| Backgrouds and objectives:Defects of soft tissue may often arise from trauma, tumor resection, congenital malformation and so on, and how to effectively repair soft tissue defects is the emphasis and difficult point in the field of tissue engineering. With the extensive study and development of stem cell technology, application of stem cells as seed cells for repairing soft tissue defects is the most promising way at present. Owing to their advantages of abundant resource, convenient obtain, simple cultivation and little ethical limits, adipose derived mesenchymal stem cells (Adipose stem cells, ASCs) become the most popular seed cells in tissue engineering. However, the adipose tissue derived stromal vascular fractions (SVF), which can be obtained from adipose tissue by a series of collagenase digestion, filtration, centrifugation procedures, are not pure ASCs, but a mixed cell populations, and will be purified after2-3times of passage. The cells currently used in the basic and clinical studies are mostly this kind of imperfect cell populations. In recent years, there are more and more studies concerning the in situ origin of ASCs in adipose tissue and the multipotential differentiation of subpopulations in SVF. Nevertheless, due to the lack of specific surface markers for mesenchymal stem cells, there is no precise conclusion for the in situ localization of ASCs in adipose tissue. Thus, further research on this question is essential for better application of ASCs in soft tissue engineering.A large number of studies show that, adipogenesis is a very delicate and complex process regulated by a variety of transcription factors, growth factors and signaling pathway. One of the transcription factor, peroxisome proliferator-activated receptor gamma (PPARY), plays a crucial role in the beginning of this process, and it is indispensable for adipose tissue formation, as knockdown of PPARY gene in3T3-L1cells may lead to a loss of their adipogenic differentiation ability, and mouse embryonic with the lack of PPARY gene can not form fat tissue. Wnt signaling is an important pathway for cell proliferation and osteogeneis, adipogenesis balance of mesenchymal stem cells, whether PPARY can regulate Wnt signaling pathway to promote adipogenic differentiation and how to regulate Wnt signaling pathway are still unknown. Additionally, insulin/insulin-like growth factor-1(IGF1) is also essential for adipogenic differentiation. Although there are reports saying that IGF1combines to its specific receptor on the membrane of target cells to promote the target cell proliferation and adipogenic differentiation, whether IGF1can influence Wnt signaling and the surrounding target cells in adipgenesis process, is also not clear so far. Therefore, the investigation of these events will contribute to more effective application of IGF1in the adipose tissue regeneration.In this study, we obtain two subpopulations of CD31-/CD34+/CD146+and CD31-/CD34+/CD146-from adipose SVF by FACS sorting, and intend to compare their proliferation, adipogenesis and osteogenesis capacities and focus on potential mechanisms, to look for seed cells with stronger adipogenic ability. Also, through the mechanism and application studies of IGF1in promoting adipogenesis, we aim to explore some more effective methods for adipose tissue regeneration. This study includes three parts:the comparison of in vitro proliferation, multiple differentiation abilities of different subpopulations in SVF; mechanism study of PPARY-Axin2-Wnt signaling pathway in adipogenesis; the influence of IGF1on Wnt signaling pathway and subpopulations in SVF, and the application of IGF1in fat tissue regeneration.Materials and Methods:(1) Prepared frozen sections with fresh human adipose tissue, and the distributions of CD31, CD34and CD146in adipose tissue were determined by immunoflurescence assays. SVF were isolated from adipose tissue and cultured, and then, cells were divided into two subpopulations CD31-/CD34+/CD146+and CD31-/CD34+/CD146-by flow cytometry sorting. Their percentages in SVF, in vitro proliferation, adipogenesis and osteogenesis abilities were compared and analyzed, and at the same time, the expressions of differentiation related genes were detected.(2) The activities of Wnt/β-catenin signaling in these two subpopulations were compared. The regulations of PPARY and Wnt on each other were carried out, and then the relationship between PPARY and Axin2, inhibitor of Wnt signaling, was further studied via observing changes of Axin2expression with overexpressed or interfered PPARY. There was binding site of PPARY on Axin2promoter. After transfected PPARY into ASCs, the expression changes of Axin2were detected when binding sited existed and when it was cut off. And also, the effect of Axin2interference on adipogenic differentiation of ASCs was investigated.(3) The influences of IGF1on proliferation, migration and adipogenic differentiation of ASCs in vitro were investigated, and the changes of subpopulations percentage in SVF and genes expression with treatment of IGF1were analyzed. IGF1was transplanted into C57B16mice inguinal fat pad using PLGA scaffold with microspheres, and after4weeks, the growth of adipose tissue in scaffolds was detected.Results:(1) Immunofluorescence results showed that, CD34+cells were mainly distributed in the surrounding of blood vessels in adipose tissue; CD146+cells existed in the blood vessel wall and were widely distributed in the connective tissue of fat, and there was co-localization of CD34and CD146in blood vessels; CD31+cells were mainly distributed in the vascular cavity, and only a small amount of them co-located with CD146. Thus, there were indeed two groups of cells, CD31-/CD34+/CD146+and CD31-/CD34+/CD146-, in surrounding of blood vessels in adipose tissue.(2) Flow cytometry results showed that in the first passage of SVF, the percentage of CD31-/CD34+/CD146+cells was much higher than that of CD31-/CD34+/CD146-cells, but both of them would decrease with cell passage. While in the population of CD31-CD34+, the percentage of CD146-cells decreased with CD146+cells increased.(3) CD31-/CD34+/CD146+cells had stronger proliferation and osteogenesis abilities than CD31-/CD34+/CD146-cells, while CD31-/CD34+/CD146-owned stronger adipogenic differentiation ability, and these were confirmed by the expressions of adipogenic genes and osteogenic genes..(4) Wnt/β-catenin signaling pathway has an important role in cell proliferation and differentiation. Results demonstrated that Wnt signaling pathway was more activated in CD31-/CD34+/CD146+cells than in CD31-/CD34+/CD146-cells activity. Add of Wnt3a in adipogenesis of subpopulations would lead to down-regulation of PPARY, and overexpression of PPARY could inhibit the activity of Wnt/β-catenin signaling. Therefore, PPARY and Wnt/β-catenin pathway had a negative feedback effect on each other in adipogenic differentiation process.(5) In early stage (day1to day4) of adipogenesis of CD31-/CD34+/CD146+and CD31-/CD34+/CD146-subpopulations, we discovered a obvious linear correlation between PPARY and Axin2. There was binding site of PPARY on Axin2promoter. Over expression or interference of PPARY could up-regulate or down-regulate the expression of Axin2. In addition, PPARY could directly bind the PPARY binding site of Axin2promoter through ChIP analysis. Interference of Axin2could obviously inhibit the adipogenesis of ASCs. These results suggested that up-regulation of Axin2by PPARY can inhibit Wnt signaling pathway, thereby promoting the adipogenic differentiation.(6) In vitro study, IGF1can promote ASCs proliferation, migration, adipogenic differentiation and induced the lineage bias of CD31-/CD34+/CD146+to CD31-/CD34+/CD146-cells. In the in vivo studies, transplantation of IGF1microsphere with PLGA scaffold could induce the homing of endogenous ASCs to promote local adipose tissue regeneration.Conclusions:To find the optimal seed cells for tissue engineering is one of the key points for application of stem cells in this field. CD31-/CD34+/CD146-population in SVF has perfect adipogenesis potential, and PPARY-Axin2-Wnt signaling pathway plays an important role for adipogenesis, that is, PPARY can up-regulate Axin2expression, resulting in the inhibit of Wnt signaling, and finally promote adipogenic differentiation. Additionally, IGF1can promote in vivo fat tissue regeneration, and this could be related to its contribution to ASCs proliferation, migration and induction of lineage bias of CD31-/CD34+/CD146+to CD31-/CD34+/CD146-subpopulation. |
PDF Full Text Request