Treatment Effect Of Bone Marrow Mesenchymal Stem Cells On Photoaging Of Guinea Pig Skin

Mesenchymal stem cells (mesenchymal stem cell, MSC) is a class of pluripotent stem cells, derived from the early embryo development, which have a wide range of self-renewal and differentiation potential。At 2005, such Klyushnenkoval found that BMSCs have a weak immunogenicity, these cells are difficult to be identified by the immune system。After infused into the host with different genes, the BMSCs can evade the host immune surveillance system after transplantation in the long-term survival in the host. Therefore, it is regarded by tissue engineering and gene Vector Engineering as ideal seed cells. It is found that bone marrow stem cell transplantation in patients with chronic injury and damage reconstruction has an important effect Three days after the inoculation of the isolated and purified BMSCs into the epidermal cells, the cell shape changes from the spindle into round or oval, with the cell size varied, arranged in a shape of typical paving stones. Further induction of cell growth showed complex differentiation. Induction derived cell surface marker expression of epidermal cells CK19 and CK10, and CK19 the differentiation rate of 60%, shows that the majority of the induction differentiation cells are epidermal stem cells, and on the other hand, BMSCs could participate the reconstruction of the dermis organization in vitro and in vivo[40, 41]. In dermal fibroblasts induced system, ultra structure of BMSCs shows deposition of micro-fibers of the extracellular collagen, observation on morphology shows:BMSCs is induced to the dermal fibroblast cells, the cell bodies show spindle or triangle shape, with close array as typical swirl or radial extension; RT-PCR proved that differentiation of cells can secrete collagen proteinⅠ; Scanning electron microscope observation collagen membrane shows collagen fibers connected with each other into porous network structure. BMSCs distribute all over the dermal layer after being transplanted in the first seven days , but less in the epidermis cells; 14 days after experimental transplant, epidermis cells obviously increased, and BMSCs in the dermal layer have trend of migration to the epidermis. Histological observation transplantation experiments in the experimental group after 21 days, with epidermal thickening, epidermal layer become clear, we can see that the basal layer, spine and granular layer, the stratum corneum are clearly divided, the basal cells are closely arranged. Dermis layer shows scar tissue formation, for the performance of neutrophil decrease fibroblast increased, and increased collagen fibers arranged in an orderly manner.Although lots of research on the photo aging of the skin mechanism and BMSCs transplantation in the skin injury have been carried out, but using BMSCs in the treatment of photoaging injury have no report. Therefore this paper using BMSCs treat photoaging skin injury, research on its repair mechanism, to provide a theoretical basis and experimental data for the skin-aging prevention and treatment .Research Methods1. Adherent BMSCs isolated and cultured in vitro;2. MTT assays the growth characteristics of guinea pigs BMSCs;3. Flow cytometry analysis BMSCs performance of the cell cycle and cell markers CD34;4. Build model of Guinea pig skin photoaging 5. General observation, HE staining, masson special staining and Sirius red staining of BMSCs to the skin of guinea pigs photoaging prevention and treatment;6. Immunohistochemistry, Sirius red staining and ELISA Detection and PCR identification of the Y chromosome on BMSCs to the prevention and treatment of the photoaging mechanism.The results showed1 BMSCs separation, purification and the morphological features in vitro Microscopy found that the inoculated bone marrow cell subculture 2 to 3 generations, cells shape become more consistent, most of which are slender spindle cells, with the rest flat-shaped cells like fibroblast cells, cell growth can be interspersed, with a clear spindle cell colonies growth. Cell-generation after 6 ~ 7 have no obvious morphological changes, but the large flat-shaped cells increased, with the spindle cells reduced. MTT assay well growing cells generation of 1,3,5 were counted for six days, take nurture time as horizontal coordinate, cells number as vertical axis, plot growth curve. An extensive line of the curve pass through zero. FCM show that 85.87 percent of BMSCs in the G0-G1 phase basically express no CD34.2 Preparation and characterization of guinea pig photoaging skin model Select 250-300 g female guinea pigs, use UVA, UVB ,two wavelength ultraviolet (6-7) irradiation, irradiation two hours a day, till 60 days of exposure. Skin elasticity loss, a deep wrinkles appear and becoming deeper, leather-like appearance, accompanied by dander, with pigment deposition change. HE, Masson staining show epidermis thinning, dermis papilla increasing , dermis thickening, density increasing, inflammatory cells and mast cells number decreasing, and elastic fibers decresing, collagen fibers increasing in the BMSCs treatment group. Expansion of hair follicles alleviating and sebaceous gland atrophy; sweat glands are inordinate, decrease in the number of secretary,cells shrink and tubal chamber expand.3. Remedy effect of BMSCs to the photoaging of the skinAfter two weeks irradiation with UVA and UVB ultraviolet radiation, injecting BMSCs multi-point in the local area skin of guinea pig , once a week, a total of four weeks to prevention and treatment. Clearly observed with eyes, compared with the model group, prevention and treatment group shows deeper wrinkles, leather-like appearance, dander, pigment deposition changed significantly reduced. HE staining showed, compared with the model group, the treatment group BMSCs prevent skin thinning, increasing the number of dermal papilla. Dermal incrassated, density increased, inflammatory cells and mast cells decreasing, elastic fibers decreasing and collagen fibers increased. Expansion of hair follicle reduce, sweat glands increase in the number. By Masson staining, compared with the model group, in the BMSCs treatment group skin thickness become significantly thinner, dermal layer elastic fibers decrease significantly and collagen fibers increase observably.4. BMSCs on the skin photo ageing treatmentAfter ultraviolet irradiation 60 days, injecting BMSCs multi-point in the irradiated area skin of guinea pig, once a week, a total of 30 days. Clearly observed with eyes, compared with the model group, the natural recovery groups change symptoms alleviatory, such as skin wrinkle, leather-like appearance, dander, pigment deposition. In the BMSCs treatment group these symptoms disappeared, near normal guinea pig skin; HE staining showed compared to the model group, natural recovery group although skin becoming thinner, epidermal and dermal junction are still flat; collagen fibers increasing, but which are still disorder. In BMSCs prevention and the treatment group, skin become thinner, the number of dermal papilla increasing. Dermal incrassated, density increased, inflammatory cells and mast cells decreasing, elastic fibers decreasing and collagen fibers increased. Expansion of hair follicle reduce, sweat glands increase in the number. Masson staining showed, compared with the model group, in the BMSCs treatment group skin thickness become significantly thinner, dermal layer elastic fibers decrease significantly and collagen fibers increase observably. And that in BMSCs prevention and treatment group, skin thickness become significantly thinner, dermal layer elastic fibers decreased significantly, collagen fibers increased significantly, which near normal skin organizational structure.5. Research of the mechanism on BMSCs in the photo ageing skin treatmentAfter ultraviolet irradiation 60 days, multi-point injecting BMSCs with DAPI marker in the irradiated area skin of guinea pig. Observed through laser scanning confocal microscope, BMSCs appear in the dermis layer after injection 24 hours; five days after injection , BMSCs appear in the dermis layer as well as organizations such as the hair follicle; 10 days after injection BMSCs widespread disperse in the skin layers, including the stratum corneum; whether injecting BMSCs from two weeks to 60 days after exposure to treatment or injecting BMSCs 30 days after exposure to radiation treatment 60 days, PCR detection of irradiated skin both contain different Y chromosome transplanting gene; Immunohistochemical detection of collagen protein expression, compared with control group, the model group and the natural recovery groupⅠcollagen protein expression increased significantly. In the BMSCs treatment group and the BMSCs preventive treatment group, the expression of collagen protein decreased, near normal control group; Sirius red staining showed that the model group and the natural recovery group collagen typeⅠsignificantly increased, collagen typeⅢreduced. BMSCs preventive treatment group and the treatment group collagen typeⅠsignificant reduction, collagen typeⅢincreased; Detection the guinea pig photo ageing skin tissue , IL-1, IL-6 and TNF–αexpression in the model group and the natural recovery group both higher than in the control group, there were significant differences (p <0.01). Compared with the model group and the natural recovery group, the BMSCs preventive treatment group and the BMSCs treatment group, those three factors expression decline, the difference was significant (p <0.01). Conclusion1. This experiment successfully separated, purified and amplified BMSCs in vitro. And after repeated passage culture BMSCs can still maintain its original shape and differentiated characteristics, which indicating BMSCs have stem cells'characteristics; Low-density vaccination can obtain more cells and clones than high-density vaccination, which confirm BMSCs have strong proliferation ability.2. This experiment through general observation and HE staining showed that after ultraviolet irradiation 60 days, guinea pig skin wrinkled, pigment deposition, skin thickening and collagen disorders are increasing, which approved guinea pig photoaging skin model has been successfully established.3. Results of this study confirmed that BMSCs can delay the development of photoaging process, BMSCs still have preventive role on photoaging.4. Results of this study confirmed that BMSCs have treatment effect on photoaging.5. Results of this study confirmed that BMSCs injected into guinea pig model can be enriched in the region of photoaging skin, suggesting that BMSCs may be through settled at injury region to repairing photoaging, which might be one of BMSCs possible repair mechanism.6. Results of this study confirmed that BMSCs may be treat photoaging by regulating expression ofⅠ,Ⅲcollagen type.7. Results of this study confirmed that BMSCs injected into the photoaging skin have the influence onIL-1, IL-6 and TNF-α, may be one of BMSCs possible repair mechanisms on photoaging.8. Results of this study confirmed that donor BMSCs can survive in the guinea pig photoaging model organization for a long term, suggesting donor BMSCs may be involved in the repair of photoaging.Innovation:1. this experiment applied BMSCs to the skin photoaging treatment;2. This experiment observed BMSCs can delay the occurrence of skin photo-aging process;3. This experiment observed BMSCs have remedial effect ion ;4. This experiment discussed the possible mechanisms of BMSCs on the skin photoaging treatment.