ABSTRACT
Objective:To identify the influence of age factors on biological characteristics of human adipose-derived stem cells (ADSCs), and to compare the differences in morphology, aging characteristics, differentiation potential, surface marker expression, and proliferation ability of human ADSCs with different ages and the effect of human ADSCs of different ages on proliferation and migration of fibroblasts.Methods:Adipose tissues from 27 individuals of healthy beauty treatments, aged 0-59 years, were collected from Department of Burns and Plastic Surgery, the 940th Hospital of Joint Logistics Support Force from January 2020 to December 2020. According to the age of the donor, they were divided into 6 groups. 0-9 years old group (4 cases), 10-19 years old group (4 cases), 20-29 years old group (5 cases), 30- 39 years old group (5 cases), 40-49 years old group (5 cases), and 50-59 years old group (4 cases). We obtained and cultured the ADSCs of each group, subcultured to obtain the ADSCs, and observed the morphological characteristics of each group; the proliferation ability of each group of ADSCs was detected by CCK-8 method; the differentiation potential of each group of ADSCs was detected by in vitro adipogenesis and osteogenic induction. Flow cytometry was used to detect the surface marker expression levels of each group of ADSCs, and qPCR was used to detect the expression levels of HGF, PPAR-γ, type Ⅲ collagen and DbX2 in each group. We also collected the skin tissues of a healthy person from Department of Burns and Plastic Surgery of the 940th Hospital of Joint Logistics Support Force in 2020, obtained and cultured fibroblasts, subcultured to obtain the second generation of fibroblasts, and prepared each group of ADSCs conditioned medium (ADSCs-CM); the proliferation ability of fibroblasts cultured in each group of ADSCs-CM was detected by CCK-8 method, and the migration ability of fibroblasts cultured in each group of ADSCs-CM was detected by cell scratch method. SPSS 21.0 software was used for data analysis and P<0.05 was considered statistically significant. Results:The cells of all age groups were in a typical spindle shape. The cells of the younger age group showed small nuclei and the cell morphology were uniform and similar; the cells of the older age group had larger nuclei and uneven morphology. With aging, the proliferation ability of ADSCs gradually decreased. In vitro adipogenic differentiation induction results showed that with the increase in age, the ability of ADSCs to differentiate into adipocytes gradually decreased. In vitro osteogenic differentiation induction results also demonstrated that with the increase in age, the ability of ADSCs to differentiate into osteoblasts gradually decreased. The expression rates of positive surface markers of ADSCs in each group of ADSCs were all above 93%. With aging, the expression levels of PPAR-γ, HGF, and type Ⅲ collagen decreased, and the expression levels of DbX2 showed an upward trend with age. The number of β-galactosidase staining positive cells increased with age, while the difference between groups A, B and C was not statistically significant, and the difference between groups A and D, E, and F was statistically significant ( P<0.05). The ADSCs conditioned medium of each group was able to promote the proliferation of fibroblasts, but the difference between the groups was not statistically significant. With aging, the ability of ADSCs conditioned medium to promote the migration of fibroblasts gradually decreased. Conclusions:The proliferation ability and multidirectional differentiation ability of human ADSCs and the ability to secrete a variety of cytokines that promote the proliferation and migration of fibroblasts show a downward trend, but the adipogenic differentiation ability of ADSCs declines significantly after the age of 40 years. ADSCs in all age groups could promote the proliferation and migration of fibroblasts.
ABSTRACT
Objective: To investigate the optimal culture conditio ns for adipose tissue-derived stromal cells(ADSCs) and for the induction of these cells to differentiate into osteogenic cells. Methods: ADSCs were cultured with routine methods,bFGF at 20 ng/ml was added into the medium and the proliferative of ADSCs was examined by cell counting. 0.1 ?mol /L of dexamethasone,10 mmol/L of ?-glycerophosphate and 50 ?mol/L of ascorbic acid were adapted to induce the cells to differentiate into osteogenic cells, ADSCs were identified by immunocytochemistry and differentiated osteogenic cells were identified by alkaline phosphatase(AP) staining and immunocytochemistry. Result: A population of ADSCs could be isolated from adul t human adipose tissue,the cells were fibroblast-like and could be maintaine d in vitro for extended periods with stable population doubling.The cells w ere expanded as undifferentiated in culture for more than 10 passages, indicati ng their proliferative capacity.bFGF stimulated the cell proliferation.Dexameth asone,?-glycerophosphate and ascorbic acid induced (40?8.6)% of ADSCs to ex press alkaline phosphatase(AP) ,(35?10.6)% of AP positive ADSCs were found to be collagen I positive. Calcification plaques were occasionally found in the cul tures. Conclusion:The data support the hypothesis that adu lt human adipose tissue contains stem cells capable of diffferentiating into ost eogenic cells.