CN106011056B - Preparation method of mesenchymal stem cells for treating skin acne and product thereof - Google Patents
Preparation method of mesenchymal stem cells for treating skin acne and product thereof Download PDFInfo
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Abstract
The invention provides a preparation method of mesenchymal stem cells for treating skin acne, wherein the method comprises the steps of constructing gene segments of peroxisome proliferator-activated receptors alpha and gamma, recombining the gene segments onto a virus expression vector, co-transfecting the mesenchymal stem cells with the peroxisome proliferator-activated receptors alpha and gamma, highly expressing proteins of the peroxisome proliferator-activated receptors alpha and gamma, exerting the anti-skin inflammatory reaction of the two and inhibiting the abnormal keratinization of cells; meanwhile, the mesenchymal stem cells are used for self proliferation and differentiation and regulating immune function, participate in skin repair and anti-inflammatory reaction and have the function of treating skin acne; the mesenchymal stem cells provided by the invention have good treatment and beauty effects on skin acne.
Description
Technical Field
The invention relates to a method for preparing mesenchymal stem cells for treating skin acne and mesenchymal stem cells for treating skin acne obtained by the method, in particular to a method for constructing recombinant viral vectors of Peroxisome proliferator-activated receptors alpha and gamma (PPAR alpha and gamma), so that the mesenchymal stem cells can stably express the Peroxisome proliferator-activated receptors alpha and gamma, exert the effects of resisting skin inflammatory reaction and inhibiting abnormal cell keratinization and treating skin acne; meanwhile, the mesenchymal stem cells for treating skin acne keep good proliferation capacity, have the functions of proliferation differentiation and immunity regulation, participate in skin repair and anti-inflammatory reaction, have no tumor formation, and are expected to achieve better treatment and beautifying effects on skin acne.
Background
Acne is a common dermatosis, also called acne, whelk and pimple, is a common inflammatory dermatosis of hair follicle sebaceous gland, is mostly developed on the face, and seriously affects the beautiful appearance of young men and women. The blockage of the pilosebaceous opening is an important factor in pathogenesis. In the case of hair follicle occlusion, propionibacterium acnes multiply in number, causing inflammation, forming the most fundamental damaging inflammatory papules of acne. Inside the occluded pilosebaceous gland, a large amount of sebum and a large amount of pus cells destroy the pilosebaceous structure to form nodules, cysts and atheroma, and finally destroy the skin and even form scars. Sebaceous duct hyperkeratosis is also an important factor in the development of acne.
Continued follicular occlusion and propionibacterium acnes proliferation lead to inflammatory cell invasion, which secretes large amounts of inflammatory factors, causing keratinocyte dyskeratosis of the follicular infundibulum. Therefore, the compound preparation can inhibit inflammatory factors and improve follicular keratosis abnormality, and is a central link for improving acne lesions and pathological changes. Oral traditional Chinese medicine and local external application, wherein the local external application intervention acne model is taken as the main direction: for example, the external medicines such as compound reversal powder, compound Zijin cream, coptis oyster paste, fruit acid and the like have stronger keratosis resistance and can effectively improve acne skin damage. The oral herba Hedyotidis Diffusae and Saviae Miltiorrhizae radix mixture mainly comprises herba Hedyotidis Diffusae, Saviae Miltiorrhizae radix, Scutellariae radix, fructus forsythiae, herba Leonuri, and fructus crataegi, can regulate androgen level, promote wound repair, and has antibacterial and inflammatory factor release inhibiting effects.
The acne is mostly treated by the traditional Chinese medicine in clinic, and has a certain treatment effect. The invention uses the mesenchymal stem cells as the protein expression vector and the functions thereof, and has better effect on treating acne. Mesenchymal Stem Cells (MSCs) have been used in the field of clinical therapy, have achieved good therapeutic effects in the treatment of neurological and orthopedic diseases, have no limitations in the aspects of immunological rejection, ethics, tumorigenicity, and the like, and have good clinical application prospects. Therefore, the invention develops the anti-inflammatory and anti-proliferative biological functions of the peroxisome proliferator-activated receptor gamma without tumorigenesis by constructing a recombinant viral vector of the peroxisome proliferator-activated receptor gamma, transfecting mesenchymal stem cells. The expressed peroxisome proliferator activated receptor gamma and mesenchymal stem cells create a microenvironment beneficial to skin acne repair, and inhibit inflammatory cytokines secreted by inflammatory cells, so that the hyperkeratosis of epithelial cells at the mouth of a hair follicle is prevented, and the better effects on skin acne treatment and beauty are expected to be generated.
Disclosure of Invention
The applicant finds that the effect of PPAR alpha on epidermal barrier and epidermal proliferation and differentiation can increase the expression of epidermal acanthosphere/granular layer structural protein, increase apoptosis, reduce proliferation and accelerate the recovery of epidermal barrier function through intensive research. PPAR γ is mainly distributed in adipose tissue, but 3 subtypes of PPAR are also present in human keratinocytes as confirmed by semiquantitative fluorescent quantitative PCR detection, and PPAR γ expression increases with keratinocyte differentiation; and found that the PPAR γ content in the skin lesions of patients with psoriasis is reduced compared to the PPAR γ content in the skin lesions.
PPAR α, γ have been shown to have partial anti-inflammatory properties, and specific PPAR γ ligands down-regulate inflammatory cytokines in T cells, monocytes, mast cells, and negatively regulate macrophage activation. In many benign and malignant cells, PPAR γ ligands restrict the proliferation of these cells and promote their differentiation. Experiments prove that the PPAR alpha can increase the expression of differentiation related genes such as coat protein and can also down regulate inflammatory mediator to achieve the anti-inflammatory effect. Prolonged response to inflammatory mediator stimulation in PPAR α deficient mice; topical application of PPAR α agonists prevents the inflammatory response of the skin following UVB radiation. In the acne group skin lesions PPAR α, γ levels were reduced compared to the non-lesion area. Activation of PPAR α inhibits keratinocyte proliferation, resulting in a decrease in stratum corneum thickness.
Thus, PPAR α and PPAR γ play a very critical role in the stabilization of the skin's internal environment, regulating the differentiation, proliferation and inflammatory response of the skin.
The peroxisome proliferator-activated receptor gamma belongs to a nuclear receptor superfamily, has various biological functions, plays an important role in aspects of participating in carbohydrate metabolism, lipid metabolism, differentiation of fat cells, insulin resistance, inflammatory reaction, periodic regulation and control of ovarian tissues and the like, and can also inhibit the secretion of inflammatory factors of human umbilical vein endothelial cells, namely Tumor necrosis factor (Tumor necrosis factor-alpha, TNF-alpha), Interleukin 6(Interleukin 6, IL-6) and the like.
The invention utilizes the characteristics of proliferation differentiation and 'homing' of mesenchymal stem cells, co-transfects MSCs with the constructed recombinant viral vectors of PPAR alpha and PPAR gamma gene segments, can highly express PPAR alpha and PPAR gamma, plays the biological effects of anti-inflammation and anti-proliferation of PPAR alpha and PPAR gamma and the functions of mesenchymal stem cells, improves the local microenvironment of skin, inhibits inflammatory cytokines secreted by inflammatory cells, and thus prevents hyperkeratosis of epithelial cells at the mouth of hair follicle.
The inventor of the invention unexpectedly finds that the mesenchymal stem cells highly express PPAR alpha and PPAR gamma, can maintain the permeability of skin barriers, inhibit the growth of keratinocytes, promote the terminal differentiation of the keratinocytes and regulate skin inflammation, and simultaneously have the protection effect on human hair follicle epithelial stem cells.
The invention provides a preparation method of mesenchymal stem cells for treating skin acne, which is characterized by comprising the steps of constructing a gene segment of peroxisome proliferator-activated receptor gamma, recombining the gene segment onto a virus expression vector, packaging the virus into virus, and co-transfecting the mesenchymal stem cells to prepare the mesenchymal stem cells capable of highly expressing PPAR alpha and PPAR gamma, improving the mesenchymal stem cells to play a better role in inhibiting skin inflammatory reaction, preventing follicular epithelial cell dyskeratosis and improving skin repair, and can be used for treating skin acne.
The peroxisome proliferator activated receptor gamma gene fragment is amplified and recombined on an expression plasmid by a PCR (polymerase chain reaction) technology, is connected to a virus expression vector through an enzyme cutting site, and is transfected by the gene recombination virus vector to obtain the mesenchymal stem cell for treating skin acne.
The mesenchymal stem cells of the invention are derived from waste umbilical cord, umbilical cord blood, placenta, autologous bone marrow or fat, and the like, preferably, derived umbilical cord. Because the mesenchymal stem cells are convenient to source, the cell culture is simple to obtain, and the mesenchymal stem cells can be obtained from individuals and can be applied individually.
The virus vector can stably proliferate and express target genes in cells, mainly comprises lentiviruses, adenoviruses, retroviruses and the like, and preferably selects a lentivirus expression system.
The gene sequences of human PPAR alpha and PPAR gamma are amplified from the genome DNA of normal human peripheral blood by PCR, the target DNA fragments of PPAR alpha and PPAR gamma are respectively connected to an expression plasmid to construct a recombinant plasmid, and the constructed plasmid can be stored in an ultra-low temperature refrigerator at the temperature of-80 ℃ after positive clone PCR identification and sequencing identification. And cloning the PPAR alpha and PPAR gamma gene segments onto a lentivirus vector, packaging the recombinant PPAR alpha and PPAR gamma lentiviruses, and co-transfecting mesenchymal stem cells to prepare the PPAR alpha and PPAR gamma lentivirus co-transfected mesenchymal stem cells.
The luminescence condition of Green Fluorescent Protein (GFP) is observed under a fluorescence microscope 72 hours after infection, the GFP color is over 90 percent, which indicates that the virus carrying the PPAR alpha and the PPAR gamma genes successfully transfects the mesenchymal stem cells, and the mesenchymal stem cells continuously expressing the PPAR alpha and the PPAR gamma are obtained.
After the MSCs transfected with the PPAR alpha and PPAR gamma virus vectors are subcultured to the 10 th generation, the genes of the PPAR alpha and the PPAR gamma are expressed by more than 100 times higher than those of the untransfected MSCs by a fluorescent quantitative PCR kit according to the detection of an instruction.
The mesenchymal stem cell flow cytometry detection shows that the markers CD90, CD105, CD73 and CD44 are expressed by more than 90%;
the MSCs transfected with recombinant PPAR α and PPAR γ viruses had the following properties: inhibiting inflammatory reaction of skin, preventing keratinization abnormality of hair follicle epithelial cells, and improving skin repair; and has no tumor.
The mesenchymal stem cells for treating skin acne can be subjected to in vitro subculture, namely recombinant PPAR alpha and PPAR gamma lentivirus transfected MSCs are subjected to in vitro subculture by using complete culture solution alpha-MEM (containing 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% fetal bovine serum), when the cells are cultured for 5-6 days, the cells grow and fuse to more than 90%, and the cells need to be digested and subcultured by using pancreatin (containing 0.05% EDTA) with the mass-volume ratio of 0.25%.
Taking the cell morphology observed under an inverted microscope of the MSCs cultured to 3 generations and 10 generations, the untransfected MSCs have similar cell morphology with other MSCs transfected with target genes.
The mesenchymal stem cells for treating skin acne overexpress PPAR alpha and PPAR gamma, and the tumor formation phenomenon is not found in-vivo and in-vitro tumor formation experiments, so that the safety of the mesenchymal stem cells is ensured.
In the rabbit ear acne model transplantation of the mesenchymal stem cells for treating skin acne, the rabbit ear acne curative effect can reach that hair follicle angle plugs are obviously reduced, hair follicles with punctate depressions are left after partial angle plugs fall off, and the pimple is flattened and reduced; and significantly reduced degrees of keratinization, substantial return to normal, and significantly reduced infiltration of inflammatory cells in the superficial dermis. After the recombinant PPAR alpha and PPAR gamma lentivirus cotransfected MSCs are transplanted to treat a rabbit auricular acne model, the secretion of TNF-alpha of inflammatory factors of the rabbit auricular acne model is inhibited, and the pathological histological grading analysis of the recombinant PPAR alpha and PPAR gamma shows that the keratinization degree, the epidermal thickening degree, the quantity of follicular interior substances, the hair follicle expansion degree and the inflammatory cell infiltration degree are all obviously better than those of single MSCs.
The invention also provides a prepared kit product of the mesenchymal stem cells for treating skin acne, which is characterized by comprising the following components in parts by weight:
1) a mesenchymal stem cell basic culture medium;
2) packaged PPAR alpha and PPAR gamma high expression viruses;
3) an enzyme that digests the cells;
4) a cytokine and;
5) instructions for use;
wherein the instructions for use comprise the methods described above.
The invention also provides application of the mesenchymal stem cells for treating skin acne, which ensures that the mesenchymal stem cells with enough quantity are obtained by in vitro culture, has the functions of stably expressing PPAR alpha and PPAR gamma proteins, playing the roles of resisting inflammation, resisting cell proliferation and the like of skin acne, repairing damaged skin cells in the skin by the mesenchymal stem cells and is expected to be used for treating the skin acne.
Drawings
FIG. 1 shows a flow cytogram of MSCs obtained for in vitro culture
FIG. 2 is a photograph showing immunofluorescence of recombinant PPAR α and PPAR γ lentiviruses after transfection of MSCs
FIG. 3 is a graph showing the expression levels of PPAR γ genes of recombinant PPAR α and PPAR γ lentiviruses after transfection of MSCs
FIG. 4 shows the improvement of rabbit ear acne after rabbit ear acne model in vivo experiment
FIG. 5 shows HE histochemical patterns improved in vivo after rabbit auricular acne model
FIG. 6 is a graph showing the in vivo secretion level of inflammatory factor TNF-alpha after rabbit auricular acne model experiment
Detailed Description
The invention provides a preparation method of mesenchymal stem cells for treating skin acne, which is characterized by comprising the steps of constructing gene segments of peroxisome proliferator activated receptors alpha and gamma, recombining the gene segments onto a virus expression vector, packaging the virus into the virus, and co-transfecting the mesenchymal stem cells to prepare the mesenchymal stem cells capable of highly expressing PPAR alpha and PPAR gamma, improving the mesenchymal stem cells to better inhibit skin inflammatory reaction, preventing follicular epithelial cell dyskeratosis and improving skin repair, and can be used for treating skin acne.
The mesenchymal stem cells are convenient to source, namely waste umbilical cord, umbilical blood, placenta, autologous bone marrow or fat and the like, preferably, derived from umbilical cord. Because the mesenchymal stem cells are convenient to source, the cell culture is simple to obtain, and the mesenchymal stem cells can be obtained from individuals and can be applied individually.
The preparation method of the mesenchymal stem cells comprises the following steps: the discarded 1-2cm umbilical cords were washed three times with 1 XPBS (pH 7.4) containing 2-fold penicillin and streptomycin double antibody, and the umbilical cords were cut to 1mm using an ophthalmic scissors3About, after resuspension with 1 XPBS (pH 7.4) containing 2 times of penicillin and streptomycin double antibody, centrifugation is carried out at 1000rpm, supernatant is then resuspended with mesenchymal stem cell culture medium (i.e., alpha-MEM containing 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% fetal bovine serum), 10ml is aspirated and added to 75cm2In a cell culture flask, 5% CO at 37 ℃2The cells were cultured in a cell incubator for 48 hours. Replacement of fresh cell-containing factorThe seed mesenchymal stem cell culture medium is continuously cultured again until the cells climb out of the tissue. When the cells grow densely, the cells are digested by 0.25% (mass to volume) pancreatin (containing 0.02% EDTA), resuspended in 10ml of mesenchymal stem cell culture medium containing cell factors, and transferred to a new 75cm2In a culture flask, 5% CO at 37 ℃2Culturing in a cell culture box until the growth fusion reaches about 90%, performing digestion subculture by using 0.25% pancreatin (containing 0.02% EDTA), namely 1 bottle of passage 2 bottle, supplementing fresh mesenchymal stem cell culture medium containing cell factors, continuously culturing, and when culturing to the 5 th generation, digesting the mesenchymal stem cells by using 0.25% pancreatin (containing 0.02% EDTA), namely obtaining the mesenchymal stem cells, wherein the mesenchymal stem cells can be re-suspended into fetal calf serum containing 10% dimethyl sulfoxide and frozen in liquid nitrogen.
The expression of MSCs markers CD90, CD73, CD105 and CD44 is respectively 99.08%, 99.85%, 99.88% and 99.60% by detecting the human mesenchymal stem cells by a flow cytometry; CD34, CD14, CD45 and CD19 were expressed as 0.00%, 0.21%, 0.58% and 0.04%.
The PPAR alpha and PPAR gamma gene segments are amplified and recombined on expression plasmids by a PCR technology, are connected and recombined on the expression plasmids by enzyme cutting sites, are recombined on virus expression vectors, and are packaged with 293T cells to prepare the virus of high expression target genes.
The virus vector can stably proliferate and express target genes in cells, mainly comprises lentivirus, adenovirus, retrovirus and the like, preferably selects lentivirus expression systems, is a commercial product and can be purchased from commercial companies.
Human PPAR α and PPAR γ sequences were amplified from normal human peripheral blood genomic DNA by PCR using PPAR α primers as the positive strand: 5'-GCTCGAGATGGTGGACACGGAAAGCCCA-3', antisense strand: 5'-CCATATGGTCAGTACATGTCCCTGTAG-3', respectively; PPAR γ primers were, plus strand: 5'-GCTCGAGATGACCATGGTTGACAC-3', antisense strand: 5'-CCATATGGTGAACATGATCCATATGG-3' are provided. The PPAR alpha and PPAR gamma target fragments and a pET28a vector fragment subjected to XhoI/Nde I double enzyme digestion are respectively subjected to ligation reaction at 4 ℃ for 12 hours under the action of T4 DNA ligase to prepare a cloning connecting solution, and after an escherichia coli competent cell DH5a is transformed, positive cloning PCR identification and sequencing identification are carried out; after gel electrophoresis detection and sequencing identification of a PCR product conform to the sizes and sequences of PPAR alpha and PPAR gamma, transferring a correctly sequenced bacterium liquid into 10ml of LB liquid culture medium containing an aminobenzene antibiotic, culturing at 37 ℃ overnight, performing plasmid extraction by using a Beijing Tiangen biological endotoxin-free plasmid small-extraction medium-volume kit, and placing the qualified recombinant plasmid in an ultra-low temperature refrigerator at-80 ℃ for long-term storage; the sizes of the PPAR alpha and the PPAR gamma are about 1407bp and 1434 bp;
carrying out XhoI/Nde I double enzyme digestion on the PPAR alpha and PPAR gamma DNA fragments and the GV358 vector respectively, carrying out ligation reaction on the PPAR alpha and the PPAR gamma fragments and the GV358 vector at 37 ℃ for 12 hours under the action of T4 DNA ligase to prepare a clone ligation solution, transforming Escherichia coli competent cells DH5a, and then carrying out positive clone PCR identification and sequencing identification;
taking 293T cells in good cell state and logarithmic growth phase, counting the cells, and then performing 6X 10 cell counting on each 10cm culture dish6The number of cells was plated in a petri dish at 37 ℃ with 5% CO2Culturing in an incubator overnight; removing the culture solution before the next day of transfection, and replacing 5ml of Opti-MEM culture solution; adding 9 μ g of the packaged mixed solution and 3 μ g of the lentivirus expression plasmid into 1.5ml of Opti-MEM, gently mixing, adding 36 μ l of lipofectamine2000 into 1.5ml of Opti-MEM, gently mixing, and standing at room temperature for 5 min; mixing the plasmid solution and lipofectamine2000 diluent, and standing at room temperature for 20 min; the mixed solution is slowly dripped into the 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO2Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10ml of PBS (phosphate buffer solution) for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 10% serum-containing cell culture medium 20ml, and adding 5% CO at 37 deg.C2Continuously culturing for 48-72h in the incubator;
collecting 293T cell supernatant 48h after transfection according to cell state; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting ultracentrifuge tubes with virus supernatant into an ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, setting the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, centrifuging at high speed of 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, subpackaging the packaged PPAR alpha and PPAR gamma lentivirus according to 50 mu l of 2E +8TU/ml respectively, and storing in an ultra-low temperature refrigerator at-80 ℃;
adding the recombinant PPAR alpha and PPAR gamma lentivirus into a six-well culture plate of mesenchymal stem cells at a concentration of 10 mu l1E +7TU/ml respectively, wherein the system is 2ml, mixing uniformly, and performing 5% CO treatment at 37 DEG C2After 8-12 hours of incubation in the incubator of (1), the mesenchymal stem cell medium (i.e., alpha-MEM containing 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% fetal bovine serum) was replaced; when the cell growth and fusion reached 90%, the cells were digested with 0.25% trypsin and transferred to 25cm2Growth in flasks, 1 well for 1 flask at 25cm2Continuing digestion subculture when the fusion in the culture flask reaches 90%; the growth transfections were divided into 3 groups: untransfected control group, blank lentivirus transfection group, co-transfected PPAR alpha and PPAR gamma lentivirus co-transfection group;
the luminescence condition of Green Fluorescent Protein (GFP) is observed under a fluorescence microscope 72 hours after infection, the GFP color is over 90 percent, which indicates that the virus carrying the PPAR alpha and the PPAR gamma genes co-transfects the mesenchymal stem cells successfully, and the mesenchymal stem cells which continuously express the PPAR alpha and the PPAR gamma are obtained.
Detection was performed by fluorescent quantitative PCR kit according to the instructions using PPAR α primers, sense strand 5'-GCGAACGATTCGACTCAAGC-3', antisense strand: 5'-CATCCCGACAGAAAGGCACT-3', respectively; and PPAR γ primers: sense strand 5'-TGGTGGGTTCTCTCTGAGTCTG-3', antisense strand: 5'-ATCCACGGAGCTGATCCCAA-3', respectively; after subculturing the MSCs co-transfected with the PPARalpha and PPARgamma virus vectors to 10 th generation, the genes of the PPARalpha and the PPARgamma are expressed by 145.67 and 167.89 times higher than those of the untransfected MSCs.
The mesenchymal stem cells for treating skin acne can be subcultured in vitro, namely recombinant PPAR alpha and PPAR gamma lentivirus cotransfected MSCs are subcultured in vitro by complete culture solution alpha-MEM (containing 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% fetal calf serum), when the cells are cultured to the 5 th-6 th day, the cells grow and fuse to more than 90%, and pancreatin (containing 0.05% EDTA) with the mass-volume ratio of 0.25% is required to be used for digestion and subculture;
and taking the cell morphology observed under an inverted microscope of the MSCs cultured to 3 generations and 10 generations, wherein the untransfected MSCs have similar cell morphology with the MSCs of other transfected target genes, and the cells grow in a fusiform manner.
After the mesenchymal stem cells for treating skin acne are injected subcutaneously in a nude mouse body, no tumorigenesis phenomenon is found, the safety of the nude mouse is ensured, and the inoculated breast cancer cell line MCF-7 positive control has tumorigenesis.
In the rabbit ear acne model transplantation of the mesenchymal stem cells for treating skin acne, the rabbit ear acne curative effect can reach that hair follicle angle plugs are obviously reduced, hair follicles with punctate depressions are left after partial angle plugs fall off, and the pimple is flattened and reduced; and significantly reduced degrees of keratinization, substantial return to normal, and significantly reduced infiltration of inflammatory cells in the superficial dermis. After the recombinant PPAR alpha and PPAR gamma lentivirus cotransfected MSCs are transplanted to treat a rabbit auricular acne model, the secretion of TNF-alpha of inflammatory factors of the rabbit auricular acne model is inhibited, and the pathological histological grading analysis of the recombinant PPAR alpha and PPAR gamma shows that the keratinization degree, the epidermal thickening degree, the quantity of follicular interior substances, the hair follicle expansion degree and the inflammatory cell infiltration degree are all obviously better than those of single MSCs.
The invention also provides a prepared kit product of the mesenchymal stem cells for treating skin acne, which is characterized by comprising the following components in parts by weight:
1) a mesenchymal stem cell basic culture medium;
2) packaged PPAR alpha and PPAR gamma high expression viruses;
3) an enzyme that digests the cells;
4) a cytokine and;
5) instructions for use;
wherein the instructions for use comprise the methods described above.
The invention also provides application of the mesenchymal stem cells for treating skin acne, which can be expected to be used for treating skin acne by ensuring that the mesenchymal stem cells with enough quantity are obtained by in vitro culture, stably expressing PPAR alpha and PPAR gamma proteins, exerting the functions of maintaining the permeability of skin barriers, inhibiting the growth of keratinocytes, promoting the terminal differentiation of keratinocytes and regulating skin inflammation and repairing damaged skin cells in the skin by the mesenchymal stem cells.
Specific examples of the present invention will be described below, but the technical scope of the present invention is not limited to these examples.
Example 1 construction and packaging of recombinant PPAR α and PPAR γ Lentiviral vectors
Human PPAR α and PPAR γ sequences were amplified by PCR from normal human peripheral blood genomic DNA using PPAR α primers as the positive strand: 5'-GCTCGAGATGGTGGACACGGAAAGCCCA-3', antisense strand: 5'-CCATATGGTCAGTACATGTCCCTGTAG-3', respectively; PPAR γ primers were, plus strand: 5'-GCTCGAGATGACCATGGTTGACAC-3', antisense strand: 5'-CCATATGGTGAACATGATCCATATGG-3', respectively; synthesized by Beijing Nosai company. The objective fragments of PPAR α and PPAR γ were separately digested with XhoI/Nde I (TAKARA, Japan) and separately digested with pET28a vector (Invitrogen, USA), ligated at 4 ℃ for 12 hours with T4 DNA ligase (TAKARA, Japan) to prepare a clone-ligated solution, and E.coli competent cell DH5a (Invitrogen, USA) was transformed and then PCR-verified and sequencing-verified for positive clones; the sizes of the PPAR alpha and PPAR gamma gene fragments are identified to be about 1407bp and 1434bp, which are in accordance with the sizes and sequences of the PPAR alpha and PPAR gamma. Transferring the correctly sequenced bacterium liquid into 10ml LB liquid culture medium containing aminobenzene antibiotics, culturing overnight at 37 ℃, extracting plasmids by using a Beijing Tiangen biological endotoxin-free plasmid miniprep medium kit, and placing the qualified recombinant plasmids in an ultra-low temperature refrigerator at-80 ℃ for long-term storage.
Carrying out XhoI/Nde I double digestion on the PPAR alpha and PPAR gamma DNA fragments and a GV358 vector (Shanghai Jikai), respectively carrying out ligation reaction on the PPAR alpha and PPAR gamma fragments and the GV358 vector at 37 ℃ for 12 hours under the action of T4 DNA ligase to prepare clone ligation liquid, transforming Escherichia coli competent cells DH5a, and then carrying out positive clone PCR identification and sequencing identification to ensure that the DNA fragments are correct.
293T cells (Invitrogen, USA) in a good cell state and a logarithmic growth phase were collected, and after counting, the cells were counted at 6X 10 in 10cm dishes6The number of cells was plated in a petri dish at 37 ℃ with 5% CO2Culturing in an incubator overnight; removing the culture solution before the next day of transfection, and replacing 5ml of Opti-MEM culture solution; adding 9 μ g of the packaged mixture and 3 μ g of the lentiviral expression plasmid into 1.5ml of Opti-MEM, gently mixing, adding 36 μ l of lipofectamine2000 (Invitrogen, USA) into 1.5ml of Opti-MEM, gently mixing, and standing at room temperature for 5 min; mixing the plasmid solution and lipofectamine2000 diluent, and standing at room temperature for 20 min; the mixed solution is slowly dripped into the 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO2Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10ml of PBS (phosphate buffer solution) for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 10% serum-containing cell culture medium 20ml, and adding 5% CO at 37 deg.C2Continuously culturing for 48-72h in the incubator;
collecting 293T cell supernatant 48h after transfection according to cell state; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting ultracentrifuge tubes with virus supernatant into an ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, setting the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, centrifuging at 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, subpackaging the packaged PPAR alpha and PPAR gamma lentivirus according to 50 mu l of 2E +8TU/ml, and storing in an ultra-low temperature refrigerator at-80 ℃.
Example 2 preparation of human mesenchymal Stem cells
Collecting 1-2cm waste umbilical cord, and treating with 1 XPBS containing 2 times of penicillin and streptomycin double antibody(pH 7.4) three washes, and the umbilical cord was minced to 1mm using an ophthalmic scissors3About, after resuspension with 1 XPBS (pH 7.4) containing 2 times of penicillin and streptomycin double antibody, centrifugation is carried out at 1000rpm, supernatant is then resuspended with mesenchyme in cell culture medium (i.e., alpha-MEM containing 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 5-100ng/ml epidermal growth factor and 5-10% fetal calf serum), 10ml is aspirated and added to 75cm2In a cell culture flask, 5% CO at 37 ℃2The cells were cultured in a cell incubator for 48 hours. And replacing the fresh mesenchymal stem cell culture medium containing the cell factors and continuously culturing until the cells climb out of the tissues.
When the cells grow densely, the cells are digested by 0.25% (mass to volume) pancreatin (containing 0.02% EDTA), resuspended in 10ml of mesenchymal stem cell culture medium containing cell factors, and transferred to a new 75cm2In a culture flask, 5% CO at 37 ℃2Culturing in a cell culture box until the growth fusion reaches about 90%, performing digestion subculture by using 0.25% pancreatin (containing 0.02% EDTA), namely 1 bottle of passage 2 bottle, supplementing fresh mesenchymal stem cell culture medium containing cell factors, continuously culturing, and when culturing to the 5 th generation, digesting the mesenchymal stem cells by using 0.25% pancreatin (containing 0.02% EDTA), namely obtaining the mesenchymal stem cells, wherein the mesenchymal stem cells can be re-suspended into fetal calf serum containing 10% dimethyl sulfoxide and frozen in liquid nitrogen.
Counting 1.0X 10 trypan blue staining6MSC cells, in three groups, the first group was added with 20 μ L FITC-mouse anti-human CD34 mab (BioLegend, usa); 20 μ L of PE-mouse anti-human CD90 mab (BioLegend, USA); the second group was supplemented with 20. mu.L of FITC-mouse anti-human CD14 monoclonal antibody (BioLegend, USA); 20 μ L of PE-mouse anti-human CD73 mab (BioLegend, USA); the third group was supplemented with 20. mu.L of FITC-mouse anti-human CD45 monoclonal antibody (BioLegend, USA); 20 μ L of PE-mouse anti-human CD44 mab (BioLegend, USA); the fourth group was supplemented with 20. mu.L of FITC-mouse anti-human CD19 monoclonal antibody (BioLegend, USA); 20 μ L PE-murine anti-human CD105 mAb (BioLegend, USA); the fifth group was isotype control, added to 20. mu.L FITC-labeled murine IgM and 20. mu.L PE-labeled murine IgM, respectively. Placing in a refrigerator at 4 ℃ for dyeing for 30 minutes, and then using 1mLWashed three times with 1 XPhosphate buffered saline (PBS), and finally resuspended washed cells with 0.5mL of 1 XPBS, and the resulting washed cells were detected using a FACS Calibur flow cytometer (BD Co., USA).
The results in fig. 1 show that the expression of MSCs markers CD90, CD73, CD105, and CD44 were 99.08%, 99.85%, 99.88%, and 99.60%, respectively, when human mesenchymal stem cells were detected by flow cytometry; CD34, CD14, CD45 and CD19 were expressed as 0.00%, 0.21%, 0.58% and 0.04%.
Example 3 preparation of recombinant PPAR α and PPAR γ lentiviruses for transfection of human mesenchymal stem cells
Adding 10 mul of 1E +7TU/ml of recombinant PPAR alpha and PPAR gamma lentivirus into a six-hole culture plate of 50 mul g/ml of human mesenchymal stem cells, wherein the system is 2ml, mixing uniformly, and performing reaction at 37 ℃ and 5% CO2After 8-12 hours of incubation in the incubator of (a) mesenchymal stem cell culture medium (i.e., alpha-MEM containing 2mM L-glutamine, 20ng/ml basic fibroblast growth factor, 20ng/ml epidermal growth factor and 10% fetal bovine serum) was replaced; when the cell growth and fusion reached 90%, the cells were digested with 0.25% trypsin and transferred to 25cm2Growth in flasks, 1 well for 1 flask at 25cm2Continuing digestion subculture when the fusion in the culture flask reaches 90%; the growth transfections were divided into 3 groups: untransfected control group, blank lentivirus transfected group, co-transfected with PPAR α and PPAR γ lentivirus transfected group.
FIG. 2 is a photograph of immunofluorescence of recombinant PPAR α and PPAR γ lentiviruses after co-transfection of MSCs. The A picture is the MSC cell picture observed under a fluorescence inversion microscope bright field, the B picture is the MSC cell picture observed under a fluorescence inversion microscope dark field, and due to the fact that the recombinant PPAR alpha and PPAR gamma lentivirus vectors carry Green Fluorescent Protein (GFP), MSCs co-transfected with the recombinant PPAR alpha and PPAR gamma lentiviruses are green under the fluorescence inversion microscope, the construction success of the MSCs co-transfected with the recombinant PPAR alpha and PPAR gamma lentiviruses is shown.
FIG. 3 shows the expression levels of PPAR α and PPAR γ in 10 th generation MSCs co-transfected with recombinant PPAR α and PPAR γ lentiviruses. The PCR primer was used as a PCR primer, sense strand 5'-GCGAACGATTCGACTCAAGC-3', antisense strand: 5'-CATCCCGACAGAAAGGCACT-3', respectively; and PPAR γ primers: sense strand 5'-TGGTGGGTTCTCTCTGAGTCTG-3', antisense strand: 5'-ATCCACGGAGCTGATCCCAA-3', respectively; synthesized by Beijing Nosai company. After the MSCs transfected with the recombinant PPAR alpha and PPAR gamma lentiviral vectors are subcultured to the 10 th generation, the genes expressing PPAR alpha and PPAR gamma are higher than those of the untransfected 1 st generation MCCs 145.67 and 167.89 times.
Example 4 kit product of mesenchymal stem cells for skin acne treatment prepared
The kit of mesenchymal stem cells for skin acne treatment comprises:
1) human mesenchymal stem cell basal medium, namely alpha MEM medium;
2) packaged PPAR alpha and PPAR gamma high expression viruses;
3) enzyme for digesting cells, 0.25% (mass to volume) pancreatin (containing 0.02% by mass to volume of EDTA);
4) cytokines and fetal calf serum, i.e., 2mM L-glutamine, 5-100ng/ml basic fibroblast growth factor, 20ng/ml epidermal growth factor and 10% fetal calf serum;
5) instructions for use;
wherein the instructions for use comprise the methods described in examples 1-3.
Example 5 rabbit ear acne model in vivo skin experiment
In 18 experimental New Zealand white rabbits, male, clean grade purchased from the animal center of the military medical academy of sciences, each rabbit was treated with a 2% coal tar solution 1 time per day, 1-0.Sml each time, in a range of about 2cm by 2cm at the opening of the canal of the inner side of the left ear of each rabbit, and the animal acne model was prepared for 2 consecutive weeks. The right ear was not smeared as a normal control. After the rabbit ears are coated with the medicine for 1 week, the skin of the ears thickens and becomes hard, pores become large, and white comedones and black keratotic plugs can be seen; after 2 weeks of application, the pores at the application part become bigger, the hair follicle mouth has obvious black angle plug, the hair follicle mouth is in a black head powder thorn shape, meanwhile, the hair follicle mouth is raised to have more red papules and a few pustules, the whole rabbit ear has rough surface, and the degree is from light to medium (grade I-II) according to the classification of acne. While the right ear of a normal rabbit is thin and soft, the hair follicle mouths on the normal rabbit are regularly arranged, and no acne, pimple, pustule and the like are seen.
After successful modeling, the model was randomly divided into A, B groups and C groups, each group containing 6 individuals, and group A was a group of transplanted recombinant PPAR α and PPAR γ lentivirus-co-transfected MSCs prepared in example 3, and two skin sites were removed from the back and abdomen of nude mice and injected subcutaneously into the dermis at 5X 105Cells, volume 50 μ l; b, injecting the mesenchymal stem cells into the mesenchymal stem cell transplantation group prepared in the example 2 by the same scheme as the A group; group C was a normal saline control group, and 50. mu.l of normal saline was also injected.
After the last administration, the modeling parts and normal right ears of three groups of rabbit modeling ears are observed by naked eyes, pathological biopsy is taken, fixed by 10% formalin, embedded by paraffin, sliced, stained by hematoxylin-eosin (HE), and the hematoxylin-eosin staining kit produced in Beijing Biyun is applied according to the instruction for use, and histological change is observed under the light mirror.
The rabbit ears before molding are pink, thin and soft, clear capillary vessels can be seen on the rabbit ears, hair follicle mouths are arranged in order, black angle plugs appear at hair follicle mouths of external auditory canals of the rabbits 4-5 days after molding, the hair follicle mouths are in a black powder thorn shape, the hair follicle mouths are raised in a papule shape, and then the hair follicle mouths are gradually increased. Fig. 4A is a left ear acne HE histochemistry of saline control rabbits showing a significant increase in comedonelike black plugs and papules in the follicular orifice of the external auditory canal. FIG. 4B is a graph showing that after 2 weeks of transplantation of MSCs co-transfected with PPAR α and PPAR γ lentiviruses, follicular keratocytes were significantly reduced, partial keratoses had fallen off leaving punctate depressed follicles, and papules were flattened, as compared to the MSCs transplanted and saline control group; the PPARalpha and PPARgamma lentivirus cotransfection MSCs express the PPARalpha and the PPARgamma, so that the appearance of rabbit auricular acne is better improved, and the beautifying effect is achieved.
Normal right ear histological appearance: epidermal tissue is 2-4 layers, hair follicle epithelium is slightly thickened, and mononuclear cell infiltration is occasionally seen in the dermal layer. FIG. 5A is hyperkeratosis of the left ear, irregular thickening of the epidermis and hair follicle epithelium in rabbits of the saline control group, in which thickening of the stratum granulosum is evident; hair follicle expansion, in which keratinizing substances are filled, and adjacent hair follicles are fused with each other; dermal superficial inflammatory cell infiltration. FIG. 5B shows that after 2 weeks of transplantation of PPAR α and PPAR γ lentivirus-co-transfected MSCs, the degree of keratinization was significantly reduced, and the normal skin was substantially restored, with a small amount of inflammatory cell infiltration in the superficial dermis, compared to the MSCs transplanted and normal saline control group; the PPARalpha and PPARgamma lentivirus cotransfected MSCs express the PPARalpha and the PPARgamma to better inhibit the keratinization and inflammatory reaction of the epithelial cells of the hair follicle opening.
Example 6 pathologic histology grading analysis of rabbit auricular acne model
In the rabbit auricular acne model in example 5, in vivo experiments using recombinant PPAR α and PPAR γ lentiviruses prepared in example 3 to co-transfect MSCs, rabbit peripheral blood was collected through the central auricular artery 2 weeks after transplantation, and serum was obtained after centrifugation at 1000rpm for 10 minutes. TNF-alpha secretion levels (purchased from R & D, USA) were measured using a TNF-alpha enzyme-linked immunosorbent assay (ELISA) kit, and were measured according to the instructions for use of the TNF-alpha ELISA kit from R & D, USA. In the graph of fig. 6, the TNF-alpha secretion of the PPAR alpha and PPAR gamma lentivirus co-transfected MSCs transplantation group is significantly lower than that of the MSCs transplantation combination and the physiological saline control group, and the mean values are 266.93pg, 449.36pg and 967.92pg respectively; the PPARalpha and PPARgamma lentivirus cotransfected MSCs are shown to inhibit the TNF-alpha secretion of inflammatory factors of rabbit auricular acne models.
Histopathological grading analysis of rabbit ear acne model in example 5 after 2 weeks of transplantation, namely:
degree of keratinization: same as normal tissue (-); mild thickening (+); moderate thickening (2 +); most notably thickening (3 +);
degree of epidermal thickening: same as normal tissue (-); the number of cell layers is 4-5 (+); 6-7 layers (2 +); 8-9 layers (3 +);
amount of keratinized substance in hair follicle: same as normal tissue (-); keratinizing material is increased but not dense and underfilled in the hair follicle (+); keratinized material dense but not filling the follicle, or not dense but filling the follicle (2 +); keratinized material dense, filling the hair follicle (3 +);
degree of expansion of hair follicle: same as normal tissue (-); mild dilation (+); moderate dilation (2 +); hyperextension (3 +);
degree of inflammatory cell infiltration: same as normal tissue (-); infiltration of focal inflammatory cells, scattered cells (+); infiltration of focal inflammatory cells, denser cells, or multiple infiltrates (2 +); diffuse inflammatory cell infiltration (3 +). Normal histological appearance: epidermal tissue is 2-4 layers, hair follicle epithelium is slightly thickened, and mononuclear cell infiltration is occasionally seen in the dermal layer.
TABLE 1 pathological changes in the rabbit ear acne model
As can be seen from Table 1, the PPAR α/γ -MSCs transplanted group and the MSCs transplanted group are significantly better than the normal saline control group in the five aspects of keratinization degree, epidermal thickening degree, keratinized substance amount, hair follicle expansion degree and inflammatory cell infiltration (p is less than 0.05). And compared with the MSC transplantation group, the PPAR alpha/gamma-MSCs transplantation group is also obviously reduced in the five aspects, is well improved and has significant difference (p is less than 0.05). This result indicates that the PPAR α/γ -MSCs, after expressing PPAR γ by transplantation, inhibit cell keratinization and inflammatory reaction, thereby improving the therapeutic effect on acne.
Claims (6)
1. A preparation method of Mesenchymal Stem Cells (MSCs) for treating skin acne is characterized by comprising the steps of constructing gene segments of peroxisome proliferator-activated receptors alpha (PPAR alpha) and gamma (PPAR gamma), recombining the gene segments onto a virus expression vector, packaging into viruses, co-transfecting the mesenchymal stem cells, and preparing the mesenchymal stem cells capable of highly expressing the PPAR alpha and the PPAR gamma; wherein, the luminescent condition of green fluorescent protein GFP (green fluorescent protein) is observed under a fluorescent microscope 72 hours after infection, and the GFP color development is more than 90 percent;
detecting according to the instruction of the fluorescent quantitative PCR kit, after the MSCs transfected with the PPAR alpha and PPAR gamma virus vectors are subcultured to the 10 th generation, the genes expressing the PPAR alpha and the PPAR gamma are more than 100 times higher than those of the untransfected MSCs; the mesenchymal stem cells are detected by flow cytometry, and the expression of markers CD90, CD105, CD73 and CD44 is more than 90%; the MSCs transfected with recombinant PPAR α and PPAR γ viruses had the following properties: inhibiting inflammatory reaction of skin, preventing keratinization abnormality of hair follicle epithelial cells, and improving skin repair;
the mesenchymal stem cells for treating skin acne overexpress cell growth factors, and in vivo and in vitro experiments show that the mesenchymal stem cells have no tumor.
2. The method of claim 1, wherein the peroxisome proliferator-activated receptor α (PPAR α) and γ (PPAR γ) gene fragments are amplified from a normal human genome by Polymerase Chain Reaction (PCR) to obtain corresponding cDNA sequences, which are recombined onto an expression plasmid, ligated to a viral expression vector via a cleavage site, and the recombinant viral vector is transfected into mesenchymal stem cells.
3. The method of any of claims 1-2, wherein the mesenchymal stem cells are derived from waste umbilical cord, cord blood, placenta, autologous bone marrow, or fat.
4. The method of claim 1, wherein the human PPAR α and PPAR γ sequences are amplified by PCR from normal human peripheral blood genomic DNA using PPAR α primers as the positive strand: 5'-GCTCGAGATGGTGGACACGGAAAGCCCA-3', antisense strand: 5'-CCATATGGTCAGTACATGTCCCTGTAG-3', respectively; PPAR γ primers were, plus strand: 5'-GCTCGAGATGACCATGGTTGACAC-3', antisense strand: 5'-CCATATGGTGAACATGATCCATATGG-3', respectively; respectively carrying out double enzyme digestion on the PPAR alpha target fragment and the PPAR gamma target fragment and a pET28a carrier fragment of XhoI/Nde I under the action of T4 DNA ligase at 4 ℃ for 12 hours to prepare a cloning ligation solution, and carrying out positive cloning PCR identification and sequencing identification after transforming an escherichia coli competent cell DH5 a; after gel electrophoresis detection and sequencing identification of PCR products conform to the sizes and sequences of PPAR alpha and PPAR gamma, transferring a correctly sequenced bacterium liquid into 10ml of LB liquid culture medium containing an aminobenzene antibiotic, culturing at 37 ℃ overnight, performing plasmid extraction by using an endotoxin-free plasmid small-extraction medium-amount kit, and placing the qualified recombinant plasmid in an ultra-low temperature refrigerator at-80 ℃ for long-term storage; the sizes of the PPAR alpha gene fragment and the PPAR gamma gene fragment are 1407bp and 1434 bp;
carrying out XhoI/Nde I double enzyme digestion on the PPAR alpha and PPAR gamma DNA fragments and the GV358 vector respectively, carrying out ligation reaction on the PPAR alpha and PPAR gamma fragments and the GV358 vector digested under the action of T4 DNA ligase at 37 ℃ for 12 hours to prepare a clone ligation solution, transforming Escherichia coli competent cells DH5a, and carrying out positive clone PCR identification and sequencing identification;
taking 293T cells in good cell state and logarithmic growth phase, counting the cells, and then performing 6X 10 cell counting on each 10cm culture dish6The number of cells was plated in a petri dish at 37 ℃ with 5% CO2Culturing in an incubator overnight; removing the culture solution before the next day of transfection, and replacing 5ml of Opti-MEM culture solution; adding 9 μ g of the packaged mixed solution and 3 μ g of the lentivirus expression plasmid into 1.5ml of Opti-MEM, gently mixing, adding 36 μ l of lipofectamine2000 into 1.5ml of Opti-MEM, gently mixing, and standing at room temperature for 5 min; mixing the plasmid solution and lipofectamine2000 diluent, and standing at room temperature for 20 min; the mixed solution is slowly dripped into the 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO2Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10ml of PBS (phosphate buffer solution) for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 10% serum-containing cell culture medium 20ml, and adding 5% CO at 37 deg.C2Continuously culturing for 48-72h in the incubator;
collecting 293T cell supernatant 48h after transfection according to cell state; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting ultracentrifuge tubes with virus supernatant into an ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, setting the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after centrifugation is finished, removing supernatant, removing liquid remained on the tube wall, adding virus preservation liquid, and repeatedly blowing and resuspending; after full dissolution, centrifuging at a high speed of 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, subpackaging the packaged PPAR alpha and PPAR gamma lentivirus according to 50 mu l of 2E +8TU/ml, and storing in an ultra-low temperature refrigerator at-80 ℃;
the recombinant PPAR alpha and PPAR gamma lentivirus are added into a six-well culture plate of mesenchymal stem cells at 10 mu l of 1E +7TU/ml for co-transfection, the system is 2ml,mixing, 37 deg.C, 5% CO2After incubation for 8-12 hours in the incubator, the complete culture solution alpha-MEM was replaced; when the cell growth and fusion reached 90%, the cells were digested with 0.25% trypsin and transferred to 25cm2Growth in flasks, 1 well for 1 flask at 25cm2Continuing digestion subculture when the fusion in the culture flask reaches 90%; the growth transfections were divided into 3 groups: untransfected control group, blank lentivirus transfected group and PPAR α and PPAR γ transfected lentivirus cotransfected group;
observing the luminescence condition of Green Fluorescent Protein (GFP) under a fluorescent microscope 72 hours after infection, wherein the GFP is developed by more than 90%;
detection was performed by fluorescent quantitative PCR kit according to the instructions using PPAR α primers, sense strand 5'-GCGAACGATTCGACTCAAGC-3', antisense strand: 5'-CATCCCGACAGAAAGGCACT-3', respectively; and PPAR γ primers: sense strand 5'-TGGTGGGTTCTCTCTGAGTCTG-3', antisense strand: 5'-ATCCACGGAGCTGATCCCAA-3', respectively; after the mesenchymal stem cells transfected with the co-transfected PPAR alpha and PPAR gamma lentiviruses are subcultured to 10 th generation, the expression of PPAR alpha and PPAR gamma genes is 145.67 times and 167.89 times higher than that of untransfected MSCs respectively.
5. A method of producing a compound according to any one of claims 1 to 2,
the recombinant PPAR alpha and PPAR gamma lentivirus transfected MSCs are subjected to in-vitro subculture by using complete culture solution alpha-MEM, when the cells are cultured for 5 to 6 days, the cells grow and fuse to more than 90 percent, and pancreatin with the mass-volume ratio of 0.25 percent is used for digestion and subculture;
taking the cell morphology observed under an inverted microscope of the MSCs cultured to 1 generation and 10 generations, wherein the untransfected MSCs have similar cell morphology with the MSCs of other transfected target genes, and the cells grow in a fusiform manner.
6. The method of any one of claims 1-2, 4, wherein the mesenchymal stem cells are prepared by the steps of:
washing 1-2cm of waste umbilical cord with 1 XPBS containing 2 times of penicillin and streptomycin double-antibodyNext, the umbilical cord was cut into pieces of 1mm using an ophthalmic scissors3Resuspending with 1 XPBS containing 2 times of penicillin and streptomycin double antibody, centrifuging at 1000rpm, resuspending with mesenchymal stem cell culture medium after supernatant, sucking 10ml, and adding to 75cm2In a cell culture flask, 5% CO at 37 ℃2Culturing in a cell culture box for 48 hours; replacing a fresh mesenchymal stem cell culture medium containing the cell factors and continuously culturing until the cells climb out of the tissues;
when the cells grow densely, the cells are digested by pancreatin with the mass volume ratio of 0.25 percent, and the cells are transferred to a new 75cm after being resuspended by 10ml of mesenchymal stem cell culture medium containing cell factors2In a culture flask, 5% CO at 37 ℃2Culturing in a cell culture box until the growth fusion reaches 90%, performing digestion subculture by using 0.25% pancreatin, namely culturing in 1 bottle of passage 2 bottle, supplementing a fresh mesenchymal stem cell culture medium containing cell factors, continuously culturing, and digesting the mesenchymal stem cells by using 0.25% pancreatin when culturing to the 5 th generation, thereby obtaining the mesenchymal stem cells; resuspending into fetal calf serum containing 10% dimethyl sulfoxide, and freezing in liquid nitrogen;
counting 1.0X 10 trypan blue staining6MSC cells, divided into three groups, the first group is added with 20 μ L FITC-mouse anti-human CD34 monoclonal antibody; 20 μ L of PE-mouse anti-human CD90 monoclonal antibody; the second group was added with 20. mu.L of FITC-mouse anti-human CD14 monoclonal antibody, respectively; 20 μ L of PE-mouse anti-human CD73 monoclonal antibody; the third group is added with 20 μ L of FITC-mouse anti-human CD45 monoclonal antibody; 20 μ L of PE-mouse anti-human CD44 monoclonal antibody; the fourth group was added with 20. mu.L of FITC-mouse anti-human CD19 monoclonal antibody, respectively; 20 μ L PE-murine anti-human CD105 mab; the fifth group is isotype control, and 20 μ L of FITC-labeled murine IgM and 20 μ L of PE-labeled murine IgM were added respectively; staining for 30 min in a refrigerator at 4 ℃, then washing three times with 1mL of 1 XPhosphate buffer solution (PBS), finally resuspending the washed cells with 0.5mL of 1 XPBS, and detecting the washed cells with a FACS Calibur flow cytometer;
the results show that the expression of MSCs markers CD90, CD73, CD105 and CD44 is 99.08%, 99.85%, 99.88% and 99.60% respectively when the human mesenchymal stem cells are detected by the flow cytometry; CD34, CD14, CD45 and CD19 were expressed as 0.00%, 0.21%, 0.58% and 0.04%.
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