不同温度对体外模拟体内生理环境寄养断肢系统的影响研究

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

目的探讨不同温度对体外模拟体内生理环境寄养断肢系统的影响。方法健康成年雄性巴马小型猪6只,体重22～30 kg。取6只猪24个肢体随机分为4组(n=6),将肢体置于体外模拟体内生理环境寄养断肢系统,A组于常温(26℃)下,B、C、D组分别于4、10、18℃低温下体外寄养断肢。12 h后取各组肢体骨骼肌,行组织学及透射电镜观察骨骼肌结构,实时定量RT-PCR检测TNF-α及IL-1βmRNA表达。结果组织学观察示,A组肌纤维变性,肌细胞严重破坏,断裂肌纤维间有大量炎性细胞浸润;B、C、D组组织间轻度水肿,肌膜完整,血管周围偶见炎性细胞浸润,其中C组细胞形态良好。透射电镜观察示,A组肌纤维排列紊乱,可见多处局灶溶解性坏死;B组肌纤维排列较整齐,肌膜尚完整,线粒体轻度肿胀,絮状变性;C组可见大量肌纤维排列整齐规则,肌节清晰;D组肌纤维排列紊乱、不规则,肌纤维溶解。实时定量RT-PCR示,A组TNF-α、IL-1βmRNA表达水平均显著高于B、C、D组(P<0.05);B、C组表达均低于D组,C组低于B组,比较差异亦均有统计学意义(P<0.05)。结论在体外模拟体内生理环境寄养断肢系统中,温度对断肢的保存具有重要作用,其中10℃保存离断肢体可显著抑制再灌注致骨骼肌细胞损伤。

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 foster-ing 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 （P 〈 0.05）. 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.