Anti-photoaging Effect And Mechanism Of Adipose-derived Stem Cells Transplantation

Several studies on the pathophysiology of photo-aging have found correlations with certain aspects of wound healing. In histologic pathology, it is mainly appeared as change of skin dermal matrix composition, like decrease of collagenous component, degeneration and deposition of elastic fibers. External aging is affected by complicated environmental aspects, including smoking, exposure to chemical substances and UV rays. The UV irradiation caused the decrease of collagen I through two interact signaling pathways: irritates the degration of collagen and restrains the synthetize of collagen. Hence, the decrease of synthetize of UV induced pro-collagen I (COLI) is a key factor of skin photoaging of pathphysiology.Several studies have found that the most significant skin rejuvenation proess for photo-damaging is collagen remodeling, and the cell that is most important in the production of collagen remodeling is the dermal fibroblast.Nowadays, cell and laser treatment have been broadly applied to the treatment of skin photoaging. Cytokines and growth factors that secreted by Adipose derived stem cells (ADSCs), not only irritates the synthesis of collagen but also improves the migration of human dermal fibroblast (HDF), is used to treat skin photoaging. Similarly, Carbon dioxide fractional laser (CO2) is generally acknowledged as the golden standard treatment of facial skin photoaging, it is used to reach treatment goal via partical photothermy theory, improve the photoaging of animal modeling.Transforming growth factor (TGF-β), as the main growth factor that secreted by ADSCs, irritates the proliferation of dermal fibroblast, increases the synthesis and secretion of collagen proteins, including ECM protein. Literature reports that the persistent high expression of β-catenin in human dermal fibroblast karyon will help the proliferation and migration of HDF, in the meantime, it will feedback and activate TGF-β signaling pathway. Some research had approved that Wnt/β-catenin will help wound healing. Among them, as the extracellular ligand, Wnt3a in order to rely on P-catenin and the way of it up regulate TGF-β through Smad2, and induce the differentiation of muscle HDF. Some research even shows that proper Wnt signaling pathway activation will induce the potency of regeneration of skin and cutaneous appendages. However, wnt/β-catenin signaling pathway is rarely used to research for photoaging. And there is no report about the mechanism of migration of ADSCs to cure skin photoaging. Based on above research background, this study will use Hematoxylin-Eosin (H&E) staining, VVG staining, COLI immunohistochemical, measurement of thickness of derma, SOD/MDA indicators to verify the co-effects of two clinical treatment to skin photoaging, namely, ADSCs migration and CO2carbon dioxide fractional laser. At the same time, check the expression of wnt3a,β-catenin,TGF-β2,COLI in photoaging tissues and normal skin tissues, and further verify and activate whether Wnt/β-catenin signaling pathway involves the process of treatment of ADSCs to skin photoaging. Furthermore, prepare HDF in vitro to prepare skin photoaging model, use ADSCs culturing medium to co-culture HDF, add wnt3a inhibitor SFRP2to ADSCs culturing medium, compare the expression levels of Wnt/β-catenin signaling pathway before and after add of inhibitor, and discuss whether Wnt/p-catenin signaling pathway involves in the process of ADSCs improves HDF proliferation to cure skin photoaging.Part Ⅰ experiment:The preparation of skin photoaging model and transplantation of ADSCs, CO2fractional laser combination treatmentObjective:ADSCs transplant skin photoaging is a international frontier technology, its application is still being explored, however, CO2fractional laser has been generally acknowledged as the golden standard measures to cure facial skin photoaging. The combination of these two treatments, being researched by scientific cases, H&E staining, VVG staining, COLI immunohistochemical,measurernent of sickness of derma, SOD/MDA enzymatic indicator, to verify the pathophysiological effects of combination of these two clinical skin photoaging treatments and combined effects.Methods:1. enzyme digestion method separate ADSCs;2. flow cytometry identify ADSCs symbol;3. MTT method to measure ADSCs cell proliferation ratio;4. Adipogenesis\osteogenic induction;5. Prepare rat skin photoaging model for5groups:①control group;②model group;③ADSCs treatment group;④ADSCS+CO2fractional laser group;⑤CO2fractional laser group;6. ADSCs14days after DAPI spike;7. chemical check the distribution difference of COLI among the5groups in immune tissues:8. Compare with normal skin tissue, observe changes of tissue after treatments to5groups via H&E staining and VG staining;9. Use enzymatic indicator to test skin anti-oxidation capacity.Results:After H&E staining, compared with ADSCs+CO2fractional laser group and model group, there is increase of Collagen I fiber, newborn, dense and slender collagen fiber appears on superficial layers of dermal, degenerative curly fiber metabolize, the treatment effects is quite obvious. Compared with ADSCs group and CO2fractional laser group, ADSCs plus CO2fractional laser group has a better result of thicker dermal. Compared VVG staining and normal group rats, rat’s collagen fiber ratio decreased, staining fades, elastic fiber increase and become thicker, part of them broke and twisted. The results of COLI immunohistochemical are visible in the ADSCs+CO2fractional laser group, after COLI treatment, the increase is most obvious, similarly, SOD level significantly increased than model group, MDA level decreased distinctly.Conclusion:Based on results of pathology immunohistochemical and staining, the preparation of rat photoaging model was successful, and compared with ADSCs transplant treatment group, CO2fractional laser, and ADSCs plus CO2fractional laser group, the combined treatment group has the most obvious result. As a consequence of above results, it can be inferred that ADSCs injection plus with CO2fractional laser has an overlapping effect on skin photoaging, and hence provides a new way of photoaging treatment. Part Ⅱ The differentiation expression of Wnt/β-catenin signaling pathway before and after photoaging treatmentObjective:this experiment aims to compare wnt3a, β-catenin of wnt signaling pathway among normal skin tissue, photoaging skin and treated tissue, and the correlation of TGF-(32expression,to discuss the effect and importance of wnt signaling pathway in ADSCs skin photoaging treatment process.Methods:Use Q-PCR, Western Blot and immunohistochemical to test expressions of wnt3a, β-catenin, TGF-β2in skin tissue, model skin tissue, model tissue before and after treatment. Results:The results of Q-PCR and Western Blot showed that, compared with normal skin tissue, the expression volume of wnt3a and β-catenin in skin photoaging decreased on some degree, and it has significant difference (P<0.01) from normal skin tissue. However, in ADSCs treatment transplant group, they significantly increased depressed expression, and they have positive correlation with expression of TGF-β2. The increase of expression volume is very obvious in ADSCs+CO2fractional laser group.Conclusion:Based on test results of molecular and protein level, Wnt/β-catenin may be involved in the process of ADSCs treatment of skin photoaging. Judging from results of pathology immunohistochemical and staining, there is a huge possibility that Wnt/β-catenin takes part in the process of formation of ECM protein and proliferation of dermal fibroblast, thus improves the formation of collagen. It is very possible to take Wnt/β-catenin signaling pathway as the target of skin photoaging, and provides a new way of skin photoaging treatment. Part Ⅲ Down regulate wnt3a expression’s effect on human dermal fibroblast in the process of ADSCs transplantationObjective:Study on protection effects of ASFM on human dermal fibroblast after irradiation of UV rays in different times in vitro. Take a series of tests and analysis of cells, molecules, protein levels, to find the influence and effect of decrease of wnt3a expression in the treatment of ADSCs transplantation in human dermal fibroblast photoaging model.Methods:Prepare cell photoaging model HDF, collect and prepare adipose derived stem cells-serum-free culturing medium (ASFM), co-culture12h HDF photoaging cell model groups to24h until48h. Use MTT and cell cycle experiment,compared with UVB group,the effect of HDF proliferation after ASFM co-cultured.Q-PCR and Western Blot showed that the change of wnt3a,β-catenin,TGF-β2,COLI mRNA and protein level expression between ASFM co-culture photoaging HDF before and after.And compared with ASFM injection with SFRP2,the effect of intervention to wnt3a,and β-catenin,TGF-β2,COLI protein expression level. Results:Flow cytometry showed that,after ASFM co-cultured HDF photoaging cell model,the cell proliferation rate is higher than UVB HDF modeling(p<0.05).Q-PCR and Western Blot showed that after ASFM co-cultured until48h,compared with simple High-DMEM cultured UVB induced HDF modeling, wnt3a,β-catenin,TGF-β2,COLI protein level HDF increased gradually in HDF photoaging model.After injected SFRP2,compared with unintervened group,wnt3a,β-catenin,TGF-p2,COLI protein level significantly decreased.Conclusion:Wnt/p-catenin signal is involved in controlling proliferation, death, differentiation and conglutination of lots of cells. The research found that the expressions of wnt3a,β-catenin,TGF-β,COLI in HDF photoaging model are notably lower than normal HDF. Just to the opposite, after the usage of ASFM to co-culture HDF photoaging model, there is a notable increase in those four proteins mentioned above. After using wnt3a inhibitor SFRP2to.intervene expression of wnt3a, expressions of β-catenin,TGF-β2,COLI in ASFM co-cultured photoaging model down regulated significantly. Hint:there is a huge possibility that wnt/β-catenin signaling pathway involves in the ADSCs transplantaion treated photoaging mechanism, thus boosts the synthesis of extracellular stroma. The results of experiment provides us with new mechanism of ADSCs treatment of skin photoaging, and also provided with some basis of cell therapy of skin photoaging in regenerative medicine.