CN105343894B - The recombination mescenchymal stem cell of efficient secretion prostaglandin E2 (PGE2) and its application - Google Patents
The recombination mescenchymal stem cell of efficient secretion prostaglandin E2 (PGE2) and its application Download PDFInfo
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Abstract
The invention discloses the recombination mescenchymal stem cell of efficient secretion prostaglandin E2 (PGE2) and its applications.The obtained recombination mescenchymal stem cell C3-shM cell secretion PGE2 ability of the present invention is strong, thus the ability enhancing for inhibiting CD3+T to be proliferated.Therefore, the mescenchymal stem cell of efficient secretion PGE2 of the invention is that the research of mescenchymal stem cell and application are laid a good foundation.
Description
Technical field
The present invention relates to the recombination mescenchymal stem cell of efficient secretion prostaglandin E2 (PGE2) and its applications.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is present in almost all of tissue, and
It is isolated from marrow, adipose tissue, umbilical cord, placenta and amniotic fluid.MSCs has powerful self-renewal capacity and multidirectional
Differentiation potential is the renewable source cell that fields of implantation has a extensive future；Meanwhile MSCs is that a kind of important immunological regulation is thin
Born of the same parents, MSCs show very strong inhibiting effect to immune system after inflammatory cytokine stimulation, so MSCs is applied to reduce
Immunological rejection extends graft survival time, related immune deficiency disorder is treated, such as autoimmune disease etc..MSCs is in inflammation
Inflammation factor stimulation is lower can to secrete a large amount of chemotactic factor (CF) and the prostaglandin E2 (PGE2) with immunosuppressive properties.In chemotactic
Under the action of the factor, immunocyte is inhibited around chemotactic to MSCs and by the PGE2 of its high local concentrations.
PGE2 is a kind of important regulatory molecule, is usually acted synergistically with other immune-regulating factors such as iNOS or IDO,
Inhibit the proliferation of immunocyte and the generation of inflammatory factor.Mysm1 is MYb-like, SWIRM and MPN domain-
The abbreviation of containing protein-1, also known as 2A-DUB or KIAA1915/MYSM1 are that histone H2A removes ubiquitin
Change one of enzyme.Mysm1 can specificity adjusting histone H2A single ubiquitination, to change histone modification, and then influence
The transcription of gene.
Summary of the invention
Technical problem to be solved by the invention is to provide being capable of efficient secretion prostaglandin E2 in the case where inflammatory factor stimulates
Recombination mescenchymal stem cell application.
T cell is inhibited to increase in preparation in order to solve the above technical problems, present invention firstly provides recombination mescenchymal stem cells
The application in product and/or immunosuppressor grown.
Recombination mescenchymal stem cell provided by the present invention inhibits product and/or the immunosupress of T cell proliferation in preparation
In application in agent, the recombination mescenchymal stem cell is prepared as follows: reducing Mysm1 in receptor mescenchymal stem cell
The expression quantity of gene obtains recombination mescenchymal stem cell；The receptor mescenchymal stem cell is in vitro mescenchymal stem cell；Institute
Stating Mysm1 is histone H2A deubiquitinating enzymes.
In above-mentioned application, the recombination mescenchymal stem cell has following 1) -6) at least one of characteristic:
1) proliferative capacity of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell；
2) differentiation capability of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell；
3) ability to express of the skeletonization marker gene RUNX2 of the recombination mescenchymal stem cell is higher than the receptor mesenchyma
Stem cell；
4) ability to express at rouge marker gene PPAR γ of the recombination mescenchymal stem cell is filled between being higher than the receptor
Matter stem cell；
5) the prostaglandin E2 secretion capacity of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell；
6) immunosuppression capability of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell.
In above-mentioned application, the immunosuppression capability can be the ability for inhibiting T cell proliferation, concretely inhibit CD3+T
The ability of cell Proliferation.
Recombination mescenchymal stem cell of the invention is compared with the receptor mescenchymal stem cell, the table of the Mysm1 gene
It is reduced up to amount；The expression quantity of the Mysm1 gene can be the mRNA level in-site of Mysm1 gene.
In above-mentioned application, the Mysm1 is the protein that amino acid sequence is SEQ ID No.1.SEQ ID No.1 by
819 amino acid compositions.
In above-mentioned application, the Mysm1 gene is the gene for encoding protein shown in SEQ ID No.1, cDNA sequence
For shown in SEQ ID No.2.
In above-mentioned application, the expression quantity for reducing Mysm1 gene in receptor mescenchymal stem cell includes filling between receptor
The substance for inhibiting Mysm1 gene expression described in the receptor mescenchymal stem cell is imported in matter stem cell.
In above-mentioned application, under the substance for inhibiting Mysm1 gene expression described in the receptor mescenchymal stem cell is
State 1) -10) in any biomaterial:
1) shRNA is the short hairpin RNA to form loop-stem structure；
2) expression vector of shRNA described in expression 1)；
3) the recombinant microorganism cell of shRNA described in expression 1)；
4) the recombinant animal cell of shRNA described in expression 1)；
5) the recombinant plant cell of shRNA described in expression 1)；
6) 1) siRNA that the shRNA generate；
7) expression vector of the 6) siRNA is expressed；
8) the recombinant microorganism cell of the 6) siRNA is expressed；
9) the recombinant animal cell of the 6) siRNA is expressed；
10) the recombinant plant cell of the 6) siRNA is expressed.
In above-mentioned application, in the biomaterial 1) described in the nucleotide sequence of shRNA be SEQ ID No.3.
In above-mentioned application, the substance for inhibiting Mysm1 gene expression described in the receptor mescenchymal stem cell can be
Express the recombinant slow virus LV-shmMysm1 of the shRNA.
In above-mentioned application, the recombinant slow virus LV-shmMysm1 and the receptor mescenchymal stem cell are co-cultured, and are inhibited
The expression of Mysm1 gene in the receptor mescenchymal stem cell.
In above-mentioned application, the recombinant slow virus LV-shmMysm1 is prepared as follows: will express SEQ ID
Recombinant plasmid, plasmid pMDLg/pRRE, plasmid pRSV-Rev and the plasmid pMD2.G cotransfection lactation of shRNA shown in No.3 is dynamic
Object cell obtains the recombinant slow virus after cultivating cell.
In above-mentioned application, the recombinant plasmid concretely plasmid of shRNA shown in the expression SEQ ID No.3
pLKO.1-shmMysm1。
In above-mentioned application, the mammalian cell concretely 293T/17 cell
In above-mentioned application, the infective dose MOI of the recombinant slow virus can be (5-40), concretely 5.
Above, the in vitro mescenchymal stem cell can be the in vitro mescenchymal stem cell of mammal, specifically may be used
For the in vitro mescenchymal stem cell (embryonic mesenchymal stem cells or mesenchymal stem cell) of mouse.
In order to solve the above technical problems, the present invention also provides the products of following B1 or B2:
B1, the product for inhibiting T cell proliferation, active constituent are the recombination mescenchymal stem cell；
B2, immunosuppressor, active constituent are the recombination mescenchymal stem cell.
Above, the T cell concretely CD3+T cell.
It is demonstrated experimentally that the obtained recombination mescenchymal stem cell C3-shM cell secretion PGE2 ability of the present invention is strong, thus
Inhibit the ability enhancing of CD3+T proliferation.Under the stimulation of TNF-α and IFN-γ, PGE2 base in the C3-shM cell of Fiber differentiation
It is 5 times of PGE2 gene expression dose in the C3-G cell of Fiber differentiation because expression significantly increases；It is non-induced culture and
Under inducing culturing condition, C3-shM cell secretes the level of PEG2 obviously higher than C3-G cell and C3H/10T1/2 cell, C3-
G cell and C3H/10T1/2 cell secrete being on close level for PEG2.C3-shM cell is bright to the Proliferation Ability ability of CD3+T cell
It is aobvious to be better than C3H/10T1/2 cell and C3-G cell, the Proliferation Ability of C3H/10T1/2 cell and C3-G cell to CD3+T cell
Ability is suitable.When the quantity ratio of C3-shM cell and CD3+T cell is 1:10, the proliferation rate of CD3+T cell only has 33.2%,
It is better than under the same terms C3-G cell (57.7%) to CD3+T cell inhibitory effect ability；The proliferation rate of CD3+T cell is merely
85.7%.With the mRNA of Mysm1 gene in C3H/10T1/2 cell relative to β-actin gene mrna expression amount be 1, C3-G
The mRNA of Mysm1 gene is Mysm1 base in 0.96, C3-shM cell relative to the mrna expression amount of β-actin gene in cell
The mRNA of cause is 0.56 relative to the mrna expression amount of β-actin gene.The proliferation energy of C3H/10T1/2 cell and C3-G cell
Power is suitable, and the proliferative capacity of C3-shM cell is significantly stronger than C3H/10T1/2 cell and C3-G cell, the BrdU of C3-shM cell
Positive cell ratio is that the BrdU positive cell ratio of 24.1%, C3-G cell is 15.3%.Skeletonization is related in C3-shM cell
The expression quantity of gene RUNX2 and thin obviously higher than C3H/10T1/2 cell and C3-G at the expression quantity of lipid phase correlation gene PPAR γ
Born of the same parents are skeletonization dependency basis in 1, C3-shM cell with expression quantity of the Bone formation-related gene RUNX2 in C3-G cell with respect to β-actin
Expression quantity because of RUNX2 with respect to β-actin is 23；With the table in C3-G cell at lipid phase correlation gene PPAR γ with respect to β-actin
It is in 1, C3-shM cell up to amount at the lipid phase correlation gene PPAR γ expression quantity of β-actin is relatively 15.Therefore, height of the invention
The mescenchymal stem cell of effect secretion PGE2 is that the research of mescenchymal stem cell and application are laid a good foundation.
Detailed description of the invention
Fig. 1 is the immunophenotypic characterization result figure for recombinating mescenchymal stem cell.C3-G cell and C3-shM cellular immunity table
Abscissa and ordinate in type qualification figure and the abscissa and ordinate phase in C3H/10T1/2 Immunophenotyping qualification figure
Together.
Fig. 2 is the mrna expression amount result that real-time fluorescence quantitative PCR detects Mysm1 gene in cell.Wherein, A C3-G
Mrna expression amount testing result of the mRNA of Mysm1 gene relative to β-actin gene in cell and C3H/10T1/2 cell；B
For mrna expression amount testing result of the mRNA relative to β-actin gene of Mysm1 gene in C3-G cell and C3-shM cell.
Fig. 3 is the proliferative capacity result that flow cytomery recombinates mescenchymal stem cell.Wherein, A be C3-G cell and
The proliferative capacity testing result of C3H/10T1/2 cell；B is the proliferative capacity testing result of C3-G cell and C3-shM cell.
Fig. 4 is the differentiation capability testing result for recombinating mescenchymal stem cell.Wherein, A is C3H/10T1/2 cell and C3-G
Bone formation-related gene RUNX2 and the expression quantity result at lipid phase correlation gene PPAR γ with respect to β-actin in cell；B is that C3-G is thin
Bone formation-related gene RUNX2 and the expression quantity result at lipid phase correlation gene PPAR γ with respect to β-actin in born of the same parents and C3-shM cell.
Fig. 5 is the gene expression dose of PGE2 in fluorescence quantitative PCR detection cell.Wherein, A is the C3-G of non-induced culture
The gene expression dose of cell and the C3H/10T1/2 cell PGE2 of non-induced culture；B is that C3-shM cell and C3-G cell exist
The gene expression dose of PGE2 under induction and non-induced condition of culture.
Fig. 6 is that C3-shM cell and C3-G cell are inducing and ELISA detection PGE2 secretion level after non-induced culture.
Fig. 7 is the suppression result of C3-shM cell, C3-G cell and C3H/10T1/2 cell to CD3+T cell Proliferation.Its
In, A is the suppression result of C3H/10T1/2 cell and C3-G cell to CD3+T cell Proliferation；B is that C3-shM cell and C3-G are thin
Suppression result of the born of the same parents to CD3+T cell Proliferation.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Mouse MSC cell line C3H/10T1/2 cell in following embodiments is Chinese Academy of Sciences Shanghai life science
The product of institute's cell resource center, catalog number SCSP-506.
α-MEM culture solution (Gibco company, cat.no.12000-022) in following embodiments, DMEM-HG culture solution
(Gibco company, cat.no.10566-024), fetal calf serum (Gibco company)；The PBS buffer solution of 0.01M, pH7.2-7.4
(PBS buffer solution hereinafter), dexamethasone, phosphorylated Vit C, β-phosphoglycerol, pancreatin, TRIZOL, insulin, 3- are different
Butyl -1- methyl xanthine (3-Isobutyl-1-Methylxanthine, IBMX) and Indomethacin (indomethacin) are equal
For the product of Sigma company；TNF-α and INF- γ are the product of Perotech company；PGE2ELISA kit is Beijing
The product of Haicheng Yuan Hong Science and Technology Ltd.；Polybrene (Polybrene) is the product of Santa Cruz company；CD3ε
MicroBead reagent is the product of Mei Tian Ni Bioisystech Co., Ltd of Germany；Reverse Transcriptase Reagents kit and SYBR Green examination
Agent is the product that (Shanghai) Biotechnology Co., Ltd (TOYOBO) spins in Japan；Propylene glycol methyl ether acetate (PMA) and
Ionomycin is the product of eBioscience company；CFSE is the product of Invitrogen company；BrdU detection kit
For the product of BD company；PEI (polyethylenimine) is the product of Polysciences Inc company, and catalog number is
Cat.No.23966)。
The antibody of anti-mouse CD44, CD105, Sca-1 and MHC II in following embodiments is the product of BD company.
Plasmid pLKO.1-puro eGFP shRNA in following embodiments is the product of Sigma company, catalog number
For SHC005V.
Plasmid pLKO.1-shmMysm1 in following embodiments, specifying information are shown in: http: //Www.broadinstitute.org/rnai/public/clone/details? cloneId=TRCN0000085873。
Plasmid pMDLg/pRRE (slow virus packaging plasmid in following embodiments；Addgene, Cat.No.12251), tool
Body information is shown in that http://www.addgene.org/12251/, the public can be from Academy of Military Medicine, PLA's bases
Plinth Institute for Medical Research obtains the biomaterial, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as it
Its purposes uses.
Plasmid pRSV-Rev (slow virus packaging plasmid in following embodiments；Addgene, Cat.No.12253), specifically
Information is shown in that http://www.addgene.org/12253/, the public can be from Academy of Military Medicine, PLA bases
Institute for Medical Research obtains the biomaterial, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other
Purposes uses.
Plasmid pMD2.G (envelope plasmid in following embodiments；Addgene, Cat.No.12259), specifying information is shown in
Http:// www.addgene.org/12259/, the public can grind from Academy of Military Medicine, PLA's preclinical medicine
Study carefully and obtain the biomaterial, which only attaches most importance to used in the related experiment of duplicate invention, and not can be used as other purposes makes
With.
Embodiment 1, the expression quantity for reducing Mysm1 gene in receptor mescenchymal stem cell obtain recombination mescenchymal stem cell
The present embodiment is using mouse MSC cell line C3H/10T1/2 cell as receptor mescenchymal stem cell, between this receptor
The shRNA recombinant slow virus that expression is directed to Mysm1 gene is imported in mesenchymal stem cells, has been obtained and receptor mescenchymal stem cell phase
Than the recombination mescenchymal stem cell that the expression quantity of Mysm1 gene reduces.The recombination mescenchymal stem cell and receptor mesenchyma are dry thin
Cell phase ratio, proliferative capacity and differentiation capability increase.The specific method is as follows:
1, pass through Mysm1 gene silencing preparation and reorganization mescenchymal stem cell
LV-shmMysm1 recombinant slow virus: by 293T/17 cell (also known as HEK293T/17 cell；ATCC Number:
CRL-11268TM it) is inoculated in 10cm culture dish, is placed in 37 DEG C, 5%CO2Constant incubator in cultivate, reach to cell
Transfection mixture 1 when 85% fusion collects supernatant, as LV-shmMysm1 recombinant slow virus liquid after continuing culture 60-72 hours
(expression that Mysm1 gene can be interfered).
The preparation method of mixture 1: by 10 μ g plasmid pLKO.1-shmMysm1,5 μ g plasmid pMDLg/pRRE, 2.5 μ g matter
Grain pRSV-Rev, 3 μ g plasmid pMD2.G are mixed with 80 μ g transfection reagent PEI, obtain transfection mixture, and transfection mixture is common
After transfecting 293T/17 cell, expression is obtained for the recombinant slow virus of the shRNA of Mysm1 gene.
PLKO.1-shmMysm1 is the table for the shRNA (entitled shm Mysm1) that expression reduces Mysm1 gene expression amount
Up to carrier, Mysm1 is amino acid sequence protein as shown in SEQ ID No.1, and the nucleotide sequence of shRNA is SEQ ID
No.3。
LV-GFP recombinant slow virus: by 293T/17 cell inoculation in 10cm culture dish, 37 DEG C, 5%CO are placed in2Perseverance
It is cultivated in warm incubator, the transfection mixture 2 when cell reaches 85% fusion, culture collected supernatant after 60-72 hours, as
LV-GFP recombinant slow virus liquid (can interfere GFP gene expression).
The preparation method of mixture 2: by 10 μ g plasmid pLKO.1-puro eGFP shRNA, 5 μ g plasmid pMDLg/pRRE,
2.5 μ g plasmid pRSV-Rev, 3 μ g plasmid pMD2.G are mixed with 80 μ g transfection reagent PEI, obtain transfection mixture, and transfection is mixed
After conjunction object transfects cell jointly, expression is obtained for the recombinant slow virus of the shRNA of eGFP gene.
Plasmid pMDLg/pRRE, plasmid pRSV-Rev and plasmid pMD2.G are the plasmid for preparing slow virus, plasmid
PLKO.1-shm Mysm1 is the plasmid for expressing the shRNA of Mysm1 gene, and plasmid pLKO.1-puro eGFP shRNA is expression
The plasmid of the shRNA of eGFP gene.
Mouse MSCs cell line C3H/10T1/2 is suspended in the training of the α-MEM containing 10% (volumn concentration) fetal calf serum
In nutrient solution, C3H/10T1/2 cell suspension is obtained.C3H/10T1/2 cell suspension is pressed 2 × 105/cm2Density be inoculated in 6 holes
In plate, it is placed in 37 DEG C, 5%CO2Constant incubator in cultivate, when cell reaches 80% fusion, be added LV-shmMysm1 weight
Group slow virus liquid (MOI=5), and polybrene (working concentration is 8 μ g/mL) is added, former training is absorbed after continuing culture 6 hours
Nutrient solution is added isometric α-MEM culture solution containing 10% (volumn concentration) fetal calf serum, is placed in 37 DEG C, 5%CO2's
Secondary culture in constant incubator obtains the C3H/10T1/2 cell of Mysm1 gene knockout, is named as experimental group recombination mesenchyma
Stem cell (abbreviation C3-shM cell).
Control group is in addition to replacing with LV-GFP recombinant slow virus liquid for LV-shmMysm1 recombinant slow virus liquid, remaining condition
It is constant, it obtains control group mescenchymal stem cell (abbreviation C3-G cell).
Two, the identification of mescenchymal stem cell is recombinated
1, immunophenotypic characterization
The C3-shM obtained using the α-MEM culture solution incubation step one containing 10% (volumn concentration) fetal calf serum
Cell is placed in 37 DEG C, 5%CO2Constant incubator in cultivate, disappeared when the cell grows to logarithmic phase using 0.25% pancreatin
Change, collect cell, is cleaned 3 times using PBS buffer solution, cell finally is resuspended with PBS buffer solution, and with blood cell counting plate meter
Number.With PBS buffer solution diluting cells suspension, C3-shM cell liquid is obtained, cell density is 2 × 10 in C3-shM cell liquid6A/
mL.Antibody is added to 100 μ L C3-shM cell liquid, every kind of antibody, which is individually added into cell liquid, to be detected, and antibody used is respectively anti-
The antibody of mouse CD44, CD105, Sca-1 and MHCII, 4 DEG C of shaking tables are protected from light 30 minutes, are then centrifuged under the conditions of 400g
5min is collected cell precipitation and is cleaned 2 times with PBS, fixed using 1% paraformaldehyde, 200 μ L, carries out flow cytometer showed.
The immunophenotypic characterization method of C3H/10T1/2 cell and C3-G cell is the same as C3-shM cell.
Experimental result is as follows: C3-shM cell and the equal high efficient expression Sca-1 molecule of C3-G cell, CD105 molecule and CD44
Molecule, cell proportion is relatively low in the expression quantity of 90% or more, MHCII molecule, the table with C3H/10T1/2 cell
Type qualification result is consistent (Fig. 1).
2, Mysm1 gene silencing efficiency identification in mescenchymal stem cell is recombinated
The C3-shM cell of culture to the third generation is cracked using Tripure reagent, cell pyrolysis liquid is collected, extracts RNA
And reverse transcription is cDNA, is detected by real-time fluorescence quantitative PCR (Quantitative Real-time PCR, QRT-PCR) thin
The mrna expression amount of Mysm1 gene in born of the same parents is indicated with the mrna expression amount relative to β-actin gene, to detect the gene
Silence efficiency, experiment in triplicate.
Wherein, Mysm1 gene primer pair:
Forward:5 '-GATGCAGAAGCAGCATACCA-3 '；
Reverse:5 '-CCTCCACAGACAAATGCTCA-3 '.
The Mysm1 gene silencing efficiency identification method of C3H/10T1/2 cell and C3-G cell is the same as C3-shM cell.
Experimental result is as follows: the mRNA with the mRNA of Mysm1 gene in C3H/10T1/2 cell relative to β-actin gene
Expression quantity be Mysm1 gene in 1, C3-G cell mRNA relative to β-actin gene mrna expression amount be 0.96, C3-shM
The mRNA of Mysm1 gene is 0.56 (Fig. 2) relative to the mrna expression amount of β-actin gene in cell；In C3-shM cell
The expression quantity of the mRNA of Mysm1 gene is significantly lower than C3-G cell and C3H/10T1/2 cell
3, the proliferative capacity detection of mescenchymal stem cell is recombinated
For the C3-shM cell inoculation of logarithmic growth phase in 6 orifice plates, cell density is (1-1.5) × 105/ hole, to thin
Intracellular growth is added the BrdU that working concentration is 10 μm of ol/L, says after 1 hour according to BrdU detection kit to when 80% fusion
Bright book carries out the detection of ability of cell proliferation, and experiment is in triplicate.
The proliferative capacity detection method of C3H/10T1/2 cell and C3-G cell is the same as C3-shM cell.
Flow cytomery result as shown in figure 3, the proliferative capacity of C3H/10T1/2 cell and C3-G cell is suitable,
The proliferative capacity of C3-shM cell is significantly stronger than C3H/10T1/2 cell and C3-G cell, the BrdU positive cell of C3-shM cell
Ratio is that the BrdU positive cell ratio of 24.1%, C3-G cell is 15.3%, the results showed that, after interference Mysm1 expression,
The proliferative capacity of mesenchymal stem cells is remarkably reinforced.
4, the differentiation capability detection of mescenchymal stem cell is recombinated
Cell Osteoinductive differentiation complete culture solution: DMEM-HG culture solution contains 10% (percent by volume) fetal calf serum,
Concentration is 10-7The dexamethasone of M, the phosphorylated Vit C that the β-phosphoglycerol and concentration that concentration is 10mM are 50 μM.
Cell adipogenic induction breaks up complete culture solution: DMEM-HG culture solution contains 10% (percent by volume) fetal calf serum,
Concentration is 10-6The dexamethasone of M, concentration are the insulin of 10ng/mL, the Yin that the IBMX and concentration that concentration is 0.5 μM are 200 μM
Diindyl U.S. is pungent.
By C3-shM cell, C3-G cell and C3H/10T1/2 cell use respectively Osteoinductive differentiation complete culture solution and at
Rouge induction differentiation complete culture solution carries out Fiber differentiation 7 days, obtain respectively the C3-shM cell of osteogenic induction, C3-G cell and
C3H/10T1/2 cell, C3-shM cell, C3-G cell and the C3H/10T1/2 cell of adipogenic induction.
Realtime fluorescent quantitative PCR experiment is carried out as follows, is tested in triplicate:
Collect Fiber differentiation 7 days after cell, with TRIZOL extracted total RNA and with Reverse Transcriptase Reagents kit reverse transcription at
cDNA.CDNA be used as template addition TOYOBO brand SYBR Green reagent determined using real-time fluorescence quantitative PCR it is specific
The expression of gene is indicated with expression quantity relative to β-actin gene.
Wherein, mouse β-actin gene primer pair:
Forward:5 '-CTTCCGCCTTAATACTTC-3 '；
Reverse:5 '-AAGCCTTCATACATCAAG-3 '；
Mouse Bone formation-related gene RUNX2 primer pair:
Forward:5 '-CCACAAGGACAGAGTCAGAT-3 '；
Reverse:5 '-GATAGGAGGGGTAAGACTGG-3 '；
Mouse is at lipid phase correlation gene PPAR γ primer pair:
Forward:5 '-TTGATTTCTCCAGCATTTCT-3 '；
Reverse:5 '-GCACTTTGGTATTCTTGGAG-3 '.
As a result as shown in figure 4, in C3H/10T1/2 cell and C3-G cell the expression quantity of Bone formation-related gene RUNX2 and at
The expression quantity of lipid phase correlation gene PPAR γ is suitable；The expression quantity of Bone formation-related gene RUNX2 and at lipid phase in C3-shM cell
The expression quantity of correlation gene PPAR γ is obviously higher than C3H/10T1/2 cell and C3-G cell.With skeletonization dependency basis in C3-G cell
Because expression quantity of the RUNX2 with respect to β-actin is expression of the Bone formation-related gene RUNX2 with respect to β-actin in 1, C3-shM cell
Amount is 23；With in C3-G cell at lipid phase correlation gene PPAR γ with respect to β-actin expression quantity be 1, C3-shM cell at rouge
Expression quantity of the related gene PPAR γ with respect to β-actin is 15.
5, the identification of mescenchymal stem cell efficient secretion prostaglandin E2 (PGE2) is recombinated
Culture solution 1: containing 10% (volumn concentration) fetal calf serum, the TNF-α that concentration is 5ng/mL and concentration is 5ng/
α-MEM the culture solution of the IFN-γ of mL.
1) gene expression dose of fluorescence quantitative PCR detection PGE2
The C3-G after C3-shM cell and recombinant virus infection 72h after the recombinant virus infection 72h that step 1 is obtained
Cell carries out Fiber differentiation with culture solution 1 respectively, according to (1-1.5) × 105Cell density be inoculated in 6 orifice plates, be placed in 37
DEG C, 5%CO2Constant incubator in 12h is cultivated under the stimulation of TNF-α and IFN-γ, respectively obtain Fiber differentiation C3-
The C3-G cell of shM cell and Fiber differentiation.C3-shM cell, recombination after the recombinant virus infection 72h that step 1 is obtained
C3-G cell and C3H/10T1/2 cell after virus infection 72h are respectively with the α-for containing 10% (volumn concentration) fetal calf serum
MEM culture solution culture, according to (1-1.5) × 105Cell density be inoculated in 6 orifice plates, be placed in 37 DEG C, 5%CO2Constant temperature training
It supports in case and cultivates 12h, obtain the C3-shM cell of non-induced culture, the C3-G cell of non-induced culture and non-induced culture respectively
C3H/10T1/2 cell.
Real-time fluorescence quantitative PCR reaction is carried out as follows, is tested in triplicate:
With the total serum IgE of TRIZOL extracting cell and with Reverse Transcriptase Reagents kit reverse transcription at cDNA.CDNA is used as mould
Plate addition TOYOBO brand SYBR Green reagent determines the expression of specific gene using real-time fluorescence quantitative PCR, with opposite
It is indicated in the expression quantity of β-actin gene.
Wherein, mouse β-actin gene primer:
Forward:5 '-CTTCCGCCTTAATACTTC-3 '；
Reverse:5 '-AAGCCTTCATACATCAAG-3 '；
The gene primer of PGE2 gene:
Forward:5 '-AAGGCCATGAATGACCAGGG-3 '；
Reverse:5 '-TGTTCGGTACACGTTGGGAG-3 '.
As a result as shown in figure 5, the expression of PGE2 gene is apparently higher than non-lure in the C3-shM cell of non-induced culture
Lead the C3-G cell of culture and the C3H/10T1/2 cell of non-induced culture, the C3-G cell of non-induced culture and non-induced culture
C3H/10T1/2 cell PGE2 gene expression it is similar.Under the stimulation of TNF-α and IFN-γ, Fiber differentiation
PGE2 gene expression dose significantly increases in C3-shM cell, is PGE2 gene expression dose in the C3-G cell of Fiber differentiation
5 times.
2) ELISA detects PGE2 secretion level
C3-shM cell after the recombinant virus infection 72h that step 1 is obtained, the C3-G after recombinant virus infection 72h are thin
Born of the same parents carry out Fiber differentiation with culture solution 1 respectively, according to (1-1.5) × 105Cell density be inoculated in 6 orifice plates, be placed in 37 DEG C,
5%CO2Constant incubator in cultivate 12h under the stimulation of TNF-α and IFN-γ, collect the supernatant in culture hole, respectively
Obtain the supernatant of the supernatant of the C3-shM cell of Fiber differentiation and the C3-G cell of Fiber differentiation.The weight that step 1 is obtained
C3-shM cell after group virus infection 72h, the C3-G cell after recombinant virus infection 72h and C3H/10T1/2 cell difference
With contain 10% (volumn concentration) fetal calf serum α-MEM culture solution culture, according to (1-1.5) × 105Cell density connect
Kind is placed in 37 DEG C, 5%CO in 6 orifice plates2Constant incubator in cultivate 12h, collect the supernatant in culture hole, obtain respectively
Obtain the supernatant of the C3-shM cell of non-induced culture, the supernatant of the C3-G cell of non-induced culture and non-induced culture
The supernatant of C3H/10T1/2 cell.
Each cell supernatant that will be collected, horizontal with PGE2ELISA kit detection PGE2, experiment is repeated twice.
As a result as shown in fig. 6, under non-induced culture and inducing culturing condition, C3-shM cell secretes the level of PEG2
It is apparently higher than C3-G cell and C3H/10T1/2 cell, C3-G cell and C3H/10T1/2 cell secrete being on close level for PEG2.
6, recombination mescenchymal stem cell inhibits T cell proliferation experiment
After mouse anesthesia, spleen taking-up is placed in PBS buffer solution, spleen is ground to and used the screen to filtrate, is centrifuged, cracking
Red blood cell, dead cell are removed using density gradient method.CD3+T cell is divided according to process using CD3 ε MicroBead suit
Choosing, obtains CD3+T cell.CD3+T cell after sorting is protected from light label 7 minutes with CFSE room temperature, is added after cleaning 2 times and contains PMA
1640 complete culture solutions of (50ng/mL) and ionomycin (1 μ g/mL), are placed in 37 DEG C, 5%CO2Constant incubator in train
After supporting 12 hours, according to the quantity of mescenchymal stem cell and CD3+T cell than being respectively 1:160,1:80,1:40,1:20 and 1:
10 quantity ratio is separately added into C3-shM cell and C3-G cell, co-cultures 48 hours；It is thin with CD3+T according to mescenchymal stem cell
The quantity of born of the same parents co-cultures 48 than being respectively that the quantity ratio of 1:80 and 1:20 are separately added into C3H/10T1/2 cell and C3-G cell
Hour detects the proliferative conditions of CD3+T cell using the fluorescence decay density of CFSE, and experiment is in triplicate.
As a result as shown in fig. 7, C3-shM cell to be significantly stronger than C3H/10T1/2 to the Proliferation Ability ability of CD3+T cell thin
Born of the same parents and C3-G cell, C3H/10T1/2 cell and C3-G cell are suitable to the Proliferation Ability ability of CD3+T cell.When C3-shM is thin
When the quantity ratio of born of the same parents and CD3+T cell is 1:20, the proliferation rate of CD3+T cell only has 44.7%, is better than C3-G under the same terms
Cell (67.6%) is to CD3+T cell inhibitory effect ability；When the quantity ratio of C3-shM cell and CD3+T cell is 1:10,
The proliferation rate of CD3+T cell only has 33.2%, is better than under the same terms C3-G cell (57.7%) to CD3+T cell inhibitory effect
Ability；The proliferation rate of simple CD3+T cell is 85.7%.
Claims (3)
1. recombinating application of the mescenchymal stem cell in preparation immunosuppressor, the recombination mescenchymal stem cell is according to such as lower section
Method preparation: reducing the expression quantity of Mysm1 gene in receptor mescenchymal stem cell, obtains recombination mescenchymal stem cell；The receptor
Mescenchymal stem cell is in vitro mescenchymal stem cell；The Mysm1 is histone H2A deubiquitinating enzymes；
The Mysm1 is amino acid sequence protein as shown in SEQ ID No.1；
The expression quantity for reducing Mysm1 gene in receptor mescenchymal stem cell includes that suppression is imported into receptor mescenchymal stem cell
Make the substance of Mysm1 gene expression described in the receptor mescenchymal stem cell；
The substance for inhibiting Mysm1 gene expression described in the receptor mescenchymal stem cell is shRNA；
The nucleotide sequence of the shRNA is SEQ ID No.3.
2. application according to claim 1, it is characterised in that: the recombination mescenchymal stem cell has following 1) -6) in
At least one characteristic:
1) proliferative capacity of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell；
2) differentiation capability of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell；
3) it is dry thin to be higher than the receptor mesenchyma for the ability to express of the skeletonization marker gene RUNX2 of the recombination mescenchymal stem cell
Born of the same parents；
4) ability to express at rouge marker gene PPAR γ of the recombination mescenchymal stem cell is dry higher than the receptor mesenchyma
Cell；
5) the prostaglandin E2 secretion capacity of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell；
6) immunosuppression capability of the recombination mescenchymal stem cell is higher than the receptor mescenchymal stem cell.
3. application according to claim 1 or 2, it is characterised in that: the in vitro mescenchymal stem cell is mammal
In vitro mescenchymal stem cell.
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