantity in umbilical cord mesenchymal stem cells than distinguishing Significantly 2~3 times of up-regulation.LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 identification differentiations P1~ P10 for umbilical cord mesenchymal stem cells and P11~P20 is respectively 0.655 for the AUC of umbilical cord mesenchymal stem cells, 0.636, 0.671.Using umbilical cord mesenchymal stem cells algebraically as dependent variable (assignment：P1~P10 generations=0, P11~P20=1), LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 are independent variable (continuous variable), carry out binary Logistic regression analyses, the results show that Logit (P)=- 2.104+4.413 × LncRNA LINC01140+3.790 × LncRNA MALAT-1+4.035 × LncRNA LINC01116, identification distinguish P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 is 0.915 for the AUC of umbilical cord mesenchymal stem cells, and cutoff value is 2.771 (ROC curve is shown in Fig. 4).LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 joint identifications are distinguished P1~P10 and are done carefully for umbilical cord mesenchyma Born of the same parents and P11~P20 for the AUC of umbilical cord mesenchymal stem cells be noticeably greater than LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 individually identify that distinguish P1~P10 does for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord mesenchyma The AUC of cell.
Verification is concentrated, respectively by each sample LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116 Relative expression levels substitute into binary Logistic regression equations, and the P11~P20 that is predicted as higher than cutoff value is done for umbilical cord mesenchyma Cell is predicted as P1~P10 for umbilical cord mesenchymal stem cells, and compared with practical algebraically, as a result this method less than cutoff value Predictablity rate is 92.75%.
2nd, hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p and its joint identification distinguish P1~ P10 is for umbilical cord mesenchymal stem cells and P11~P20 for the value of umbilical cord mesenchymal stem cells
P1~P10 is randomly selected often to do carefully for umbilical cord mesenchyma for each 20 pipes and P11~P20 for umbilical cord mesenchymal stem cells Born of the same parents are often for each 20 pipe as training set；It is another randomly select P1~P10 for umbilical cord mesenchymal stem cells often for each 20 pipes and P11~ P20 often collects for umbilical cord mesenchymal stem cells for each 20 pipe as verification.
In training set, hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p are in P11~P20 for navel With the relative expression quantity in mescenchymal stem cell than being shown respectively for the relative expression quantity in umbilical cord mesenchymal stem cells in P1~P10 Write 2.5~3.5 times of up-regulation.P1~P10 is distinguished in hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p identification For the AUC of umbilical cord mesenchymal stem cells it is respectively 0.665,0.684,0.679 for umbilical cord mesenchymal stem cells and P11~P20.With Umbilical cord mesenchymal stem cells algebraically is dependent variable (assignment：P1~P10 generations=0, P11~P20=1), hsa-miR-532-3p, Hsa-miR-146a-5p, hsa-miR-223-5p are independent variable (continuous variable), carry out binary Logistic regression analyses, The results show that Logit (P)=- 3.023+3.885 × hsa-miR-532-3p+5.025 × hsa-miR-146a-5p+4.157 × hsa-miR-223-5p, identification are distinguished P1~P10 and are done for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord mesenchyma The AUC of cell is 0.925, and cutoff value is 2.998 (ROC curve is shown in Fig. 5).hsa-miR-532-3p、hsa-miR-146a-5p、 Hsa-miR-223-5p joint identifications are distinguished P1~P10 and are done carefully for umbilical cord mesenchyma for umbilical cord mesenchymal stem cells and P11~P20 The AUC of born of the same parents be noticeably greater than hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p individually identify distinguish P1~ P10 is for umbilical cord mesenchymal stem cells and P11~P20 for the AUC of umbilical cord mesenchymal stem cells.
Verification is concentrated, respectively by each sample hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p phase Binary Logistic regression equations are substituted into expression, the P11~P20 that is predicted as higher than cutoff value is done carefully for umbilical cord mesenchyma Born of the same parents are predicted as P1~P10 for umbilical cord mesenchymal stem cells, and compared with practical algebraically, as a result this method is pre- less than cutoff value It is 93.25% to survey accuracy rate.
3rd, hsa_circ_0052766, hsa_circ_0019389, hsa_circ_0077559 and its joint identification are distinguished P1~P10 is for umbilical cord mesenchymal stem cells and P11~P20 for the value of umbilical cord mesenchymal stem cells
P1~P10 is randomly selected often to do carefully for umbilical cord mesenchyma for each 20 pipes and P11~P20 for umbilical cord mesenchymal stem cells Born of the same parents are often for each 20 pipe as training set；It is another randomly select P1~P10 for umbilical cord mesenchymal stem cells often for each 20 pipes and P11~ P20 often collects for umbilical cord mesenchymal stem cells for each 20 pipe as verification.
In training set, hsa_circ_0052766, hsa_circ_0019389, hsa_circ_0077559 are in P11~P20 For the relative expression quantity in umbilical cord mesenchymal stem cells than dividing in P1~P10 for the relative expression quantity in umbilical cord mesenchymal stem cells 3.0~4.0 times are not raised not significantly.Hsa_circ_0052766, hsa_circ_0019389, hsa_circ_0077559 identify area Point P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 is respectively 0.705 for the AUC of umbilical cord mesenchymal stem cells, 0.724、0.717.Using umbilical cord mesenchymal stem cells algebraically as dependent variable (assignment：P1~P10 generations=0, P11~P20=1), Hsa_circ_0052766, hsa_circ_0019389, hsa_circ_0077559 are independent variable (continuous variable), carry out two First Logistic regression analyses, the results show that Logit (P)=- 0.395+5.003 × hsa_circ_0052766+4.751 × Hsa_circ_0019389+4.805 × hsa_circ_0077559, identification distinguish P1~P10 for umbilical cord mesenchymal stem cells With P11~P20 for umbilical cord mesenchymal stem cells AUC for 0.939, cutoff value is 3.682 (ROC curve is shown in Fig. 6).hsa_ Circ_0052766, hsa_circ_0019389, hsa_circ_0077559 joint identification distinguish P1~P10 for umbilical cord mesenchyma Stem cell and P11~P20 are noticeably greater than hsa_circ_0052766, hsa_circ_ for the AUC of umbilical cord mesenchymal stem cells 0019389th, hsa_circ_0077559, which is individually identified, distinguishes P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord The AUC of mescenchymal stem cell.
Verification is concentrated, respectively by each sample hsa_circ_0052766, hsa_circ_0019389, hsa_circ_ 0077559 relative expression levels substitute into binary Logistic regression equations, and P11~P20 is predicted as umbilical cord higher than cutoff value Mescenchymal stem cell is predicted as P1~P10 for umbilical cord mesenchymal stem cells, and compared with practical algebraically less than cutoff value, knot Fruit this method predictablity rate is 94.00%.
4th, reagents of the P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord mesenchymal stem cells is distinguished in identification Box
It is a kind of to identify the examination for distinguishing P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord mesenchymal stem cells Agent box, containing LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116, internal reference U6 qPCR primers, also Contain reverse transcription reagents, qPCR reagents.LncRNA LINC01140, LncRNA MALAT-1, LncRNA LINC01116, internal reference The qPCR primers of U6 are as shown in the table.
It is a kind of to identify the examination for distinguishing P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord mesenchymal stem cells Agent box, containing hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p, internal reference U6 qPCR primers, also contain There are reverse transcription reagents, qPCR reagents.Hsa-miR-532-3p, hsa-miR-146a-5p, hsa-miR-223-5p, internal reference U6 QPCR primers are as shown in the table.
It is a kind of to identify the examination for distinguishing P1~P10 for umbilical cord mesenchymal stem cells and P11~P20 for umbilical cord mesenchymal stem cells Agent box, containing hsa_circ_0052766, hsa_circ_0019389, hsa_circ_0077559, internal reference U6 qPCR primers, Also containing reverse transcription reagents, qPCR reagents.hsa_circ_0052766、hsa_circ_0019389、hsa_circ_0077559、 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 gene detecting kit of the identification mesenchymal stem cell passage number based on long-chain non-coding RNA, It is characterized in that：Including being used to measuring LncRNA CASC2, LncRNA BX357664 and LncRNA LINC00520 expressions QPCR primers；The identification mesenchymal stem cell passage number refer to identification distinguish P1~P5 for mesenchymal stem cell and P6~P10 is for mesenchymal stem cell.
2. gene detecting kit according to claim 1, it is characterised in that：Measure LncRNA CASC2, LncRNA BX357664 and LncRNA LINC00520 expressions refer to measure LncRNA CASC2, LncRNA BX357664 and LncRNA LINC00520 relative to internal reference U6 relative expression levels.
3. gene detecting kit according to claim 2, it is characterised in that：
The qPCR sense primers of LncRNA CASC2 are 5'-CGACTGGTGAGCTTGCTGATGATTC-3'；
The qPCR downstream primers of LncRNA CASC2 are 5'-AAGTGTTCCTGTTGCGCCTCTTGAT-3'.
4. gene detecting kit according to claim 2, it is characterised in that：
The qPCR sense primers of LncRNA BX357664 are 5'-AGTCGTATAGCACTTGATAGAGAGGC-3'；
The qPCR downstream primers of LncRNA BX357664 are 5'-CGCAGTTCTTCACCGTCGGTAGGAAA-3'.
5. gene detecting kit according to claim 2, it is characterised in that：
The qPCR sense primers of LncRNA LINC00520 are 5'-ATGCTCAGGTATATAACGATGAGTCGC-3'；
The qPCR downstream primers of LncRNA LINC00520 are 5'-CACACAAACATAGACGGGGACCATG-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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