【作者】 黄菁；

【导师】 刘承宜；

【作者基本信息】 华南师范大学 ， 生理学， 2007， 硕士

【摘要】 目的：糖皮质激素(glucocorticoid,GC)是体内重要的代谢调节激素。病理浓度GC引起的骨骼肌蛋白降解是肌肉萎缩、肌肉延迟性酸痛、类固醇肌病等发生过程的重要步骤。地塞米松(dexamethasone, DEX)广泛地运用在导致骨骼肌蛋白降解的在体和离体实验中。本实验采用小鼠骨骼肌C2C12成肌细胞分化形成C2C12肌管，用DEX刺激，拟建立GC引起蛋白含量下降的细胞模型。在此基础上分别采用发光二极管阵列(light emitting diode array,LED)所产生的红光(640±17nm)【red light(640±17nm)from LED, RLED】照射和他汀类药物处理，观察其对GC引起的蛋白含量下降的调节作用。实验方法：1．用含2％的马血清的培养基诱导C2C12成肌细胞分化2-9天，用二辛可宁酸(bicinchoninic acid，BCA)法研究分化过程中C2C12细胞蛋白总量的变化特性。用分化8天的C2C12肌管开展以下实验。2．为了建立GC引起蛋白含量下降的细胞模型，分别用含0、25nM、100nM、1μM和10μM的DEX的培养基孵育C2C12肌管24h。用BCA法和Western Blot分别测定蛋白总量和α-肌动蛋白(a-actin)含量，建立GC引起蛋白含量下降的离体模型。3．为了研究RLED照射对DEX引起的蛋白含量下降的光生物调节作用，C2C12肌管加入1μM DEX后即刻用RLED(3.5mW／cm~2)照射，照射时间分别为0、110s、300s、900s和1800s，继续孵育24h。用BCA法和Western Blot分别测定蛋白总量和a-actin含量。4．为了研究辛伐他汀对DEX引起的蛋白含量下降的调节作用，用加入不同浓度辛伐他汀(0、100nM、1μM和10μM)与DEX(100nM)共同孵育C2C12肌管24h，用BCA法测定蛋白总量。实验结果：1．C2C12成肌细胞分化后2-9天蛋白总量不断增加，其中分化4天至9天增幅变缓，蛋白总量维持在一个相对稳定的水平。2．浓度为25nM、100nM、1μM和10μM的DEX孵育分化8天的C2C12肌管24h均可使C2C12肌管蛋白总量显著下降，具有剂量依赖性。a-actin的蛋白印迹显示出相似的结果。100nM、1μM和10μM浓度的DEX各组对蛋白含量下降的影响彼此无显著性差异。3．不同时间(110s、300s、900s和1800s)RLED(3.5mW／cm~2)照射能部分抑制DEX导致的C2C12肌管蛋白总量下降，光照效应近似正弦曲线。光照300s和900s照射组能显著的抑制DEX引起的蛋白总量和a-actin下降。4．100nM，1μM和10μM的辛伐他汀能显著地刺激C2C12肌管蛋白总量下降，这种效应呈剂量依赖性，且能显著地促进100nM的DEX对C2C12肌管蛋白含量下降的效应，但不同浓度辛伐他汀和DEX共同作用组与辛伐他汀单独作用组相比，蛋白总量没有显著性差异。结论：DEX可以显著地引起C2C12肌管蛋白含量下降。DEX的这种效应可以被RLED照射显著抑制，但被辛伐他汀显著促进。更多还原

【Abstract】 Purpose: Glucocorticoid (GC) inhibits protein synthesis and stimulates protein degradation inskeletal muscle and is an important factor in the development of muscle wasting invarious catabolic conditions such as atrophy and steroid myopathy. Dexamethasone(DEX), a synthetic GC, has been widely used to induce muscle proteolysis either in vivoor in cell culture. This study was to establish a cellular model of DEX induced decreaseof protein content, and to study the biomodulation of red light (640±17nm) from lightemitting diode array (RLED) and statins on protein content on mice C2C12 myotubes,respectively.Method:1. The total protein content of C2C12 myoblasts differentiated for 2 to 9 days wasevaluated by bicinchoninic acid (BCA) assay. The C2C12 myotubes differentiated for 8days were used to do following experiments.2. In order to establish a cellular model of DEX induced decrease of protein content,C2C12 myotubes were incubated with DEX at 0, 25nm, 100nM, 1μM and 10μM,respectively, in medium of 2% horse serum. The content of total protein and a-actinprotein were evaluated by BCA assay and Western Blot, respectively.3. In order to study the modulation of RLED on DEX induced decrease of protein content,C2C12 myotubes were irradiated by RLED at 3.5mW/cm~2 for 0, 110s, 300s, 900sand1800s, respectively, just after incubated with DEX at 1μM, and then were incubatedwith DEX for 24h. The total content of total protein and a-actin protein were evaluatedby BCA assay and Western Blot, respectively.4. In order to study the biomodulation of simvastatin on DEX induced decrease of proteincontent, C2C12 myotubes were treated with simvastatin at 100nM, 1μM and 10μM inmedium of DEX at 0 nM and 100 nM for 24h, respectively. The protein content wasevaluated by BCA assay.Result:1. The protein content of differentiated C2C12 myotubes increased gradually in the first 4days and became stable after day 4.2. Protein content of C2C12 myotubes incubated with DEX decreased significantly (P＜0.05) in a DEX concentration dependence.3. The DEX induced decrease of protein content of C2C12 myotubes were inhibited by RLED at 3.5mW/cm~2 in a sine function of radiation time.4. Simvastain at 100nM, 1μM and 10μM significantly promoted the DEX induceddecrease of protein content of C2C12 myotubes.Conclusion:DEX at different concentration could induce the decrease of protein content of C2C12myotubes. The DEX induced decrease of protein content was inhibited by RLED at3.5mW/cm~2 in a sine function of radiation time, but was promoted by simvastatinsignificantly in a concentration dependence.更多还原