CN105586310B - Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation - Google Patents
Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation Download PDFInfo
- Publication number
- CN105586310B CN105586310B CN201610084921.XA CN201610084921A CN105586310B CN 105586310 B CN105586310 B CN 105586310B CN 201610084921 A CN201610084921 A CN 201610084921A CN 105586310 B CN105586310 B CN 105586310B
- Authority
- CN
- China
- Prior art keywords
- mscs
- differentiation
- oct4
- regulation
- proliferation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 14
- 230000033228 biological regulation Effects 0.000 title claims abstract description 11
- 230000004663 cell proliferation Effects 0.000 title claims abstract description 11
- 230000024245 cell differentiation Effects 0.000 title claims abstract description 9
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims description 17
- 230000004072 osteoblast differentiation Effects 0.000 claims description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 12
- 230000004069 differentiation Effects 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 230000001737 promoting effect Effects 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000003827 upregulation Effects 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims 2
- 102000039446 nucleic acids Human genes 0.000 claims 2
- 210000004271 bone marrow stromal cell Anatomy 0.000 abstract description 30
- 241000700605 Viruses Species 0.000 abstract description 24
- 230000035755 proliferation Effects 0.000 abstract description 16
- 210000004027 cell Anatomy 0.000 abstract description 14
- 239000013641 positive control Substances 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 4
- 239000013604 expression vector Substances 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000002659 cell therapy Methods 0.000 abstract description 2
- 230000008844 regulatory mechanism Effects 0.000 abstract 1
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 28
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 23
- 239000013612 plasmid Substances 0.000 description 21
- 238000001890 transfection Methods 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 14
- 108091027967 Small hairpin RNA Proteins 0.000 description 10
- 230000029087 digestion Effects 0.000 description 10
- 239000004055 small Interfering RNA Substances 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 108010042407 Endonucleases Proteins 0.000 description 7
- 102000004533 Endonucleases Human genes 0.000 description 7
- 238000004064 recycling Methods 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 102000012410 DNA Ligases Human genes 0.000 description 5
- 108010061982 DNA Ligases Proteins 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000013467 fragmentation Methods 0.000 description 5
- 238000006062 fragmentation reaction Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000001172 regenerating effect Effects 0.000 description 5
- 238000001712 DNA sequencing Methods 0.000 description 4
- 108010010803 Gelatin Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000006160 differential media Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 238000000879 optical micrograph Methods 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000003954 umbilical cord Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102000002584 Octamer Transcription Factor-3 Human genes 0.000 description 1
- 108010068425 Octamer Transcription Factor-3 Proteins 0.000 description 1
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2331—Interleukin-31 (IL-31)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/603—Oct-3/4
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention discloses application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation.The present invention is the achievement for finding cell factor IL-31 for the first time based on inventor and the self-renewing of MSCs and multi-lineage potential capable of being promoted to obtain.The present invention constructs the expression vector of IL-31, shIL-31, Oct4 and shOct4 by the method for genetic engineering, it is transfected into MSCs respectively by slow virus, it was found that IL-31 and the Oct4 as positive control can promote the proliferation and multi-lineage potential of MSCs, and shIL-31 and the proliferation and multi-lineage potential that inhibit MSCs as the shOct4 of positive control.Therefore, these results of study will provide fundamental basis for Regulation Mechanism of the research IL-31 in MSCs self-renewing and multi-lineage potential, establish necessary basis for MSCs is used for cell therapy.
Description
Technical field
The present invention relates to regenerative medicine and genetic engineering fields, and in particular to IL-31 is in regulation mescenchymal stem cell proliferation
With the application in differentiation.
Background technique
Mescenchymal stem cell (Mesenchymal stem cells, MSCs) is located away from marrow by Pittenger etc. earliest,
It later also can be isolated in the tissue such as umbilical cord and fat.MSCs has the advantages that MSCs plasticity is very high, in vivo and in vitro
The tissue such as cartilage, bone, muscle, tendon, ligament and fat can be divided under inductive condition；MSCs has ability of going back to the nest, Ke Yi
It is concentrated in organism to impaired and inflammation part；MSCs can induce or increase the formation of new blood vessel；MSCs lacks significant immune
Originality prevents autoimmunity；In addition MSCs convenient separation.In recent years, regenerative medicine was fast-developing, since embryonic stem cell is used
It is influenced in cell therapy by ethics, and has carcinogenic risk, so MSCs replaces embryonic stem cell to control for cell
Treatment has become a kind of trend.But there is also defects in regenerative medicine and organizational project application by MSCs, such as: as cell passes
The form of the increase of generation number, MSCs changes, and proliferative capacity and multi-lineage potential etc. are all gradually reduced.How to improve
The proliferative capacity and multi-lineage potential of MSCs has become urgent need to solve the problem, and MSCs will be applied to by solving these problems
Regenerative medicine and organizational project generate significant impact.It is nearest the study found that versatility gene Oct4, Sox2 and Nanog etc. couple
The proliferation and multi-lineage potential of MSCs plays a driving role, and inhibits the expression of these genes can be latent to MSCs proliferation and Multidirectional Differentiation
Inhibiting effect can be generated.MSCs proliferation is improved by the means of genetic engineering and multi-lineage potential has become the heat of research
Point.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology and deficiency, provides IL-31 in regulation mescenchymal stem cell
Application in proliferation and differentiation.
The purpose of the invention is achieved by the following technical solution: IL-31 is in regulation mescenchymal stem cell proliferation and differentiation
Application, based on inventor for the first time find transfection IL-31 can promote the proliferation of MSCs and at rouge or Osteoblast Differentiation potential.
The amino acid sequence of the IL-31 is as follows:
MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNY
TLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQF
SECMDLALKSLTSGAQQATT。
The coding nucleotide sequence of the IL-31 is as follows:
ATGGCCTCTCACTCAGGCCCCTCGACGTCTGTGCTCTTTCTGTTCTGCTGCCTGGGAGGCTGGCTGGC
CTCCCACACGTTGCCCGTCCGTTTACTACGACCAAGTGATGATGTACAGAAAATAGTCGAGGAATTACAGTCCCTC
TCGAAGATGCTTTTGAAAGATGTGGAGGAAGAGAAGGGCGTGCTCGTGTCCCAGAATTACACGCTGCCGTGTCTCA
GCCCTGACGCCCAGCCGCCAAACAACATCCACAGCCCAGCCATCCGGGCATATCTCAAGACAATCAGACAGCTAGA
CAACAAATCTGTTATTGATGAGATCATAGAGCACCTCGACAAACTCATATTTCAAGATGCACCAGAAACAAACATT
TCTGTGCCAACAGACACCCATGAATGTAAACGCTTCATCCTGACTATTTCTCAACAGTTTTCAGAGTGCATGGACC
TCGCACTAAAATCATTGACCTCTGGAGCCCAACAGGCCACCACTTAA。
The regulation is positive regulation, i.e. facilitation.
Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation, is that by IL-31 albumen or can express
The nucleic acid sequence of IL-31 is prepared as promoting the preparation of mescenchymal stem cell proliferation and differentiation.
The nucleic acid sequence is preferably DNA sequence dna.
Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation, is using IL-31 gene as target position
Point will promote the substance of IL-31 genetic transcription and expression to be prepared as promoting the preparation of mescenchymal stem cell proliferation and differentiation.
The substance is preferably Oct4 transcription factor.
The present invention has the following advantages and effects with respect to the prior art: the present invention is to be based on inventor for the first time
It was found that transfection IL-31 can promote the proliferation of MSCs and dive getable achievement at rouge or Osteoblast Differentiation.The present invention will contain IL-
31, the slow virus of shIL-31, Oct4, shOct4 and empty plasmid transfects respectively is expressed into MSCs, discovery transfection Oct4 or
IL-31 plasmid can promote the proliferation of MSCs and at rouge or Osteoblast Differentiation potential, and transfecting shOct4 or shIL-31 can inhibit
The proliferation and skeletonization of MSCs or at rouge differentiation potential.The proliferation and skeletonization of Oct4 promotion MSCs have been reported at rouge differentiation potential
Road, in our current research by as positive control.Present invention discloses cell factor IL-31 to the self-renewing of MSCs and multidirectional point
Change potential to play crucial adjustment effect and provide theoretical foundation, MSCs to be used for regenerative medicine and organizational project has established necessity
Basis.
Detailed description of the invention
Fig. 1 be in the embodiment of the present invention 1 by the slow virus recombinant vector containing Oct4, IL-31, shOct4, shIL-31 and
After slow virus empty plasmid as control infects UC-MSCs respectively, using MTT method detection cell factor IL-31 to UC-MSCs
The result figure of proliferative effect；Wherein, empty control plasmid of P < 0.01 * relative to different number of days.
Fig. 2 is in the embodiment of the present invention 2 by the slow virus recombinant vector containing shOct4, shIL-31 and as control
After slow virus empty plasmid infects UC-MSCs respectively, detection cell factor IL-31 is acted at rouge or Osteoblast Differentiation UC-MSCs's
Result figure.
Specific embodiment
Below with reference to examples and drawings, the present invention is described in further detail, but is not limited to model of the invention
It encloses.If unspecified, embodiment carries out referring to conventional laboratory conditions, or referring to the specification of kit manufacturer.
Used engineering bacteria, cell strain are commercialized bacterial strain or cell strain in embodiment.The system of competent escherichia coli cell
It is standby to be prepared according to " molecular cloning ".
The influence that embodiment 1 is proliferated UC-MSCs using MTT method detection IL-31.
(1) building of IL-31 and shIL-31 Lentiviral.
1) design of IL-31 gene primer.
With the primer of 5.0 software design of Primer amplification IL-31 gene open reading frame, primer both ends restriction enzyme site point
Not Wei BamH I and Hind III, primer synthesizes by the raw work biology Co., Ltd in Shanghai, and the sequence of primer is as follows:
IL-31 forward primer: 5 '-TAGGATCCTCCCACACGTTGCCCGT-3 '；
IL-31 reverse primer: 5 '-GCAAGCTTAGTGGTGGCCTGTTG-3 '.
2) IL-31 gene is expanded.
Using the cDNA of people Th2 cell (being purchased from Shanghai Ha Ling Biotechnology Co., Ltd) as template amplification IL-31 gene.
The reaction system of PCR amplification is as follows:
The reaction condition of PCR amplification is as follows:
3) recycling of IL-31 gene.
Above-mentioned PCR product is subjected to 1% agarose gel electrophoresis, and with Gel Extraction Kit (Omega) to PCR
Product recycling, experimental procedure are pressed kit specification, are slightly changed, steps are as follows:
A, the agarose gel containing target fragment, as far as possible agar extra around excision DNA fragmentation are cut with knife blade
Carbohydrate gum is to reduce gel volume.
B, gel piece is placed in 1.5ml microcentrifugal tube.Be added with the isometric Binding buffer of gel (×
p2).Mixture is placed in 50-55 DEG C, 7min, until gel is completely dissolved.
C, HiBind DNA centrifugal column is placed in 2ml collecting pipe, 700 μ l DNA/ agarose solutions is added into HiBind
DNA centrifugal column, at room temperature, 10,000 × g are centrifuged 1min.
D, liquid is discarded, HiBind DNA centrifugal column is placed back in into identical collecting pipe, to HiBind DNA centrifugal column
300 μ l Binding buffer (× p2) of middle addition, at room temperature, 10,000 × g are centrifuged 1min, wash HiBind DNA centrifugal column,
It discards efflux and reuses collecting pipe.
E, the diluted washing buffer of 700 μ l dehydrated alcohols is added and washs HiBind DNA centrifugal column, at room temperature 10,000
× g is centrifuged 1min.
F, liquid and maximum (top) speed void column centrifugation 2min are discarded to dry centrifugal column basement membrane.
G, HiBind DNA centrifugal column is placed in clean 1.5ml centrifuge tube, 15-30 μ l is added dropwise to centrifugal column basement membrane centre
Elution buffer is placed at room temperature for after 1min and is centrifuged 1min eluted dna with 13,000g.
4) building of IL-31 expression vector.
The IL-31 DNA fragmentation of recycling and pGL3-Basic carrier for expression of eukaryon (Promega company) are passed through into I He of BamH
The processing of III double digestion of Hind, is cloned into the pGL3- through double digestion for the IL-31 DNA fragmentation after double digestion with T4 DNA ligase
In Basic carrier for expression of eukaryon, then transfection Escherichia coli DH5 α competent cell, using forward primer and reverse primer to gram
It is grand to be screened, the positive colony that screening obtains is sent into sequencing, DNA sequencing shows that sequence is correct, is named as pGL3-IL-31.
5) building of IL-31 slow virus plasmid.
A, using BamH I single endonuclease digestion pGL3-IL-31 and slow virus carrier pLVX-IRES-ZsGreen1 (purchase Yu Hanheng
Biotechnology Co., Ltd)；
B, the slow virus that IL-31 DNA fragmentation is subcloned into the linearisation of BamH I single endonuclease digestion is turned using T4 DNA ligase
Plasmid pLVX-IRES-ZsGreen1 is moved, then transfection Escherichia coli DH5 α competent cell, passes through restriction enzyme Hind
The correct clone of identification is served the raw work biology Co., Ltd in sea and carries out DNA sequencing, survey by III single endonuclease digestion Preliminary Identification recombinant plasmid
The correct clone designation of sequence is pLVX-IL-31-ZsGreen1；Large quantity extracting plasmid is in case transfection is used.
6) building of shIL-31 slow virus plasmid.
In Shanghai, the corresponding DNA sequence dna of raw work biology Co., Ltd synthesis IL-31 shRNA neck ring structure (contains I He of Xho
I restriction enzyme site of Nco), use the corresponding DNA sequence dna of I single endonuclease digestion IL-31 shRNA neck ring structure of Xho and slow virus carrier pLVX-
The corresponding DNA sequence dna of IL-31 shRNA neck ring structure is cloned into linearisation using T4 DNA ligase by IRES-ZsGreen1
Slow virus plasmid vector pLVX-IRES-ZsGreen1, be named as pLVX-shIL-31-ZsGreen1.IL-31 shRNA neck
The corresponding DNA sequence dna of ring structure is as follows:
5’-CTCGAGAGAGGGCTACCTGGAGACACCATTG-3’。
(2) building of Oct4 and shOct4 Lentiviral (in this experiment as control group).
1) design of Oct4 gene primer.
With the primer of 5.0 software design of Primer amplification Oct4 gene open reading frame, primer both ends restriction enzyme site difference
For Xho I and Nco I.Primer is synthesized by the raw work biology Co., Ltd in Shanghai, and the primer sequence for expanding Oct4 is as follows:
Oct4 forward primer: 5 '-CCGCTCGAGAGGATGGCGGGACACCTGGCTT-3 '；
Oct4 reverse primer: 5 '-CATGCCATGGAAGGGCAGGCACCTCAGTTTG-3 '.
2) Oct4 gene is expanded.
With the cDNA (being purchased from Shanghai Si Dansai Bioisystech Co., Ltd) of human embryo stem cell for template amplification Oct4 base
Cause.The reaction condition of PCR amplification is same as above, and the reaction system of PCR amplification is as follows:
3) recycling of Oct4 gene.
Above-mentioned PCR product is subjected to 1% agarose gel electrophoresis, and to PCR product recovery purifying (step and IL- above
The recycling of 31 genes is identical).
4) building of Oct4 expression vector.
The PCR product of recycling is cloned into pGL3-Basic carrier for expression of eukaryon (Promega by Xho I and I site Nco
Company), it is named as pGL3-Oct4, DNA sequencing shows that sequence is correct.
5) building of Oct4 slow virus plasmid.
A, using Xho I single endonuclease digestion pGL3-Oct4 and slow virus carrier pLVX-IRES-ZsGreen1, (purchase is in Chinese Hang Seng
Object Science and Technology Ltd.)；
B, by Oct4 DNA fragmentation and lentivirus transfer plasmid pLVX-IRES- is linearized using T4 DNA ligase
ZsGreen1 connection, then transfection Escherichia coli DH5 α competent cell, is tentatively reflected by I single endonuclease digestion of restriction enzyme Nco
Determine recombinant plasmid, the correct clone of identification is served into the raw work biology Co., Ltd in sea and carries out DNA sequencing, is named as pLVX-Oct4-
ZsGreen1.Sequencing is correctly cloned into large quantity extracting plasmid in case transfection is used.
6) building of shOct4 slow virus plasmid.
In Shanghai, the corresponding DNA sequence dna of raw work biology Co., Ltd synthesis Oct4 shRNA neck ring structure (contains I He of Xho
I restriction enzyme site of Nco), use the corresponding DNA sequence dna of I single endonuclease digestion Oct4 shRNA neck ring structure of Xho and slow virus carrier pLVX-
The corresponding DNA sequence dna of Oct4 shRNA neck ring structure is cloned into linearisation using T4 DNA ligase by IRES-ZsGreen1
Slow virus plasmid vector pLVX-IRES-ZsGreen1, is named as pLVX-shOct4-ZsGreen1.Oct4 shRNA neck ring knot
The corresponding DNA sequence dna of structure is as follows:
5’-CTCGAGCCCTCACTTC ACTGCACTGCCATTG-3’。
(3) by the slow-virus transfection UC-MSCs containing target gene.
1) preparation of slow virus.
A, it is inoculated with human renal epithelial cell line 293T cell (being purchased from Shanghai Mei Yan Biotechnology Co., Ltd) and relies ammonia in poly
In the 100mm culture dish of acid processing, liquid is changed when cell fusion degree reaches 80%, by three plasmid (pspax2, pMD2G, pLVX-
IL-31-ZsGreen1, pLVX-shIL-31-ZsGreen1, pLVX-Oct4-ZsGreen1 or pLVX-shOct4-
ZsGreen1) cotransfection 293T cell replaces culture medium after cultivating 6h；Pspax2 and pMD2G purchase is in excellent precious biology.
B, vial supernatant is collected after transfecting 48h, is centrifuged 10min in 4 DEG C, 4000r/min；Supernatant after collecting centrifugation
And it filters；
C, 4 DEG C, 25000r/min centrifugation 2h after with serum-free medium lytic virus precipitate；
D, virus titer is measured using limiting dilution assay, packing is stored in spare in -80 DEG C of refrigerators.
2) transfection of slow virus.
Umbilical cord mesenchymal stem cells (UC-MSCs, Shanghai Bai Li Biotechnology Co., Ltd) are inoculated in six orifice plates, when
The lentiviral particle that 1ml step 1) obtains is added when Monolayer growth of cells density reaches 80%~90%, and (titre is 6 × 107TU/
Ml), and polybrene is added to final concentration of 8 μ g/ml to promote viruses adsorption.Transfected according to following 5 groups: IL-31 is to turn
The UC-MSCs of IL-31 plasmid (i.e. pLVX-IL-31-ZsGreen1) is contaminated, shIL-31 is transfection IL-31 shRNA (i.e. pLVX-
ShIL-31-ZsGreen1 UC-MSCs), Oct4 are the UC-MSCs for transfecting Oct4 plasmid (i.e. pLVX-Oct4-ZsGreen1),
ShOct4 is the UC-MSCs for transfecting Oct4 shRNA (i.e. pLVX-shOct4-ZsGreen1), is compareed to transfect the UC- of empty plasmid
MSCs。
(4) MTT detects the proliferation of UC-MSCs.
1) above-mentioned 5 groups of UC-MSCs are done into MTT experiment respectively.
A, 5 groups of UC-MSCs after transfection are incubated at 37 DEG C, 5%CO2In incubator；
B, per 3 Duplicate Samples taken out respectively in 5 groups of UC-MSCs for 24 hours, it is separately added into (the MTT kit purchase of 20 μ L MTT liquid
The biological Co., Ltd of spirit is breathed out in Shanghai), continue to cultivate 4h；
C, culture medium in hole is carefully sucked, 100 μ L DMSO, low-speed oscillation 10min, which are added, in every hole keeps crystal sufficiently molten
Solution；
D, each hole absorbance (A) value is measured at 490nm wavelength in microplate reader, is analyzed with t inspection statistics.
As the result is shown: IL-31 can promote the proliferation of UC-MSCs, and shIL-31 can inhibit the proliferation (Fig. 1) of UC-MSCs.?
Using Oct4 as positive control in this experiment, Oct4 can also promote the proliferation of UC-MSCs, and sh Oct4 can also inhibit UC-MSCs
Proliferation.
Embodiment 2 detects IL-31 to UC-MSCs into the effect of rouge and Osteoblast Differentiation.
(1) UC-MSCs at rouge break up and identify.
1) UC-MSCs breaks up at rouge；
A, UC-MSCs is transfected using the slow virus containing shOct4 or shIL-31 respectively, using the slow disease containing empty plasmid
Poison transfection is as control；
B, the UC-MSCs after above-mentioned 3 kinds transfections is pressed 3 × 104cells/cm2Density be inoculated into containing growth medium
48 orifice plates in, growth medium be containing 10% (V/V) FBS DMEM/F12 (FBS purchased from the macro neoformation technology in Guangzhou it is limited
Company；DMEM/F12 is purchased from Guangzhou Sai Ye Biotechnology Co., Ltd) inoculated culture plate is placed on 5%CO237 DEG C training
It supports and is incubated in case；
C, after culture for 24 hours, gently sop up the growth medium of UC-MSCs from each hole, be added 200 μ lUC-MSCs at
Rouge differential medium A liquid is changed into rouge differential medium B liquid after 3 days, gain A liquid again within the 4th day, so recycles, induction differentiation 21
It (is purchased from Guangzhou Sai Ye Biotechnology Co., Ltd at rouge kit)；
2) identification that UC-MSCs breaks up at rouge；
A, it after above-mentioned UC-MSCs breaks up 21 days, is sucked out into rouge differential medium, is rinsed 1 time with PBS, then with 4% poly
Formalin fixes 30min；
B, PBS is rinsed twice, with 200 μ l oil red O stain 30min；
C, it is rinsed 2-3 times with PBS；
D, it observes and takes pictures under an optical microscope image.
(2) Osteoblast Differentiation and identification of UC-MSCs.
1) Osteoblast Differentiation of UC-MSCs；
A, 0.1% enough gelatin solution is added and its bottom is completely covered into 48 orifice plates, be rotated up gelatin solution and cover
The bottom of entire culture plate is covered, at least stands 30min at room temperature；
B, gelatin solution is blotted, 30min is placed in super-clean bench, makes remaining solution evaporating completely；
C, UC-MSCs is transfected using the slow virus containing shOct4 or shIL-31 respectively, using the slow disease containing empty plasmid
Poison transfection is as control；
D, the UC-MSCs after above-mentioned 3 kinds transfections is pressed 3 × 104cells/cm2Density be inoculated into that be pre-coated with gelatin molten
In the culture plate of liquid, growth medium is the DMEM/F12 containing 10% (V/V) FBS；Inoculated culture plate is placed on 5%
CO237 DEG C of incubators in be incubated for；
E, after culture for 24 hours, growth medium is gently sopped up from each hole, and the training of 200 μ l UC-MSCs Osteoblast Differentiations is added
Support base (skeletonization kit is purchased from Guangzhou Sai Ye Biotechnology Co., Ltd)；Induction differentiation 21 days, the primary differentiation training of replacement in every 3 days
Support base；
2) identification of UC-MSCs Osteoblast Differentiation；
A, after above-mentioned UC-MSCs breaks up 21 days, osteoblast differentiation culture medium is sucked out, is rinsed 1 time with PBS, then with 4%
Paraformaldehyde solution fixes 30min；
B, PBS is rinsed twice, with 200 μ l Alizarin red staining 3-5min；
C, it is rinsed 2-3 times with PBS；
D, it observes and takes pictures under an optical microscope image.
As the result is shown: shIL-31 can reduce UC-MSCs at rouge and Osteoblast Differentiation, show that IL-31 can enhance UC-MSCs
Multi-lineage potential (Fig. 2).Using shOct4 as positive control in this experiment, shOct4 can also reduce UC-MSCs's
At rouge and Osteoblast Differentiation, the multi-lineage potential of UC-MSCs can be enhanced by showing Oct4 also.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
- Application of the 1.IL-31 in preparation regulation mescenchymal stem cell proliferation and the preparation of differentiation, it is characterised in that: be by IL- 31 albumen or the nucleic acid that can express IL-31 are prepared as promoting the preparation of mescenchymal stem cell proliferation and differentiation；Described is divided into rouge differentiation and Osteoblast Differentiation.
- 2. application according to claim 1, it is characterised in that: the amino acid sequence of the IL-31 such as SEQ ID NO.1 It is shown.
- 3. application according to claim 2, it is characterised in that: the coding nucleotide sequence of the IL-31 such as SEQ ID Shown in NO.2.
- 4. application according to claim 1, it is characterised in that: the regulation is positive regulation.
- 5. application according to claim 1, it is characterised in that: the nucleic acid is DNA sequence dna.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610084921.XA CN105586310B (en) | 2016-02-14 | 2016-02-14 | Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610084921.XA CN105586310B (en) | 2016-02-14 | 2016-02-14 | Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105586310A CN105586310A (en) | 2016-05-18 |
| CN105586310B true CN105586310B (en) | 2019-03-22 |
Family
ID=55926218
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610084921.XA Expired - Fee Related CN105586310B (en) | 2016-02-14 | 2016-02-14 | Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105586310B (en) |
-
2016
- 2016-02-14 CN CN201610084921.XA patent/CN105586310B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer;Markus F. Neurath and Susetta Finotto;《Cytokine & Growth Factor Reviews》;20110305;第22卷;第83-89页 * |
| Mucosal cytokine network in inflammatory bowel disease;Akira Andoh et al.;《World J Gastroenterol》;20080907;第14卷(第33期);第5154-5161页 * |
| The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition;Christoph Garbers et al.;《Current Opinion in Immunology》;20150306;第34卷;第75-82页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105586310A (en) | 2016-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102827254B (en) | Cell penetrating peptide hPP10 and use thereof | |
| CN103304637B (en) | Cell permeable peptide hPP3 and usage thereof | |
| CN116574172B (en) | Recombinant humanized type I collagen and preparation method thereof | |
| CN111471089A (en) | Recombinant African swine fever virus CD2V subunit protein and preparation method and application thereof | |
| CN110981968B (en) | Fusion protein containing rabies virus G protein, preparation method, application and vaccine thereof | |
| CN111393531A (en) | Subunit fusion protein CD2V-Fc and preparation method and application thereof | |
| WO2022228086A1 (en) | Myeloid-specific promoter and use thereof | |
| CN113402591A (en) | Novel coronavirus vaccine based on spinous process protein gene modified stem cells, and preparation method and application thereof | |
| Tang et al. | Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy | |
| CN105586310B (en) | Application of the IL-31 in regulation mescenchymal stem cell proliferation and differentiation | |
| CN111378017B (en) | Subunit F protein of peste des petits ruminants virus and preparation method and application thereof | |
| JP5134964B2 (en) | Isolated human cells, methods for obtaining the same and uses thereof | |
| CN104450781B (en) | A kind of cell line of overexpression CIAPIN1 albumen and its preparation method and application | |
| CN114181973A (en) | Self-constructed sT2 cell expression exogenous SLA-2 gene and preparation method thereof | |
| CN109266683B (en) | Lentiviral recombinant vector containing E4BP4 gene and preparation method and application thereof | |
| CN111378621B (en) | B lymphoma cell strain stably transfected by EB virus latent membrane protein 1, construction method and application thereof | |
| AU2020267343A1 (en) | Adenoviral polypeptide IX increases adenoviral gene therapy vector productivity and infectivity | |
| CN113855794A (en) | Novel coronavirus vaccine based on spinous process protein gene modified stem cells | |
| CN113136369A (en) | Cell modified with lipoxygenase gene and preparation method and application thereof | |
| CN105601726B (en) | Application of the Oct4 in regulation IL-31 gene expression | |
| CN101787359B (en) | Recombinant lentivirus for carrying out targeted induction on osteogenic differentiated cell apoptosis as well as preparation method and application thereof | |
| CN111321169A (en) | Genetically modified NK cell and preparation method and application thereof | |
| CN117487009B (en) | Anti-chicken PML monoclonal antibody and application thereof | |
| CN116640231B (en) | Recombinant humanized 17-type collagen polypeptide and preparation method thereof | |
| CN113201062B (en) | Method for stably expressing secreted human insulin in mesenchymal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190322 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |