Study Of Effects And Mechanisms About Adipose Stem Cells Microenvironment On Aging Fibroblasts

Aging of the skin is one of the most prominent manifestations of human aging and people have been chasing the purpose of slowing aging all alone the history. Wherein the dermal fibroblast cells are one of the main functional cells of the skin, which is also a major target cells for skin anti-aging. Adipose-derived stem cells(ASCs) are present within mature adipose tissues, which are relatively abundant. The extraction is simple, and easily accepted by patients. ASCs have been considered to have great application prospects of stem cells. Many studies confirmed that adipose stem cells exhibited paracrine of various cytokines, including inflammatory cytokines, angiogenic factors, growth factors et al. These cytokines constitute micro-environment for ASCs growth, which have effects on the surrounding tissue and cells through a variety of pathways. The number of ASCs differentiated itself is relatively small; they are more like tissue cell activity organizers, rather than participants. Many studies have shown that ASCs biologically active growth factors could co-operate with the surrounding tissue and cell signal transduction, and exhibits positive activities on wound healing, tissue repair and anti-aging aspects. However, prolonged effects of ASCs microenvironment on fibroblasts have not been reported. Whether ASCs microenvironment changes the senescence of fibroblasts, and the mechanism through which changes, these issues are not clear. Cell aging itself is a cellular self-protection, to prevent over-stimulation of cell proliferation and even the formation of tumors. The p53/p21 signal pathway plays an important role in regulating cell cycle, inducing cell senescence, and preventing tumorigenesis. This research will employ adipose stem cells and human dermal fibroblasts indirect non-contact co-culture, and further elucidate the effects of adipose stem cell microenvironment on dermal fibroblasts function. We will study senescence change of fibroblasts by measuring senescence associated beta- galactosidase, as well as expression of p53, p21 protein which indicating cellular replication and stress-related senescence through p53 / p21 pathway.Section 1: Isolation, culture, identification of the human adipose stem cell and co-culture with fibroblasts established systemObjective: To study micro-environment of ASCs on dermal fibroblasts, we need to isolate and culture of ASCs and fibroblasts, and establish an indirect co-culture system using Transwell chamber.Method:(1) Abdominal subcutaneous adipose tissue(approximately 5g) were sampled from each five cases of young people(aged <30) and the elderly(age> 60) of have CD44 immunohistochemical staining. CD44 positive cells distribution and numbers were analyzed from four fields on one slice selection.(2) Adipose tissue was harvested by vacuum aspiration or excision. Isolatioin and culture were performed as well as flow cytometry for CD34, CD44, CD90 for cell identification.(3) Human dermal fibroblasts were isolated and cultured, the indirect co-culture system using Transwell chamber with ASCs was established.Results:(1) Immunohistochemical staining of adipose tissue indicated that CD44(+) cells are mainly located in perivascular fibrous connective tissue which in close contact with the vascular endothelial cells.(2) The surface antigen expression of primary ASCs from adipose tissue cultured was CD34(-), CD44(+), CD90(+).(3) ASCs and fibroblasts indirect co-culture system was successfully established using Transwell chamber.Conclusion: CD44(+) cells are mainly located in perivascular fibrous connective tissue. Free of adipose tissue with a syringe can increase collagenase digestion efficiency, adequate primary ASCs, the antigen expression of CD34(-), CD44(+), CD90(+). Transwell chamber may be constructed with ASCs and fibroblasts indirect co-culture system.Section 2: The effects of ASCs microenvironment on dermal fibroblasts functionObjective: To study the microenvironment constituted of ASCs paracrine factors on fibroblast from different ages.Methods: In this study, fibroblasts were divided into four groups, in culture for 24 h, 48 h, 72 h cell proliferation assay with MTT(tetrazolium salt) was performed respectively; m RNA expression of type I collagen, matrix metalloproteinase-1(MMP-1) in fibroblasts were detected by quantitative real-time PCR.Results: Proliferation of co-cultured youth and elder HDFs group were significantly higher than those in the control group(P <0.01). MMP-1 m RNA expression of co-culture elder HDFs were significantly reduced than the control group after 48 hours(P <0.01). Wherein the co-culture groups MMP 1-expression decreased significantly at 48h(P <0.01), but the decline trend stopped at 72h(P> 0.05). In the control group, the expression of MMP-1 increased gradually over time(P <0.05). At the first 48 h, the expression of type I collagen in co-cultured elder HDFs showed gradual increase and then decrease slightly in 72h(P> 0.05). Co-culture youth HDFs type I collagen expression in 48 h was significantly higher(P <0.01), and decreased at 72h(P <0.05). Expression of fibroblasts type I collagen in 24 h and 48 h exhibited no significant difference in control groups(P> 0.05), while type I collagen expression of the youth control group declined at 72h(P <0.05).Conclusion: The results of this section showed that adipose stem cells microenvironment could promote the proliferation of co-cultured fibroblasts, while reducing MMP-1 production, and increasing type I collagen expression. It indicated that adipose stem cells microenvironment could improve cellular function of fibroblasts. But the promotion effects gradually reduced with the further extension of co-culture, indicating that microenvironment of ASCs could not improve the function of fibroblasts for a long time.Section 3: Changing of the senescence state of fibroblast and the effect on p53 / p21 pathway by the microenvironment from human adipose derived stem cellsObjective: To explore the effects of ASCs microenvironment on fibroblasts senescence state and p53/p21 pathway.Methods: β-galactosidase mRNA changes were detected in each fibroblast culture groups after cultured for 24 h, 48 h, and 72 h by real-time PCR assay; the p53, p21 protein expression in fibroblasts were analyzed by western blot method as well.Results: The expression of SA-β-Gal in the two fibroblasts control groups during the first 24 hours showed no significant difference(P> 0.05), and then gradually increased in each HDFs group after 24 hours(P <0.05). The SA-β-Gal in each elder HDFs group showed higher expression levels at each time point(P <0.01). The SA-β-Gal in the two co-culture groups exhibited significantly higher expression compared with the control groups(P <0.05). The expression of p53 protein in co-cultured fibroblast groups significantly increased compared with the control groups at each time points(P <0.05). The test results showed similar ascending trend between p21 protein and p53 protein(P <0.05), but the increase extent is less than the p53 protein. What more, the expression of p21 protein between co-control groups and control groups had no significant difference at 24h(P> 0.05).Conclusion: The senescence associated β-galactosidase mRNA expression in the co-cultured fibroblasts would increase with ASCs microenvironment, and with longer co-culture time, the ascending effect would be more significant. Meanwhile, the p53 and p21 proteins expression increased with the extension of the culture time, p53 protein levels elevated earlier and faster, indicating that ASCs microenvironment promoted fibroblast senescence by p53/p21 pathway.