CN106011056A - Preparation method of mesenchymal stem cells for skin acne treatment, preparation method and product thereof - Google Patents
Preparation method of mesenchymal stem cells for skin acne treatment, preparation method and product thereof Download PDFInfo
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Abstract
The invention provides a preparation method of mesenchymal stem cells for skin acne treatment. Specifically, the method includes constructing gene segments of peroxisome proliferator-activated receptor alpha and gamma, recombining the gene segments to a virus expression vector, then utilizing the two to conduct cotransfection of mesenchymal stem cells so as to obtain the mesenchymal stem cells, which can achieve high expression of the peroxisome proliferator-activated receptor alpha and gamma protein, give play to the effects of the two to resist cutaneous inflammation response and inhibit cell cornification abnormity. Also, the proliferation differentiation and regulating immune functions of mesenchymal stem cells themselves are also utilized to participate in skin repair and anti-inflammatory response and treat skin acne. The mesenchymal stem cells provided by the invention can have very good therapeutic and cosmetic effects on skin acne.
Description
Technical field
The present invention relates to a kind of method of mescenchymal stem cell for acne treatment and obtained by described method
The mescenchymal stem cell of acne must be treated, especially, the present invention relates to build peroxisome proliferator-activated receptor alpha
Restructuring with γ (Peroxisome proliferator-activated receptor α and γ, PPAR α and γ)
Viral vector so that mescenchymal stem cell can stably express peroxisome proliferator-activated receptor alpha and γ, plays both anti-
Scytitis reaction and suppression cellular keratinization are abnormal, the effect for the treatment of acne；The controlling for acne of the present invention simultaneously
The mescenchymal stem cell treated keeps good multiplication capacity, and mescenchymal stem cell has proliferation and differentiation and regulation immunologic function, ginseng
With skin repair and anti-inflammatory response, and without Tumor formation, will be expected to reach acne more preferably to treat and cosmetic result.
Background technology
A kind of common dermatosis of acne, also referred to as acne, comedo and skin sore, is a kind of common pilosebaceous inflammations
Dermatoses, is mainly in face, badly influences youth of both sexes youth beautiful image.Pilosebaceous unit opening is blocked is to send out
Key factor in pathogenesis system.In the case of hair follicle obturation, propionibacterium acnes amount reproduction, cause inflammation, form acne
Most basic infringement inflamed papules.Inside inaccessible pilosebaceous unit, a large amount of sebums, a large amount of pus cell tie pilosebaceous unit
Structure destroys, and forms tuberosity, cyst and sebaceous cyst, finally destroys skin and even forms cicatrix.Sebaceous gland duct hyperkeratosis is also acne
The key factor of morbidity.
Continuing inaccessible the breeding with propionibacterium acnes of hair follicle causes inflammatory cell to invade, thus secretes a large amount of inflammatory factor,
Cause the keratinocyte dyskeratosis in infundibulum of hair follicle portion.Thus suppress inflammatory factor and improve follicular keratosis extremely, it is to change
Kind acne lesions and the key link of pathological change.Oral Chinese medicine and local topical aspect, wherein intervene acne with local topical
Model is Main way: as the external used medicines such as compound recipe DIANDAO SAN, Compound Zi-Jin Cream, Rhizoma Coptidis Concha Ostreae cream, fruit acid all have stronger resisting
Cutinization, it is possible to be effectively improved acne lesions.Oral Acne Vulgaris is for being mainly composed of Herba Hedyotidis Diffusae, Radix Salviae Miltiorrhizae, Radix Scutellariae, company
Stick up, Herba Leonuri, Fructus Crataegi etc., it is possible to regulation estrogen and androgen level, promote wound repair, and have antibacterial, suppression inflammatory because of
The effect of son release.
At clinical treatment, the acne overwhelming majority is Chinese medicine, has certain therapeutic effect.The present invention uses mesenchyme dry thin
Born of the same parents are protein expression vector and the function of itself, and acne treatment is produced more preferable effect.Mescenchymal stem cell (human
Mesenchymal stem cells, MSCs) have been used to clinical treatment field, treat at nervous system disease and orthopaedic disease
In achieve good curative effect, the restriction of the aspects such as the most non-existent immunologic rejection, ethics and oncogenicity, there is good facing
Bed application prospect.Thus, the present invention, by building the recombinant viral vector of peroxisome proliferators activated receptor γ, transfects
Mescenchymal stem cell, performance peroxisome proliferators activated receptor γ anti-inflammatory and antiproliferative biological function, and nothing
Tumor formation.Creation is conducive to acne to repair by peroxisome proliferators activated receptor γ and the mescenchymal stem cell expressed
Multiple microenvironment, suppression inflammatory cell secretion inflammatory cytokine, thus stop follicular orifice epithelial cell hyperkeratosis, it is right to be expected to
Acne treatment and beauty treatment produce more preferable effect.
Summary of the invention
The applicant passes through discovery of concentrating on studies, the PPAR α effect to epidermal barrier, epidermal proliferation and differentiation, can increase
The expression of spinous layer of epidermis/granular layer structural protein, also increases apoptosis, reduces propagation, accelerates the recovery of epidermal barrier function.PPAR
Although γ is mainly distributed in fatty tissue, but confirm that 3 hypotypes of PPAR are also deposited by sxemiquantitative fluorescence quantitative PCR detection
It is in the keratinocyte of people, and the expression of PPAR γ can increase along with the differentiation of keratinocyte；And find
PPAR γ content at Psoriasis reduces compared with without the PPAR γ content at skin lesion.
Have proven to PPAR α, γ and there is part anti-inflammatory property, specific PPAR γ part lower T cell, mononuclear cell,
Inflammatory cytokine in mastocyte, and the activation of negativity regulation macrophage.In many benign and malignant cells,
PPAR γ part limits the propagation of these cells and promotes their differentiation.We test proof PPAR α can increase differentiation phase
Correlation gene is as draped over one's shoulders outward the expression of albumen, and the cytokine that also can lower inflammatory mediator reaches antiinflammatory action.PPAR α deficient mice
The reaction stimulating inflammatory mediator extends；Topical application PPAR alfa agonists can prevent the scytitis reaction after UVB radiation.?
In acne group skin lesion, PPAR α, γ level is to reduce compared with non-skin lesion district.The activation energy suppression keratinocyte of PPAR α
Increment, thus cause cuticle thickness to reduce.
Thus PPAR α and PPAR γ in skin environment stable in played the most crucial effect, regulate skin
Differentiation, propagation and inflammatory reaction.
Peroxisome proliferators activated receptor γ belongs to nuclear receptor superfamily, and biological function is various, participates in saccharide
The aspects such as the periodicity regulation and control of metabolism, lipid metabolism, the differentiation of adipose cell, insulin resistant, inflammatory reaction and ovary tissue
All play a significant role, it is also possible to the secretion of suppression Human umbilical vein endothelial cells inflammatory factor, i.e. tumor necrosis factor (Tumor
Necrosis factor-α, TNF-α) and interleukin 6 (Interleukin 6, IL-6) etc..
The present invention utilizes the characteristic of mescenchymal stem cell proliferation and differentiation and " going back to the nest ", by the PPAR α built and PPAR γ base
Because of the recombinant viral vector cotransfection MSCs of fragment, can high expressed PPAR α and PPAR γ, play PPAR α and PPAR γ anti-inflammatory
With antiproliferative biological action and the function of mescenchymal stem cell itself thereof, improving local skin microenvironment, suppression inflammation is thin
Intracrine inflammatory cytokine, thus stop follicular orifice epithelial cell hyperkeratosis.
Inventor has been surprisingly found that mescenchymal stem cell high expressed PPAR α and PPAR γ can maintain skin barrier to ooze
Property thoroughly, suppresses keratinocyte growth, promotes keratinocyte terminal differentiation and regulation of skin inflammation, and they are also to people's hair simultaneously
Capsular epithelium stem cell has the effect of protection.
The invention provides the preparation method of a kind of mescenchymal stem cell for acne treatment, it is characterised in that
Described method includes the genetic fragment building peroxisome proliferators activated receptor γ, recombinates on virus expression carrier,
Be packaged into after virus and cotransfection mescenchymal stem cell, be prepared as can high expressed PPAR α and the mescenchymal stem cell of PPAR γ, carry
High mescenchymal stem cell plays and more preferably suppresses scytitis reaction, prevention Hair follicle epithelial cells dyskeratosis and improve skin to repair
Multiple, can be used for treating acne.
Peroxisome proliferators activated receptor γ genetic fragment of the present invention is by round pcr amplification restructuring
On expression plasmid, it is connected on virus expression carrier through restriction enzyme site, gene recombined virus carrier transfection mescenchymal stem cell,
Obtain the mescenchymal stem cell for the treatment of acne.
Mescenchymal stem cell of the present invention includes deriving from discarded umbilical cord, umbilical blood, Placenta Hominis, autologous bone marrow or fat etc.,
Preferably, for source umbilical cord.Owing to source for mesenchymal stem cells is convenient, cell is cultivated and is obtained simply, can obtain from individuation
Can accomplish that individuation is applied.
Viral vector of the present invention, can cell inner stablity propagation and express genes of interest, predominantly slow virus,
Adenovirus, retrovirus etc., it is preferable that select slow virus expression system.
People PPAR α and PPAR gamma gene sequences are expanded from normal human peripheral blood's genomic DNA by PCR, will
After the target DNA fragment of PPAR α and PPAR γ is connected respectively to be built into recombiant plasmid on expression plasmid, through positive colony PCR
Identify and after order-checking qualification, the plasmid built can be saved in-80 DEG C of ultra cold storage freezers.Again by PPAR α and PPAR γ base
Because fragment is cloned on slow sick carrier, packaged restructuring PPAR α and PPAR γ slow virus, can be dry thin with cotransfection mesenchyme
Born of the same parents, are prepared as PPAR α and the mescenchymal stem cell of PPAR γ slow virus cotransfection.
Infect latter 72 hours fluorescence microscopy Microscopic observation green fluorescent proteins (Green fluorescent protein,
GFP) luminous situation, GFP develops the color more than more than 90%, shows to carry PPAR α and PPAR γ gene viruses transfection mesenchyme is done
Cell success, it is thus achieved that continuous expression PPAR α and the mescenchymal stem cell of PPAR γ.
Detected according to operation instructions by PCR kit for fluorescence quantitative, transfected PPAR α and PPAR γ viral vector
MSCs Secondary Culture is to after the 10th generation, and it expresses PPAR α and the PPAR γ gene MSCs more than at least 100 times higher than untransfected.
Described mescenchymal stem cell FCM analysis, its mark CD90, CD105, CD73 and CD44 express 90% with
On；
The described MSCs having transfected restructuring PPAR α and PPAR γ virus has following performance: suppression scytitis reacts, resistance
Stop Hair follicle epithelial cells dyskeratosis and improve skin repair；And without tumor.
Mescenchymal stem cell for acne treatment of the present invention, can cultivate with subculture in vitro separately, i.e. recombinate
The MSCs of PPAR α and PPAR γ slow-virus transfection passes through complete culture solution α-MEM (containing 2mM L-glutaminate, 5-100ng/ml
Basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% hyclone) subculture in vitro separately cultivation, work as cell
Cultivating by the 5-6 days, cell growth fusion reaches more than 90%, needs the pancreatin using mass volume ratio to be 0.25% (to contain
0.05%EDTA) had digestive transfer culture.
Take Secondary Culture to the cellular morphology observed under 3 generations and 10 generation MSCs inverted microscopes, the MSCs of untransfected and its
He transfects the MSCs of genes of interest and has similar cellular morphology.
The described mescenchymal stem cell process LAN PPAR α and PPAR γ for acne treatment, one-tenth tumor internal, external is real
Tumor phenomenon is not found into, it is ensured that its safety in testing.
The described mescenchymal stem cell for acne treatment is in rabbit ear acne model transplantations, and rabbit ear acne curative effect can
Significantly reducing reaching hair follicle keratotic plug, part keratotic plug leaves over the hair follicle of pitting after coming off, and pimple flattens minimizing；And keratinization
Degree substantially alleviates, substantially recovers normally to substantially reduce with high dermis cell infiltration.Restructuring PPAR α and PPAR γ slow virus
After the MSCs transplantation treatment rabbit ear acne model of cotransfection, it is suppressed that the TNF-α secretion of the rabbit ear acne modeled inflammation factor, and its
Grading of pathological histology analysis finds, keratinization degree, epidermis thickened degree, hair follicle interior angle compound matter are how many, hair follicle degrees of expansion
With cell infiltration degree all significantly better than independent MSCs transplanting.
The present invention also provides for the reagent kit product of a kind of mescenchymal stem cell for acne treatment prepared, and it is special
Levying and be, described test kit includes:
1) mescenchymal stem cell basal medium；
2) packaged PPAR α and PPAR γ high expressed virus；
3) enzyme of peptic cell；
4) cytokine and；
5) operation instructions；
Wherein, described operation instructions include above-mentioned method.
The present invention also provides for the application of the described mescenchymal stem cell for acne treatment, by ensure that external training
Support and obtain sufficient amount of mescenchymal stem cell, and there is stable expression PPAR α and PPAR γ albumen, play it to acne
Anti-inflammatory and the effect of cell proliferation etc., and mescenchymal stem cell repairs the function of the Skin Cell of damage in skin,
It is expected to be used for treating acne.
Accompanying drawing explanation
Fig. 1 is expressed as the fluidic cell figure of the MSCs that In vitro culture obtains
Fig. 2 is expressed as recombinate PPAR α and PPAR γ slow virus immunofluorescence figure after transfection MSCs
Fig. 3 is expressed as recombinate PPAR α and PPAR γ slow virus PPAR γ gene expression dose figure after transfection MSCs
Fig. 4 is expressed as rabbit ear acne improvement figure after rabbit ear acne model experiment in vivo
Fig. 5 is expressed as HE group improvement figure after rabbit ear acne model experiment in vivo
Fig. 6 is expressed as inflammatory factor TNF-α secretion level view after rabbit ear acne model experiment in vivo
Detailed description of the invention
The invention provides the preparation method of a kind of mescenchymal stem cell for acne treatment, it is characterised in that
Described method includes building peroxisome proliferator-activated receptor alpha and the genetic fragment of γ, restructuring to virus expression carrier
On, be packaged into after virus and cotransfection mescenchymal stem cell, be prepared as can the mesenchyme of high expressed PPAR α and PPAR γ dry thin
Born of the same parents, improve mescenchymal stem cell and play more preferably suppression scytitis reaction, stop Hair follicle epithelial cells dyskeratosis and improvement
Skin repair, can be used for treating acne.
Source for mesenchymal stem cells of the present invention is convenient, i.e. discards umbilical cord, umbilical blood, Placenta Hominis, autologous bone marrow or fat etc.,
Preferably, umbilical cord is derived from.Owing to source for mesenchymal stem cells is convenient, cell is cultivated and is obtained simply, can obtain from individuation
Can accomplish that individuation is applied.
Mescenchymal stem cell preparation method of the present invention, it may be assumed that take discarded 1-2cm umbilical cord through containing 2 times of penicillins and strepto-
Dual anti-1 × the PBS (pH=7.4) of element washs three times, uses eye scissors that umbilical cord scissors is broken to 1mm3Left and right, with containing 2 times of penicillins
Resuspended latter 1000 rpms of the 1 × PBS (pH=7.4) dual anti-with streptomycin is centrifuged, and trains with mescenchymal stem cell after its supernatant
(i.e. α-MEM contains 2mM L-glutaminate, 5-100ng/ml basic fibroblast growth factor, 5-100ng/ml epidermal growth to support base
The factor and 5-10% hyclone) resuspended, draw 10ml and join 75cm2In Tissue Culture Flask, in 37 DEG C, 5%CO2Cell is trained
Support in case and cultivate 48 hours.The mescenchymal stem cell culture medium changing fresh factor-containing is cultivated the most continuously, until cell from
Tissue is crawled out.When cell growth fraction is closeer, use 0.25% (mass volume ratio) pancreatin (containing mass volume ratio 0.02%
EDTA) digest, with the mescenchymal stem cell culture medium of 10ml factor-containing resuspended after transfer to new 75cm2Culture bottle
In, in 37 DEG C, 5%CO2Cell culture incubator is cultivated until growth fusion reaches about 90%, use 0.25% pancreatin (to contain
0.02% EDTA) carry out had digestive transfer culture cultivation, i.e. 1 bottle passes on 2 bottles, supplements the mescenchymal stem cell training of fresh factor-containing
Supporting base to continue to cultivate, when cultivating to 5 generation, mescenchymal stem cell uses 0.25% pancreatin (containing 0.02% EDTA) to digest, i.e. obtains
Obtain mescenchymal stem cell, can be resuspended to frozen in liquid nitrogen in the hyclone containing 10% dimethyl sulfoxide.
Human mesenchymal stem cell detects through Flow Cytometry, and MSCs mark CD90, CD73, CD105 and CD44 express
It is respectively 99.08%, 99.85%, 99.88% and 99.60%；CD34, CD14, CD45 and CD19 be expressed as 0.00%,
0.21%, 0.58% and 0.04%.
PPAR α of the present invention and PPAR γ genetic fragment are to be expanded by round pcr and recombinate on expression plasmid,
It is connected to recombinate on expression plasmid through restriction enzyme site, then recombinates on virus expression carrier, then pack 293T cell and prepare
Become the virus of high expressed genes of interest.
Viral vector of the present invention, can cell inner stablity propagation and express genes of interest, predominantly slow virus,
Adenovirus, retrovirus etc., it is preferable that select slow virus expression system, be commercially produced product, can purchase from commercial company
Buy.
People PPAR α and PPAR γ sequence expands by PCR from normal human peripheral blood's genomic DNA and obtains, use
PPAR α primer is, normal chain: 5 '-GCTCGAGATGGTGGACACGGAAAGCCCA-3 ', antisense strand: 5 '-
CCATATGGTCAGTACATGTCCCTGTAG-3’；PPAR γ primer is, normal chain: 5 '-GCTCGAGATGACCATGGTTGACAC-
3 ', antisense strand: 5 '-CCATATGGTGAACATGATCCATATGG-3 '.The fragment of PPAR α and PPAR γ mesh is respectively and XhoI/
The pET28a carrier segments of Nde I double digestion, under T4DNA ligase effect, in 4 DEG C, coupled reaction in 12 hours preparation clone
Connect liquid, carry out positive colony PCR qualification after converting competent escherichia coli cell DH5a and order-checking is identified；PCR primer gel
After electrophoresis detection and order-checking qualification meet PPAR α and PPAR γ size and sequence, the correct bacterium solution that checks order is transferred and contains in 10ml
In the LB fluid medium of ammonia benzyl antibiotic, 37 DEG C of overnight incubation, propose the examination of middle amount with sky, Beijing root biology without endotoxin plasmid is little
Agent box carries out plasmid extraction, extracts qualified recombiant plasmid and places-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer；Described PPAR α and
PPAR γ size is about 1407bp and 1434bp；
Respectively PPAR α, PPAR γ DNA fragmentation and GV358 carrier are carried out XhoI/Nde I double digestion, connect at T4 DNA
Under enzyme effect, by PPAR α and PPAR γ fragment respectively with enzyme action GV358 carrier in 37 DEG C, coupled reaction in 12 hours preparation clone
Connect liquid, carry out positive colony PCR qualification after converting competent escherichia coli cell DH5a and order-checking is identified；
Take cell state good, be in the 293T cell of exponential phase, after cell counting, according to the cultivation of each 10cm
Ware 6 × 106Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in overnight incubation；Remove before transfection in second day
Culture fluid, changes 5ml Opti-MEM culture fluid；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids add 1.5ml Opti-
In MEM, mixing gently, take 36 μ l lipofectamine 2000 and add in 1.5ml Opti-MEM, mix gently, room temperature is put
Put 5min；Mixing plasmid solution and lipofectamine 2000 diluent, put room temperature 20min；Mixed liquor be slowly added dropwise to
In the culture fluid of 293T cell, mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated；Discard containing transfection mixed after cultivating 6h
With the culture medium of thing, the PBS liquid adding 10ml cleans once, softly rocks culture dish to fall after the transfection mixture of washing remnants
Abandon；It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate 48-72h；
According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10min, removes
Cell debris；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Trim sample respectively, will be with viral supernatants
The ultracentrifugation pipe of liquid is put into one by one to supercentrifuge, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifugal
Temperature controls at 4 DEG C；After centrifugal end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus preservation liquid,
The most repeatedly blow and beat resuspended；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence spectrometry and drip
Degree, packaged PPAR α and PPAR γ slow virus according to 50 μ l 2E+8TU/ml subpackages, are stored in-80 DEG C of ultralow temperature ices respectively
Case；
Restructuring PPAR α and PPAR γ slow virus are joined the six of mescenchymal stem cell with 10 μ l 1E+7 TU/ml respectively
In well culture plate, system is 2ml, mixing, 37 DEG C, 5%CO2Incubator in hatch 8-12 hour after, change mesenchyme and do
(i.e. α-MEM contains 2mM L-glutaminate, 5-100ng/ml basic fibroblast growth factor, 5-100ng/ml table to cell culture medium
Skin growth factor and 5-10% hyclone)；When cell growth fusion reaches 90%, use 0.25% trypsinization, proceed to
25cm2Growing in culture bottle, corresponding 1 culture bottle in 1 hole, at 25cm2Culture bottle merges and when reaching 90%, continues had digestive transfer culture training
Support；Growth transfection is divided into 3 groups: untransfected matched group, blank slow-virus transfection group, cotransfection PPAR α and PPAR γ slow virus
Cotransfection group；
Infect latter 72 hours fluorescence microscopy Microscopic observation green fluorescent proteins (Green fluorescent protein,
GFP) luminous situation, GFP develops the color more than more than 90%, shows to carry PPAR α and PPAR γ gene viruses cotransfection mesenchyme
Stem cell success, it is thus achieved that continuous expression PPAR α and the mescenchymal stem cell of PPAR γ.
Detected according to operation instructions by PCR kit for fluorescence quantitative, use the PPAR α primer to be, positive-sense strand 5 '-
GCGAACGATTCGACTCAAGC-3 ', antisense strand: 5 '-CATCCCGACAGAAAGGCACT-3 '；With PPAR γ primer: positive-sense strand
5 '-TGGTGGGTTCTCTCTGAGTCTG-3 ', antisense strand: 5 '-ATCCACGGAGCTGATCCCAA-3 '；Cotransfection PPAR α
After the MSCs Secondary Culture of PPAR γ viral vector to the 10th generation, it expresses PPAR α and PPAR γ gene higher than untransfected
MSCs is 145.67 and 167.89 times.
Mescenchymal stem cell for acne treatment of the present invention, can cultivate with subculture in vitro separately, i.e. recombinate
The MSCs of PPAR α and PPAR γ slow virus cotransfection passes through complete culture solution α-MEM (containing 2mM L-glutaminate, 5-100ng/
Ml basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% hyclone subculture in vitro separately are cultivated, when carefully
Born of the same parents cultivate the 5-6 days, and cell growth fusion reaches more than 90%, need the pancreatin using mass volume ratio to be 0.25% (to contain
0.05%EDTA) had digestive transfer culture；
Take Secondary Culture to the cellular morphology observed under 3 generations and 10 generation MSCs inverted microscopes, the MSCs of untransfected and its
He transfects the MSCs of genes of interest and has similar cellular morphology, and cell becomes fusiformis to grow.
The mescenchymal stem cell of acne of the present invention treatment, in nude mouse after subcutaneous injection, does not finds into tumor
Phenomenon, it is ensured that its safety, and the breast cancer cell line MCF-7 positive control inoculated occurs in that into tumor.
The described mescenchymal stem cell for acne treatment is in rabbit ear acne model transplantations, and rabbit ear acne curative effect can
Significantly reducing reaching hair follicle keratotic plug, part keratotic plug leaves over the hair follicle of pitting after coming off, and pimple flattens minimizing；And keratinization
Degree substantially alleviates, substantially recovers normally to substantially reduce with high dermis cell infiltration.Restructuring PPAR α and PPAR γ slow virus
After the MSCs transplantation treatment rabbit ear acne model of cotransfection, it is suppressed that the TNF-α secretion of the rabbit ear acne modeled inflammation factor, and its
Grading of pathological histology analysis finds, keratinization degree, epidermis thickened degree, hair follicle interior angle compound matter are how many, hair follicle degrees of expansion
With cell infiltration degree all significantly better than independent MSCs transplanting.
The present invention also provides for the reagent kit product of a kind of mescenchymal stem cell for acne treatment prepared, and it is special
Levying and be, described test kit includes:
1) mescenchymal stem cell basal medium；
2) packaged PPAR α and PPAR γ high expressed virus；
3) enzyme of peptic cell；
4) cytokine and；
5) operation instructions；
Wherein, described operation instructions include above-mentioned method.
The present invention also provides for the application of the described mescenchymal stem cell for acne treatment, by ensure that external training
Support and obtain sufficient amount of mescenchymal stem cell, and there is stable expression PPAR α and PPAR γ albumen, play it to maintaining skin
Barrier permeability, suppresses keratinocyte growth, promotes keratinocyte terminal differentiation and the effect of regulation of skin inflammation, and
Mescenchymal stem cell repairs the function of the Skin Cell of damage in skin, is expected to be used for treating acne.
Hereinafter, the specific embodiment of the present invention is illustrated, but the technical scope of the present invention is not limited to these examples.
Embodiment 1 is recombinated PPAR α and the structure of PPAR γ slow virus carrier and packaging
People PPAR α and PPAR γ sequence are expanded by PCR from normal human peripheral blood's genomic DNA, uses
PPAR α primer is, normal chain: 5 '-GCTCGAGATGGTGGACACGGAAAGCCCA-3 ', antisense strand: 5 '-
CCATATGGTCAGTACATGTCCCTGTAG-3’；PPAR γ primer is, normal chain: 5 '-GCTCGAGATGACCATGGTTGACAC-
3 ', antisense strand: 5 '-CCATATGGTGAACATGATCCATATGG-3 '；Synthesize through Beijing Nuo Sai company.PPAR α and PPAR γ mesh
Fragment respectively with the pET28a carrier (American I nvitrogen company) of XhoI/Nde I (Japan TAKARA company) double digestion
Fragment, under T4DNA ligase (TAKARA company of Japan) effect, in 4 DEG C, coupled reaction in 12 hours preparation clone's connection liquid,
Positive colony PCR qualification and order-checking mirror is carried out after converting competent escherichia coli cell DH5a (American I nvitrogen company)
Fixed；Identify that PPAR α and PPAR γ genetic fragment size are about 1407bp and 1434bp, meet PPAR α and PPAR γ size and sequence
Row.The correct bacterium solution that checks order is transferred in 10ml contains the LB fluid medium of ammonia benzyl antibiotic, 37 DEG C of overnight incubation, use Beijing
It root biology carries middle amount test kit carry out plasmid extraction without endotoxin plasmid is little, extracts qualified recombiant plasmid and places-80 DEG C and surpass
The medium-term and long-term preservation of cryogenic refrigerator.
PPAR α and PPAR γ DNA fragmentation and GV358 carrier (Shanghai Ji is triumphant) are carried out XhoI/Nde I double digestion,
Under T4DNA ligase effect, by PPAR α and PPAR γ fragment respectively with enzyme action GV358 carrier in 37 DEG C, coupled reaction in 12 hours
Preparation clone connects liquid, carries out positive colony PCR qualification and order-checking is identified correct after converting competent escherichia coli cell DH5a.
Take cell state good, be in the 293T cell (American I nvitrogen company) of exponential phase, cell counting
After, according to the culture dish 6 × 10 of each 10cm6Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in cultivate
Overnight；Remove culture fluid before transfection in second day, change 5ml Opti-MEM culture fluid；Take 9 μ g packaging mixed liquor and 3 μ g slow virus tables
Reach plasmid to add in 1.5ml Opti-MEM, mix gently, take 36 μ l lipofectamine 2000 (American I nvitrogen
Company) add in 1.5ml Opti-MEM, mix gently, room temperature places 5min；Mixing plasmid solution and lipofectamine
2000 diluents, put room temperature 20min；Mixed liquor is slowly added dropwise to the culture fluid of 293T cell, and mixing, in 37 DEG C, 5%CO2
Cell culture incubator is cultivated；Discarding the culture medium containing transfection mixture after cultivating 6h, the PBS liquid adding 10ml cleans once,
Abandon after softly rocking the culture dish transfection mixture with washing remnants；It is slowly added to the cell culture medium containing 10% serum
20ml, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate 48-72h；
According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10min, removes
Cell debris；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Trim sample respectively, will be with viral supernatants
The ultracentrifugation pipe of liquid is put into one by one to supercentrifuge, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifugal
Temperature controls at 4 DEG C；After centrifugal end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus preservation liquid,
The most repeatedly blow and beat resuspended；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence spectrometry and drip
Degree, packaged PPAR α and PPAR γ slow virus, according to 50 μ l 2E+8 TU/ml subpackages, are stored in-80 DEG C of ultra cold storage freezers.
The preparation of embodiment 2 human mesenchymal stem cell
Take 1-2cm and discard umbilical cord through washing three times containing 1 × PBS (pH=7.4) that 2 times of penicillins and streptomycin are dual anti-, make
With eye scissors, umbilical cord scissors is broken to 1mm3Left and right, with the 1 × PBS (pH=7.4) dual anti-containing 2 times of penicillins and streptomycin resuspended after
1000 rpms are centrifuged, and after its supernatant, by mescenchymal stem cell culture medium, (i.e. α-MEM contains 2mM L-glutaminate, 5-
100ng/ml basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% hyclone) resuspended, draw
10ml joins 75cm2In Tissue Culture Flask, in 37 DEG C, 5%CO2Cell culture incubator is cultivated 48 hours.Change fresh containing cell
The mescenchymal stem cell culture medium of the factor is cultivated the most continuously, until cell is crawled out from tissue.
When cell growth fraction is closeer, use 0.25% (mass volume ratio) pancreatin (containing mass volume ratio 0.02%EDTA)
Digest, with the mescenchymal stem cell culture medium of 10ml factor-containing resuspended after transfer to new 75cm2In culture bottle, in
37 DEG C, 5%CO2Cell culture incubator is cultivated until growth fusion reaches about 90%, use 0.25% pancreatin (containing 0.02%
EDTA) carrying out had digestive transfer culture cultivation, i.e. 1 bottle passes on 2 bottles, and the mescenchymal stem cell culture medium supplementing fresh factor-containing continues
Cultivating, when cultivating to 5 generation, mescenchymal stem cell uses 0.25% pancreatin (containing 0.02% EDTA) to digest, i.e. obtains mesenchyme
Stem cell, can be resuspended to frozen in liquid nitrogen in the hyclone containing 10% dimethyl sulfoxide.
Take tongue and expect 1.0 × 10 after blue dyeing counting6MSC cell, divides three groups, and first group has been respectively added to 20 μ L
FITC-Mus antihuman CD 34 monoclonal antibody (BioLegend company of the U.S.)；(U.S. BioLegend is public for 20 μ L PE-mouse-anti people's CD90 monoclonal antibodies
Department)；Second component does not add 20 μ L FITC-mouse-anti people's CD14 monoclonal antibody (BioLegend company of the U.S.)；20 μ L PE-mouse-anti people
CD73 monoclonal antibody (BioLegend company of the U.S.)；3rd group is added the 20 μ L FITC-mouse-anti people CD45 monoclonal antibodies (U.S. respectively
BioLegend company)；20 μ L PE-Mus antihuman CD 44 monoclonal antibody (BioLegend company of the U.S.)；4th group is added 20 μ L respectively
FITC-Mus anti human CD 19 monoclonal antibody (BioLegend company of the U.S.)；20 μ L PE-mouse-anti people CD105 monoclonal antibody (U.S. BioLegend
Company)；5th group is Isotype control, has been respectively added to 20 μ L FITC labelling Mus IgM and 20 μ L PE labelling Mus IgM.It is placed in 4
DEG C refrigerator dyes 30 minutes, then washs three times with the 1 × phosphate buffer (PBS) of 1mL, finally uses the 1 × PBS of 0.5mL
Cell after resuspended washing, the cell after gained washing detects with FACS Calibur flow cytometer (U.S. company BD).
Fig. 1 result shows, human mesenchymal stem cell through Flow Cytometry detect, MSCs mark CD90, CD73,
CD105 and CD44 expresses and is respectively 99.08%, 99.85%, 99.88% and 99.60%；CD34, CD14, CD45 and CD19 table
Reach is 0.00%, 0.21%, 0.58% and 0.04%.
Embodiment 3 is recombinated PPAR α and the preparation of PPAR γ slow-virus transfection application on human skin mescenchymal stem cell
Restructuring PPAR α and each 10 μ l1E+7TU/ml of PPAR γ slow virus is joined 50 μ g/ml of human mesenchymal stem cell
Six well culture plates in, system is 2ml, mixing, 37 DEG C, 5% CO2Incubator in hatch 8-12 hour after, fill between replacing
(i.e. α-MEM contains 2mM L-glutaminate, 20ng/ml basic fibroblast growth factor, 20ng/ml epidermis to matter stem cell media
Somatomedin and 10% hyclone)；When cell growth fusion reaches 90%, use 0.25% trypsinization, proceed to 25cm2Training
Supporting in bottle and grow, corresponding 1 culture bottle in 1 hole, at 25cm2Culture bottle merges and when reaching 90%, continues had digestive transfer culture cultivation；Raw
Long transfection is divided into 3 groups: untransfected matched group, blank slow-virus transfection group, cotransfection PPAR α and PPAR γ slow-virus transfection
Group.
Fig. 2 is restructuring PPAR α and PPAR γ slow virus immunofluorescence picture after cotransfection MSCs.A figure is to fall at fluorescence
Putting the MSC cytological map observed under microscope light field, B figure is the MSC cytological map observed under fluorescence inverted microscope details in a play not acted out on stage, but told through dialogues, because of restructuring
PPAR α and PPAR γ slow virus carrier Carrying Green Fluorescent Protein (GFP), cotransfection restructuring PPAR α and PPAR γ slow virus
MSCs fluorescence inverted microscope under in green, show recombinate PPAR α and PPAR γ slow virus cotransfection MSCs successfully construct.
Fig. 3 is PPAR α and PPAR γ table in the 10th generation MSCs of cotransfection restructuring PPAR α and PPAR γ slow virus
Reach level.Detected according to operation instructions (American I nvitrogen company) by PCR kit for fluorescence quantitative, using PPAR
α primer is, positive-sense strand 5 '-GCGAACGATTCGACTCAAGC-3 ', antisense strand: 5 '-CATCCCGACAGAAAGGCACT-3 '；With
PPAR γ primer: positive-sense strand 5 '-TGGTGGGTTCTCTCTGAGTCTG-3 ', antisense strand: 5 '-ATCCACGGAGCTGATCCCAA-
3’；Synthesize through Beijing Nuo Sai company.Transfect the MSCs Secondary Culture of restructuring PPAR α and PPAR γ slow virus carrier to the 10th generation
After, it is expressed PPAR α and PPAR γ gene and is above 1st generation MCCs 145.67 of untransfected and 167.89 times.
The reagent kit product of the mescenchymal stem cell for acne treatment of embodiment 4 preparation
Test kit for the mescenchymal stem cell of acne treatment includes:
1) human mesenchymal stem cell basal medium, i.e. α MEM culture medium；
2) packaged PPAR α and PPAR γ high expressed virus；
3) enzyme of peptic cell, 0.25% (mass volume ratio) pancreatin (containing mass volume ratio 0.02%EDTA)；
4) cytokine and hyclone, i.e. 2mM L-glutaminate, 5-100ng/ml basic fibroblast growth factor,
20ng/ml epidermal growth factor and 10% hyclone；
5) operation instructions；
Wherein, described operation instructions include the method described in embodiment 1-3.
Embodiment 5 rabbit ear acne model skin experiment in vivo
Purchased from 18 experiment new zealand white rabbits of Military Medical Science Institute's animal center, male, cleaning grade, in experimental group
Every rabbit left in ear side auditive catheter opening part about 2cm × 2cm scope, is coated with 2% liquor carbonis detergens every day 1 time, is the most about coated with 1-
0.5ml, continuous 2 weeks to make animal Acne Model.Auris dextra is not smeared and is made normal control.Experimental group rabbit ear coating is after 1 week, ear
Pachyderma, hardening, pore is thicker greatly, it is seen that Whiteheads and black keratotic plug；Coating 2 weeks, it is seen that at coating, pore becomes more
Thick, see obvious black keratotic plug at its follicular orifice, in blackhead shape, the visible more red papules of follicular orifice protuberance and few simultaneously
Permitted pustule, whole rabbit ear rough surface, belonged to mild to moderate (I-II level) by acne classification.And normal rabbit auris dextra is thin, soft, on it
Follicular orifice marshalling, has no acne, pimple and pustule etc..
After modeling success, be randomly divided into A, B and C tri-groups, often group 6, A group be the restructuring PPAR α for preparing of embodiment 3 with
PPAR γ slow virus cotransfection MSCs transplantation group, take in nude mice back and abdominal part skin at two carry out skin corium subcutaneous injection 5 ×
105Cell, volume 50 μ l；The mesenchymal stem cell transplantation group of B group embodiment 2 preparation, scheme injection mesenchyme is done as A group
Cell；C group is physiology saline control group, same injection 50 μ l normal saline.
Perusal is done after being administered in three groups of rabbit modeling ear modeling positions and normal auris dextra by last, and takes pathological biopsy,
Fixing with 10% formalin, paraffin embedding, section, Hematoxylin-eosin (HE) dyes, and application buys what the green skies, Beijing produced
Hematoxylin-eosin staining kit is carried out according to operation instructions, and under light microscopic, tissues observed changes.
Before modeling, rabbit ear color is pink, poor, soft, blood capillary, follicular orifice marshalling clearly seen from it, makes
After mould the 4-5 days, there is black keratotic plug, darkly toner thorn-like in the external auditory meatus follicular orifice of rabbit, and follicular orifice swells in pimple shape, after by
Cumulative many.Fig. 4 A be physiology saline control group rabbit left ear acne HE groupization display external auditory meatus follicular orifice black acne shape keratotic plug and
Pimple significantly increases.Fig. 4 B, for after PPAR α and PPAR γ slow virus cotransfection MSCs transplants 2 weeks, transplants and physiology with MSCs
Saline control group compares, and hair follicle keratotic plug significantly reduces, and part keratotic plug leaves over the hair follicle of pitting after coming off, and pimple flattens and subtracts
Few；Show that PPAR α and PPAR γ slow virus cotransfection MSCs preferably improves rabbit ear acne after expressing PPAR α and PPAR γ
Outward appearance, reaches cosmetic result.
Normal auris dextra histological appearance: epidermal tissue's layer 2-4, follicular epithelium slightly thickens, and the accidental monokaryon of skin corium is scorching thin
Born of the same parents infiltrate.Fig. 5 A is physiology saline control group rabbit left ear hyperkeratosis, epidermis and follicular epithelium irregular thickening, wherein granular layer
Thicken substantially；Hair follicle is expanded, and is filled with keratinization material, and adjacent hair follicle mutually merges；High dermis cell infiltration.Fig. 5 B
For after PPAR α and PPAR γ slow virus cotransfection MSCs transplants 2 weeks, transplant with MSCs and saline control group compares, angle
Change degree substantially alleviates, and basic recovery is normal, a small amount of cell infiltration of high dermis；Show PPAR α and PPAR γ slow virus altogether
Transfection MSCs preferably inhibits follicular orifice epithelial cell keratinization and inflammatory reaction after expressing PPAR α and PPAR γ.
Embodiment 6 rabbit ear acne model grading of pathological histology is analyzed
In embodiment 5, rabbit ear acne model uses restructuring PPAR α and the PPAR γ slow virus cotransfection of embodiment 3 preparation
MSCs experiment in vivo, gathers rabbit peripheral blood by ear central artery after transplanting 2 weeks, obtains after being centrifuged 10 minutes by 1000rpm
Serum.Use TNF-α enzyme-linked immunosorbent assay (ELISA) test kit detection TNF-α secretion level (public purchased from U.S. R&D
Department), detect according to U.S. R&D company's T NF-α ELISA kit operation instruction.PPAR α and PPAR γ slow virus in Fig. 6
Cotransfection MSCs transplantation group TNF-α secretion is substantially less than MSCs and transplants combination and saline control group, and average is respectively
266.93pg, 449.36pg and 967.92pg；Show that PPAR α and PPAR γ slow virus cotransfection MSCs inhibits rabbit ear acne mould
The TNF-α secretion of type inflammatory factor.
Rabbit ear acne model transplantations grading of pathological histology analysis after 2 weeks in embodiment 5, it may be assumed that
Keratinization degree: identical with normal structure (-)；Keratinization material slightly thicken (+)；Moderate thickens (2+)；Work thickens
(3+)；
Epidermis thickened degree: identical with normal structure (-)；The cell number of plies 4～5 layers (+)；6-7 layer (2+)；8～9 layer (3
+)；
Hair follicle interior angle compound matter is how many: identical with normal structure (-)；Keratinization material increases, but the finest and close, and underfill hair
Capsule (+)；Keratinization material is fine and close, but underfill hair follicle, or keratinization material is the finest and close, but it is full of hair follicle (2+)；Keratinization material is fine and close,
It is full of hair follicle (3+)；
Hair follicle degrees of expansion: identical with normal structure (-)；Slight expansion (+)；Moderate distension (2+)；Highly expansion (3+)；
Cell infiltration degree: identical with normal structure (-)；Focal inflammation cellular infiltration, cell be dispersed in (+)；Focal
Cell infiltration, cell comparatively dense, or multiple infiltration stove (2+) occurs；Diffusivity cell infiltration (3+).Normal histology's table
Existing: epidermal tissue's layer 2-4, follicular epithelium slightly thickens, skin corium accidental monokaryon cell infiltration.
The pathological change situation of table 1 rabbit ear Acne Model
As it can be seen from table 1, hair follicle degrees of expansion how many at keratinization degree, epidermis thickened degree, keratinization material and scorching thin
Born of the same parents infiltrate five aspects, PPAR α/γ-MSCs transplantation group and MSCs transplantation group and are significantly better than saline control group, are respectively provided with notable
Sex differernce (p ＜ 0.05).And PPAR α/γ-MSCs transplantation group is also all decreased obviously at above-mentioned five aspects than MSC transplantation group,
And well improved, there is significant difference (p ＜ 0.05).This result shows that PPAR α/γ-MSCs transplants expression
After PPAR γ, it is suppressed that cellular keratinization and inflammatory reaction, thus improve the therapeutic effect of acne.
Claims (10)
1. the preparation method for the mescenchymal stem cell (MSCs) of acne treatment, it is characterised in that described method
Including building peroxisome proliferator-activated receptor alpha (PPAR α) and the genetic fragment of γ (PPAR γ), restructuring is to virus table
Reaching on carrier, be packaged into after virus and cotransfection mescenchymal stem cell, being prepared as can high expressed PPAR α and the mesenchyme of PPAR γ
Stem cell；Wherein, latter 72 hours fluorescence microscopy Microscopic observation green fluorescent protein GFP (Green fluorescent are infected
Protein) luminous situation, GFP develops the color more than more than 90%；
Detect according to the operation instructions of PCR kit for fluorescence quantitative, transfected PPAR α and PPAR γ viral vector
MSCs Secondary Culture is to after the 10th generation, and it expresses PPAR α and the PPAR γ gene MSCs more than at least 100 times higher than untransfected；
Described mescenchymal stem cell is through FCM analysis, and its mark CD90, CD105, CD73 and CD44 express more than 90%；
The described MSCs having transfected restructuring PPAR α and PPAR γ virus has following performance: suppression scytitis reacts, stops hair
Capsular epithelium cellular keratinization is abnormal and improves skin repair；And without tumor.
Method the most according to claim 1, it is characterised in that swashed by round pcr amplification peroxisome proliferation
Receptor alpha alive and γ cDNA sequence, recombinate on expression plasmid, be connected on virus expression carrier through restriction enzyme site, gene recombinaton
Viral vector transfection mescenchymal stem cell.
3. according to the method described in any one of claim 1-2, it is characterised in that described source for mesenchymal stem cells discards umbilicus
Band, umbilical blood, Placenta Hominis, autologous bone marrow or fat；It is preferably derived from umbilical cord.
4. the method for the mescenchymal stem cell of acne treatment, it is characterised in that by people PPAR α and PPAR γ sequence
Row are expanded from normal human peripheral blood's genomic DNA by PCR, and use PPAR α primer is, normal chain: 5 '-
GCTCGAGATGGTGGACACGGAAAGCCCA-3 ', antisense strand: 5 '-CCATATGGTCAGTACATGTCCCTGTAG-3 '；PPAR
γ primer is, normal chain: 5 '-GCTCGAGATGACCATGGTTGACAC-3 ', antisense strand: 5 '-
CCATATGGTGAACATGATCCATATGG-3’；By the fragment of PPAR α and PPAR γ mesh respectively with XhoI/Nde I double digestion
PET28a carrier segments, under T4DNA ligase effect, in 4 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, converts big
After enterobacteria competent cell DH5a, carry out positive colony PCR qualification and order-checking is identified；PCR primer detected through gel electrophoresis and survey
Sequence is identified after meeting PPAR α, PPAR γ size and sequence, and the correct bacterium solution that checks order transferred contains the LB of ammonia benzyl antibiotic in 10ml
In fluid medium, 37 DEG C of overnight incubation, carry middle amount test kit carry out plasmid extraction with without endotoxin plasmid is little, it is qualified to extract
Recombiant plasmid places-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer；Described PPAR α and PPAR γ genetic fragment size be 1407bp and
1434bp；
Respectively PPAR α, PPAR γ DNA fragmentation and GV358 carrier are carried out XhoI/Nde I double digestion, make at T4DNA ligase
Under with, by PPAR α, PPAR γ fragment and enzyme action GV358 carrier in 37 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, turn
After changing competent escherichia coli cell DH5a, carry out positive colony PCR qualification and order-checking is identified；
Take cell state good, be in the 293T cell of exponential phase, after cell counting, according to the culture dish 6 of each 10cm
×106Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in overnight incubation；Cultivation is removed before transfection in second day
Liquid, changes 5ml Opti-MEM culture fluid；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids add 1.5ml Opti-MEM
In, mixing gently, take 36 μ l lipofectamine2000 and add in 1.5ml Opti-MEM, mix gently, room temperature is placed
5min；Mixing plasmid solution and lipofectamine 2000 diluent, put room temperature 20min；Mixed liquor is slowly added dropwise to 293T
In the culture fluid of cell, mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated；Discard after cultivating 6h containing transfection mixture
Culture medium, the PBS liquid adding 10ml cleans once, softly rocks culture dish to abandon after the remaining transfection mixture of washing；
It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate 48-72h；
According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10min, removes cell
Fragment；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Trim sample respectively, by with vial supernatant
Ultracentrifugation pipe is put into one by one to supercentrifuge, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature
Control at 4 DEG C；After centrifugal end, supernatant discarded, remove and remain in the liquid on tube wall, add virus and preserve liquid, repeatedly blow and beat
Resuspended；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence spectrometry titre, packaged
PPAR α and PPAR γ slow virus, according to 50 μ l 2E+8TU/ml subpackages, are stored in-80 DEG C of ultra cold storage freezers；
Six holes that restructuring PPAR α and PPAR γ slow virus join mescenchymal stem cell with 10 μ l 1E+7TU/ml respectively are cultivated
Cotransfection in plate, system is 2ml, mixing, 37 DEG C, 5%CO2Incubator in hatch 8-12 hour after, change and cultivate completely
Liquid α-MEM；Described complete culture solution preferably containing α-MEM containing 2mM L-glutaminate, 5-100ng/ml basic fibroblast growth because of
Son, 5-100ng/ml epidermal growth factor and and 5-10% hyclone；When cell growth fusion reaches 90%, with 0.25%
Trypsinization, proceeds to 25cm2Growing in culture bottle, corresponding 1 culture bottle in 1 hole, at 25cm2When in culture bottle, fusion reaches 90%
Continue had digestive transfer culture to cultivate；Growth transfection is divided into 3 groups: untransfected matched group, blank slow-virus transfection group and transfected PPAR α and
PPAR γ slow virus cotransfection group；
Infect latter 72 hours fluorescence microscopy Microscopic observation green fluorescent proteins (Green fluorescent protein, GFP)
Luminous situation, GFP develops the color more than more than 90%；
Detected according to operation instructions by PCR kit for fluorescence quantitative, use the PPAR α primer to be, positive-sense strand 5 '-
GCGAACGATTCGACTCAAGC-3 ', antisense strand: 5 '-CATCCCGACAGAAAGGCACT-3 '；With PPAR γ primer: positive-sense strand
5 '-TGGTGGGTTCTCTCTGAGTCTG-3 ', antisense strand: 5 '-ATCCACGGAGCTGATCCCAA-3 '；Cotransfection PPAR α
After PPAR γ slow-virus transfection mescenchymal stem cell Secondary Culture to the 10th generation, it expresses PPAR α and PPAR γ gene is higher than
The MSCs of untransfected is respectively 145.67 times and 167.89 times.
5. the preparation method as described in any one of claim 1-4, it is characterised in that
The MSCs of restructuring PPAR α and PPAR γ slow-virus transfection is cultivated by complete culture solution α-MEM subculture in vitro separately, when cell is trained
Supporting the 5-6 days, cell growth fusion reaches more than 90%, needs the trypsinization using mass volume ratio to be 0.25% to pass
Generation；
Take Secondary Culture to the cellular morphology of observation, the MSCs of untransfected and other turns under 1 generation and 10 generation MSCs inverted microscopes
The MSCs of dye genes of interest has similar cellular morphology, and cell becomes fusiformis to grow.
6., according to the method described in any one of claim 1-5, it is characterised in that the preparation of described human mesenchymal stem cell, adopt
By following steps:
Take 1-2cm and discard umbilical cord through washing three times containing the dual anti-1 × PBS of 2 times of penicillins and streptomycin, use eye scissors by umbilical cord
Shred to 1mm3, it is centrifuged with resuspended latter 1000 rpms of the 1 × PBS dual anti-containing 2 times of penicillins and streptomycin, uses after its supernatant
Mescenchymal stem cell culture medium is resuspended, draws 10ml and joins 75cm2In Tissue Culture Flask, in 37 DEG C, 5%CO2Cell culture incubator
Middle cultivation 48 hours；The mescenchymal stem cell culture medium changing fresh factor-containing is cultivated the most continuously, until cell is from tissue
In crawl out；Described cell culture medium α-MEM preferably containing 2mM L-glutaminate, 5-100ng/ml basic fibroblast growth because of
Son, 5-100ng/ml epidermal growth factor and 5-10% hyclone；
When cell growth fraction is closeer, the pancreatin using mass volume ratio to be 0.25% digests, and uses 10ml factor-containing
Mescenchymal stem cell culture medium resuspended after transfer to new 75cm2In culture bottle, in 37 DEG C, 5%CO2Cell culture incubator is trained
Supporting until growth fusion reaches 90%, use 0.25% pancreatin to carry out had digestive transfer culture cultivation, i.e. 1 bottle passes on 2 bottles, supplements fresh containing
The mescenchymal stem cell culture medium of cytokine continues to cultivate, and when cultivating to 5 generation, mescenchymal stem cell uses 0.25% pancreatin
Digestion, i.e. obtains mescenchymal stem cell；Resuspended to frozen in liquid nitrogen in the hyclone containing 10% dimethyl sulfoxide；Described pancreas
Enzyme preferably contains the EDTA of mass volume ratio 0.02%；
Take tongue and expect 1.0 × 10 after blue dyeing counting6MSC cell, divides three groups, and first group has been respectively added to 20 μ L FITC-Mus
Antihuman CD 34 monoclonal antibody；20 μ L PE-mouse-anti people's CD90 monoclonal antibodies；Second component does not add 20 μ L FITC-mouse-anti people's CD14 monoclonal antibodies；20
μ L PE-mouse-anti people's CD73 monoclonal antibody；3rd group is added 20 μ L FITC-mouse-anti people's CD45 monoclonal antibodies respectively；20 μ L PE-mouse-anti people
CD44 monoclonal antibody；4th group is added 20 μ L FITC-Mus anti human CD 19 monoclonal antibodies respectively；20 μ L PE-mouse-anti people's CD105 monoclonal antibodies；5th
Group is Isotype control, has been respectively added to 20 μ L FITC labelling Mus IgM and 20 μ L PE labelling Mus IgM；It is placed in 4 DEG C of refrigerator dyeing
30 minutes, then wash three times, finally with after the resuspended washing of 1 × PBS of 0.5mL with the 1 × phosphate buffer (PBS) of 1mL
Cell, gained washing after cell FACS Calibur flow cytomery；
Result shows, human mesenchymal stem cell detects through Flow Cytometry, MSCs mark CD90, CD73, CD105 and CD44
Express and be respectively 99.08%, 99.85%, 99.88% and 99.60%；CD34, CD14, CD45 and CD19 be expressed as 0.00%,
0.21%, 0.58% and 0.04%.
7. according to the method described in any one of claim 1-5, it is characterised in that the mesenchyme of described treatment acne is dry thin
Born of the same parents, in rabbit ear acne model experiment in vivo, transplant with single MSCs and saline control group compare, it is suppressed that the rabbit ear
The TNF-α secretion of the acne modeled inflammation factor, average is respectively 266.93pg, 449.36pg and 967.92pg.
8. the test kit of the mescenchymal stem cell for acne treatment prepared as described in any one of claim 1-7
Product, it is characterised in that described test kit includes:
1) mescenchymal stem cell basal medium；
2) packaged PPAR α and PPAR γ high expressed virus；
3) enzyme of peptic cell；
4) cytokine and；
5) operation instructions；
Wherein, described operation instructions include described in any one of claim 1-7 method.
9. the preparation method as described in any one of claim 1-7, it is characterised in that fill between described treatment acne
Matter stem cell overexpressing cell somatomedin, vivo and vitro experiment shows all without tumor.
10. one kind transfected as described in any one of claim 1-9 restructuring PPAR α and PPAR γ virus mescenchymal stem cell and
Its application.
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| CN109628406A (en) * | 2019-01-03 | 2019-04-16 | 北京贝来生物科技有限公司 | A kind of mescenchymal stem cell and its preparation method and application for treating autoimmune disease |
| CN112704687A (en) * | 2019-10-08 | 2021-04-27 | 上海赛比曼生物科技有限公司 | Preparation containing mesenchymal stem cells and application thereof in psoriasis treatment |
| CN117959350A (en) * | 2024-03-29 | 2024-05-03 | 赛尔医学科技(山东)有限公司 | Osteoarthritis treatment composition based on composite stem cell SVF |
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| CN112704687A (en) * | 2019-10-08 | 2021-04-27 | 上海赛比曼生物科技有限公司 | Preparation containing mesenchymal stem cells and application thereof in psoriasis treatment |
| CN117959350A (en) * | 2024-03-29 | 2024-05-03 | 赛尔医学科技(山东)有限公司 | Osteoarthritis treatment composition based on composite stem cell SVF |
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