摘要
目的：探讨IGF-Ⅰ对反复力竭运动致大鼠骨骼肌细胞凋亡的作用及其机制。
方法：建立大鼠反复力竭动物模型及IGF-Ⅰ药物模型，雄性SD大鼠40只，随机分为4组，即安静对照组(A)、正常训练组(C)、力竭对照组(S)、力竭+IGF-Ⅰ组(Y)，S和Y组进行一周的反复力竭跑台训练，C组进行一周的正常训练。检测各组大鼠运动距离，各组大鼠骨骼肌细胞膜、肌浆网和线粒体Ca~(2+)—ATPase活性，肌浆网和线粒体游离Ca~(2+)浓度，电镜观察骨骼肌及线粒体超微结构，流式细胞仪和TUNEL染色检测腓肠肌凋亡细胞，RT-PCR检测凋亡相关基因表达的变化及葡萄糖转运蛋白-4(GULT4)的mRNA表达变化，探讨IGF-Ⅰ对力竭运动后骨骼肌细胞凋亡的抑制作用，及其促进骨骼肌细胞的存活、提高运动能力的可能机制。
结果：力竭+IGF-Ⅰ组(Y)大鼠运动能力显著强于力竭对照组(S)，力竭+IGF-Ⅰ组(Y)大鼠的总运动距离显著大于力竭对照组(S)，P＜0.05；力竭+IGF-Ⅰ组(Y)和正常训练组(C)的细胞膜、肌浆网体Ca~(2+)—ATPase活性显著高于力竭对照组(S)和安静对照组(A)，P＜0.05；力竭+IGF-Ⅰ组(Y)和正常训练组(C)的线粒体Ca~(2+)—ATPase活性显著低于力竭对照组(S)，P＜0.05；力竭+IGF-Ⅰ组(Y)和正常训练组(C)肌浆网游离Ca~(2+)浓度显著高于力竭对照组(S)和安静对照组(A)，P＜0.05；力竭+IGF-Ⅰ组(Y)和正常训练组(C)的线粒体游离Ca~(2+)浓度显著低于力竭对照组(S)，P＜0.05。透射电镜观察示力竭对照组(S)大鼠骨骼肌肌丝排列紊乱等坏死表现，核固缩等凋亡表现，线粒体肿胀和空泡变性等异常表现；正常训练组(C)和力竭+IGF-Ⅰ组(Y)线粒体数量增多、糖元颗粒增多。流式细胞仪和TUNEL染色检测示力竭对照组(S)大鼠腓肠肌细胞凋亡增多，显著高于力竭+IGF-Ⅰ组(Y)和正常训练组(C)，
P<0.05;力竭+I GF一I组(Y)和正常训练组(C)GULT4的mRNA表达
增多，高于力竭对照组(s)和安静对照组(A)。力竭+I GF一I组(Y)和
正常训练组(C)大鼠胖肠肌bel一2的mRNA表达强于力竭对照组(S)，
Bax的表达则呈现与bel一2相反的趋势。
结论:一周反复力竭离心收缩后，骨骼肌细胞膜、肌浆网C扩+--
ATpase活性减低，而线粒体c扩+一灯Pase活性出现应激性异常增高，导
致胞浆和线粒体游离C扩+浓度异常增高，引起线粒体功能障碍，可能是
介导骨骼肌凋亡的重要原因。IGF一I可促进骨骼肌细胞GULT4的mRNA
表达，促进线粒体的能量代谢，提高细胞膜和肌浆网c矛+一华汀Pase活性，
使胞浆c扩+“外流”和进入细胞内“钙库”，避免线粒体C扩+一灯Pase
活性出现应激性异常增高引起线粒体c扩十超载，保护线粒体，促进细胞
存活;并促进抗凋亡基因表达，抑制促凋亡基因表达，最终减少骨骼肌
细胞凋亡，提高运动能力。
Objective To investigate the phenomenon of apoptosis in rats' muscle and the effect of IGF-1 (Insulin-like growth factor-1) on skeletal muscles in rats which taking repeated exhausting run on the treadmill.
Methods Using SD rat repeat exhausting and IGF-1 effect model. 40 SD male rats were divided randomly into four groups: queit control group(A),normal train group(C), exhausting exercising control group(S), exhausting exercising and IGF-1 effect group(Y). The exercising groups(S and Y group) took repeating exhausting run on the treadmill for 1 weeks. The C group took normal training for 1 week.Inspect the exercise length ,and the content of free calcium of sarcoplasmic reticulum and mitochondria, the activity of Ca2+-ATPase in rats' musculi skeleti cell membrane,sarcoplasmic reticulum and mitochondria. Observe the changes of the skeletal muscle cells and mitochondria ultrastructure through the electron microscope(TEM). Apoptotic cells of gastrocnemius muscle cells of rats were measured by TUNEL dyeing and Flow cytometer(FCM). The express of the mRNA of GULT4 and apoptosis-correlating genes were detected by means of polymerase chain reaction(PCR).Investigated the possible mechanism of IGF-I inhibiting cell apop
tosis,promoting cell survival and enhance locomotivity.
Results The locomotivity and total exercise treadmill length of Y group is significant higher than S group after having 1-week exhausting exercise on treadmill, P<0.05. Y and C groups' Ca2+-ATPase activity and free calcium content of mitochondria were less than the S and A group, P< 0.05. Y and C groups' activity of Ca2+-ATPase of cellular mimbrane and free calcium content of sarcoplasmic were more than the S and A group, P< 0.05. Skeletal muscle myofilament disord, the contraction of nucleus and the
vacuolar degeneration and engorge of mitochondria could be seen under electron microscope(TEM) in S group, the number of mitochondria and glycogenosome is higher in C and Y group. The number of apoptosis cell in C group is more than Y and C group by TUNEL dyeingand FCM , P< 0.05 .The expresses of mRNA of GULT4 were powerful in the Y and C group. In the Y and C groups, the expresses of bcl-2 increased more than in S group, the expresses of Bax is crosscurrent.
Conclusions A low level of Ca2+-ATPase activity was observed in the working skeletal muscle cellular menbrane and sarcoplasmic reticulum which have taken 1 weeks repeated centrifugal contraction.but it was anomaly higher in mitochondria.that lead to the functional disturbance of mitochondria. The malfunction of the mitochondria maybe the main reason of induce the apoptosis of muscle cells. The IGF-I can promote the express of the mRNA of GULT4 and the energy metabolism of mitochondrial.enhance the activity of Ca2+-ATPase in cell membrane and sarcoplasmic reticulum,that made Ca2+ in endochylema outflow and enter calcium library. Avoided the abnomal higher of Ca2+-ATPase in mitochondria ,lead to Ca2+ overload, protect the mitochondria of muscle cells, also promote the express of the anti-apoptosis genes and reduce the express of the auxo-apoptosis genes in muscle cells, protect the mitochondria in skeletal muscle and improve the sports ability of the exhausting exercising rats on treadmill.
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