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EFFECT OF DIFFERENT TEMPERATURES ON SYSTEM OF IN VITRO PHYSIOLOGICAL ENVIRONMENT FOSTERING LIMBS

YIN Yefeng1,WANG Jiangning1,2,GAO Lei2,LIU Ning1,XIA Xiaoyong2,QIN Xinyuan2,ZUO Youwei2.1Graduate School,Dalian University,Dalian Liaoning,116622,P.R.China;2Department of Plastic and Reconstructive Surgery,Beijing Luhe Hospital of Capital Medical University.  
Objective To investigate the effects of different temperatures on the system of in vitro physiological environment fostering limbs.Methods Twenty-four limbs were harvested from 6 adult Bama mini pigs and were randomly divided into 4 groups(n=6) according to different temperatures: limbs were placed in in vitro physiological environment fostering limbs at 26℃(group A),4℃(group B),10℃(group C),and 18℃(group D).After 12 hours of perfusion,the morphology observation was done for the structure and ultrastructure changes of the skeletal muscle by light microscope and transmission electron microscope.The mRNA levels of tumor necrosis factor α(TNF-α) and interleukin 1β(IL-1β) were detected by real-time fluorescent quantitative PCR(RT-qPCR).Results Histological results showed that the skeletal muscle exhibited mild edema,integrity of the sarcolemma,and occasional perivascular inflammatory cell infiltration in groups B,C,and D,meanwhile,the cells of group C had normal morphology;however,muscle fibers degenerated,muscle cells were seriously damaged,a great number of inflammatory cells infiltrated in the fractured muscle fibers in group A.Transmission electron microscope results showed as follows: the muscle fibers arranged in disorder,and many focal solubility necrosis occurred in group A;the muscle fibers arranged in order relatively and sarcolemma was still intact,with mild swelling and flocculent degenerative mitochondria in group B;a large number of muscle fibers arranged in order and regularity with clear sarcomere in group C;and the muscle fibers arranged in disorder and irregularity and partly dissolved in group D.RT-qPCR results showed that the expressions of inflammatory factor TNF-α and IL-1β mRNA in group A were significantly higher than those in groups B,C,and D(P 0.05);the expressions were significantly lower in groups B and C than in group D,and in group C than in group B(P0.05).Conclusion In the system of in vitro physiological environment fostering limbs,temperature plays an important role in the preservation of amputated limbs.It is suggested that 10℃ can significantly attenuate the reperfusion-induced skeletal muscle cell injuries in this system.
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