Abstract: Objective To observe the effect of different pressure and duration time on the proliferation of skin fibroblast in vitro,provide theoretical basis for decreasing the necrosis of skin flap caused by skin tissue expansion. Methods The fibroblast cells were isolated and cultured in vitro from the skin of medial malleolus of a 23 years old male.The sixth generation fibroblasts were used for pressure test.The experiment included four groups:40 kPa group(continuous 40 kPa pressure),60 kPa Ⅰ group(discontinuous 60 kPa pressure),60 kPa Ⅱ group(continuous 60 kPa pressure),and control group.The level of IL-6 in nutrient fluid was also measured.The shape of mitochondria and nucleus were observed using transmission electron microscope.The average IOD(integrated optical density) of mitochondria and nucleus of each group were calculated and compared using Plus Image-Pro 6 image analysis software. Results The level of IL-6 of 60 kPa Ⅱ group increased,the amount of mitochondria decreased and cell proliferation was inhibited with significant differences compared with blank group(P＜0.05).The fibroblast proliferation of 60 kPa Ⅰ group and 40 kPa group were similar with the control group(P＞0.05). Conclusion The risk of skin flap necrosis would not increase under discontinuous 60 kPa pressure.The discontinuous 60 kPa pressure is suitable for the instant tissue expansion surgery.