| Objectives:1. Collection of serum-free cultur supernatant of mesenchyma stem cells and preparation USFM.2. Inwestigation the protective effect of the USFM on human dermal fibroblast(HDF) exposed to ultraviolet A rays, has and the protective effect in the condition of skin lesion caused by imitated sunlight(UVA95% and UVB5%).Methods:1. Mesenchyma stem cells were separated from human umbilical cord, and collect serum-free cultur supernatant of the cells to study its anti-light aging effect on the skin.2. Separate and culture HDF to build the in vitro model of cell lesion caused by UVA. Determine the toxicity of the cultur supernatant to HDF and the concentrations of promoting proliferation of HDF by MTT method.3. Firstly build the in vivo model of cutaneous photo-aging by imitated sunlight, and then observe skin texture changes by Hematoxylin-Eosin staining. Besides, detect the activity of SOD, GSH-PX, MDA in the plasma of mice by biochemical method and observe changes of collagenous fiber in corium.Results:1. The culturing supernatant of stem cells has no obvious toxic effect on HDF with the protein concentration less than 400μg/ml. When the protein concentration was 20μg/ml, the supernatant significantly promote the proliferation of HDF. In the test of HDF lesion by UVA, the survival rate was 86.4%±1.10 in the group of HDF with pre-treatment of the USFM, which has significant difference, compared to the model group(64.35%±3.22). The activity of SOD inside the cells in high dose of stem cells'secretin group was 42.95±1.15U/ml, and the activity of SOD in low dose of stem cells'secretin group was38.74±0.53 U/ml, which has significant difference, compared to the model group 21.31±1.31 U/ml.2. Test in vivo showed that light-exposed skin of mice, whose back hair was cut out, presented typical light aging, following the ultraviolet rays (UVA+UVB) exposed schedule. Observation of pathological sections of the skin showed that fibroblast injured and even broke up; elastic fibers, type I and type III collagenous fibres ruptured and disorganized; and elastic fibers-liked substances abnormally grew with the infiltration of mononuclear inflammatory cells. Biochemical test of plasma showed that the activity of SOD in vitro significantly fell, it was 7.26±2.24U/ml, and MDA accumulated abnormally, it was 25.93±1.16nmol/ml. If the skin was pre-treated with the culturing supernatant of stem cells 30 min before exposing, cutaneous light aging could be alleviated. Although collagenous fibres grew a little bit, they were in order. Increase of collagenous fibres would be good for anti-wrinkle, which was in accordance with the results of HE staining. At the same time, infiltration of inflammatory cells greatly reduced. Compared to the model group, the activity of SOD and GSH-PX in the plasma remarkably increased, but MDA remarkably decreased. The activity of SOD inside the mice in high dose of stem cells'secretin group was53.3±5.99 U/ml, and the activity of SOD in low dose of stem cells'secretin group was37.26±2.24 U/ml, which has significant difference, compared to the model group 7.26±2.24U/ml.Conclusion:1. Mesenchyma stem cells were separated from human umbilical cord.2. UVA can inhibit the growth of HDF cells, and even leads to cell apoptosis and necrosis.3. Exposed to UVA+UVB can accelerate cutaneous photo-aging, destroy the structure of dermis, weaken the defensive function of antioxidation, and enhance oxidizing lesion. When harm of ultraviolet exceeds instinctive reparing function of the skin, photo-aging occurs.4. There are plenty of antioxidants in the serum-free culturing supernatant of mesenchyma stem cells, which maybe include growth-promoting cell growth factor. They have good ability of antioxidation, which provides certain study basis to further use of the culturing supernatant of stem cells to other medical treatment. |
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