| Objective:In order to research the anti-aging effects of adipose-derived stem cells(ADSCs)and microvesicles(MVs)on skin fibroblasts in vitro,we compared the differences in anti-aging ability between MVs and ADSCs.Then the difference in this ability of single donor-derived MVs and multiple donor-derived MVs were also compared.We hope that this study will provide some experimental and theoretical basis for the application of MVs in clinical anti-aging.Methods:1.Adipose tissue was obtained from fat aspiration in three young adults aged 20 to 30 years,and the cell samples were isolated and digested by enzymatic digestion.In order to verify whether these cells were ADSCs,morphological characteristics of these cells were observed by microscope and cell surface markers CD34,CD44,CD45,CD73,CD90 and CD105 were identified.At the same time,these cell samples were induced to adipogenic,osteogenic differentiation.2.Using low-temperature ultra-high speed centrifugation,MVs were separated from the supernatant of ADSCs.Then morphology of MVs was observed by transmission electron microscopy.3.Fibroblasts were isolated from skin tissue which obtained from the elderly men in surgery.4.Skin fibroblasts were co-cultured with ADSCs or MVs through Transwell chambers.(1)In the experimental group I,fibroblasts were cultured with single donorderived MVs.(2)In the experimental group II,fibroblasts were cultured with multiple donor-derived MVs.(3)And in the positive control group,fibroblasts were cultured with ADSCs.(4)The blank control group was only added with medium.5.After co-culture for 3 days,the morphological changes of fibroblasts were observed with a microscope.Then the following experiments were implemented:(A)The CCK-8 method was used to detect the activity of fibroblasts.(B)Senescence state of each group was detected with Senescence-associated ?-Galactosidase(SA-?-gal)staining.(C)The levels of reactive oxygen species(ROS)in fibroblasts were determined using a test kit.(D)ELISA was used to determine the content of type I procollagen in supernatant of fibroblasts.(E)The expressions of p16INK4 a,p21Cip1 and P53 in fibroblasts were detected by Western Blot.Results:1.The cells extracted from adipose tissue were spindle-shaped.Moreover,the expression markers of CD44,CD73,CD90 and CD105 of these cells were positive,whereas CD34 and CD45 were negative.In addition,the cells can be successfully induced into adipogenic and osteogenic differentiation.2.After co-culture with ADSCs or MVs,part of the elderly fibroblasts gradually changed from irregular flat stellate,cord-like to regular long shuttle shape.3.The results of CCK-8 and ELISA suggested that both MVs and ADSCs could enhance cell-proliferation and synthesis of type I pro-collagen.Futhermore,the results of ELISA showed that ADSCs are better than MVs in improving protein synthesis of fibroblasts.Moreover,there is no significant difference between single donor-derived MVs and multiple donor-derived MVs in enhancing fibroblast proliferation.4.The results of SA-?-Gal staining,ROS determining and Western Blot also showed that both MVs and ADSCs could reduce cell aging related biological indicators.Futhermore,ADSCs are still superior to MVs in reducing the indicators of cell senescence.Moreover,there is no significant difference two kinds of MVs.Conclusions:1.The results suggest that MVs and ADSCs could enhance cell proliferation,synthesis of type I pro-collagen,and decrease the biological parameters of cell aging.Thus,we can determine that both MVs and ADSCs can have certain effect on fibroblasts.Futhermore,the experimental results also show that ADSCs anti-aging effect is stronger than MVs.2.Single donor-derived MVs and multiple donor-derived MVs have the same antiaging effect on fibroblast. |
PDF Full Text Request