A Research On Proliferation Of Skin Fibroblast Cultured With Mechanical Stress

Objective To mimic cell proliferation stimulated with skin soft tissue expansion and skin stretching and to study the mechanism of skin fibroblast proliferation stimulated by stress , To establish a new cell culture model with mechanical stress .To investigate the influence of mechanical stress on human being skin fibroblast proliferation , the secretion of extra-cellular matrix and the expression of proliferating cell nucleus antigen(PCNA) in vitro, and the effect on TGF 3 iand bFGF expression during cell proliferation. Method The first part of the experiment was to design and facture the cultured cell model with mechanical strain. Based on the theory of cultured cell strain unit in common use on abroad, combined with the clinical characteristic of tissue expansion and skin stretching technique, we have designed a new cell culture model with mechanical strain. Primary cultured skin fibroblast was obtained from full thickness skin residual in skin-graft surgery. The3-5 pages fibroblasts(cell density : 5000-10000/cm2), digested with trypsin, were planted on elastic membranes of the model. The extend degree was 40% in the experiment. In the second part, the sample were divided into the tested group and the control, counted the number of the cells individually 12hu 24h^ 36hu 48hu 60h > 72h after the begin of the mechanical stress, drew the proliferation curve and compared the cell proliferation between the tested group and the control one in different time with MTT. The cell cyclic was assessed with FACS. After stretching for 48h, cells were counted to quantify the protein and the expression of the PCNA was detected with western-blot. In the third part, the cell proliferation in conditioned culture medium (after stretched 48h) wasdetected with MTT. The TGF P jand bFGF expression were detected at different time point with western-blot. In the last part, the cells stretching for 48h in the test group and control were digested with trypsin and centrifuged to smear, fixed with formaldehyde and acetone(l:l) for 15 minites, dyed by imrnunohischemistry to detect the expression of collagen I and III. Results: After many times modification, the designed cell culture mechanical strain unit was successfully manufactured. It is easy to disinfect and observe during the cell culture. Skin fibroblasts were succeeded to culture in the model and stretch based on the elastic membrane. After stretching, we found the maximum percentage of S phase cell appeared 36h after the strain stimulant. The number of the cells of the test group was apparently increased compared with the number in control group. The expression of PCNA was positive stretching for 48h and the control was negative. The expression of collagen I III were enhanced. Fibroblast proliferation was much increased while cultured with supernatant of conditioned medium from the cell culture model 48h after stretching. The expression of TGF 3 i and bFGF 48h after stretching were much more enhanced. Conclusion: Skin fibroblasts grow and proliferation in the cell culture model with mechanical stress. Mechanical stress can change the cell cyclic and improve the cell proliferation and the expression of collagen I III, PCNA and TGF 3 i, bFGF. These approve that mechanical stress can stimulate cell proliferation. This study also offers a new approach to investigate the mechanism of cell proliferation with stress in the tissue expansion.