【作者】 陈世益；

【导师】 顾玉东；

【作者基本信息】 复旦大学 ， 外科学， 2002， 博士

【摘要】 第一部分胰岛素样生长因子促进骨骼肌损伤后愈合的研究目的：本部分研究胰岛素样生长因子（IGF）对SD（Sprague-Dawley）大鼠骨骼肌钝挫伤愈合进程中组织学、超微结构和局部IGF核酸表达的作用，了解IGF对骨骼肌钝挫伤愈合进程和质量的影响，探讨IGF促进骨骼肌钝挫伤愈合的可能机制。方法：1％硫贲妥钠（0.5ml/100g）麻醉后，对大鼠右下肢腓肠肌内侧面中段实施钝性打击伤（Kami,1992），而后在创伤局部实施皮下肌膜外注射药物，分：生理盐水、IGF－I 1.5ug、IGF－II 1.5ug三组，每组均作以下分析：（1）mRNA分析（n=44）：损伤后1，2，3，4，6，9，13天，硫贲妥钠麻醉和颈椎脱位处死大鼠，取右下肢腓肠肌，抽提总RNA，作逆转录(RT)、聚合酶链反应(PCR)、琼脂糖凝胶电泳，了解IGF－I和II的mRNA含量变化，2只/天，2只正常鼠作对照；（2）组织学分析（n=20）：损伤后1，4，6天，硫贲妥钠麻醉和颈椎脱位处死大鼠，取右下肢腓肠肌，甲醛固定等处理后了解组织学变化，2只/天，2只正常鼠作对照；（3）电镜超微结构（n=32）：损伤后1，4，6，9，13天，硫贲妥钠麻醉和颈椎脱位处死大鼠，取右下肢腓肠肌，戊二醛固定等处理后电镜观察超微结构改变，2只/天，2只正常鼠作对照。结果：（1）mRNA分析：伤后用生理盐水组的IGF－I mRNA含量在伤后2、3、4、6、9、13天升高，IGF－II的mRNA含量在伤后3、4、6、9天升高，4、6天明显升高；伤后用IGF－I组的IGF－I mRNA含量在伤后1、2、3、4、6、9天升高，IGF－II的mRNA含量在伤后1、2、3、4、6、9、13天均升高；伤后用IGF－II组的IGF－I mRNA含量在伤后1、2、3、4天升高，IGF－II的mRNA含量在伤后1、2、3、4、6、9、13天均升高；（2）组织学分析：本实验模型可造成大鼠腓肠肌局部充血、水肿，部分肌纤维断裂，在愈合过程中，伤后用IGF－I和IGF－II组的大鼠比伤后用生理盐水组的大鼠成肌细胞激活早，肌管形成早且丰富，肌管融合成肌纤维早，愈合情况较好。（3）超微结构：伤后用IGF－I和IGF－II组的大鼠比伤后用生理盐水组的大鼠更早形成基底板层保护膜，更早更多地形成肌丝、出现肌管融合，更早愈合。结论：本研究运用RT-PCR技术检测内源性IGF mRNA的表达情况，发现：局部注<WP=5>射胰岛素样生长因子可以使内源性的IGFmRNA表达提早；可以刺激成肌细胞增生，促进肌管形成，加速肌管融合成肌纤维，加速骨骼肌创伤后修复过程，改善愈合质量。关键词：胰岛素样生长因子;骨骼肌;损伤;愈合更多还原

【Abstract】 Part IResearch of Insulin-like Growth Factors (IGF) in Promoting the Healing Following Skeletal Muscles InjuryObjective : The study was undertaken to investigate the effect of insulin-like growth factors（IGF）on the healing course and healing quality following skeletal muscles contusion in Sprague-Dawley (SD) rats in histology、ultrastructure and molecular biology, to discuss the possible mechanisms of insulin-like growth factors in promoting the healing. Methods: The rats undergone anesthesia with 1％ thiopental sodium (0.5ml/100g）received a single impact trauma at the medial-middle gastrocnemius muscle of the right leg, and then a following injection was given at the subcutaneous part of contusion site with one of following three, 0.9% sodium chloride (S group) , IGF－I 1.5(g (I group) , IGF－II 1.5(g (II group). The rats were divided into three groups. The following observations were made. (1) mRNA analysis (n=44): The rats were sacrificed under thiopental sodium anesthesia and cervical vertebra dislocation at 1,2,3,4,6,9,13 day after the muscle trauma. (Two experimental-rats sacrificed each time and another 2 for control). The medial gastrocnemius muscles including injuried areas were resected. The corresponding area on the same side of the normal rats were resected as control. The samples were treated through reverse transcription（RT）、polymerase chain reaction(PCR) and agarose gel electrophoresis. (2) The histological analysis (n=20): The rats were sacrificed at 1,4,6 day after the muscle trauma (two rats sacrificed each time, two normal for control). The samples were fixed in 4% formaldehyde and cut at the midline-medial half was embedded in paraffin. Serial 5 (m sections were cut for morphological investigation. (3)Ultrastructural analysis (n=32): The rats were sacrificed at 1,4,6,9,13 day after the muscle trauma (2 rats sacrificed each time, two normal for control). The samples were fixed in 2.5% glutaraldehyde and then in 1% OsO4, dehydrated and emdedded in Epon. Sections were double stained with uranyl acetate and lead citrate and examined in a JEM 100 CX electron microscope.<WP=8>Results: (1) mRNA: (S) IGF-I mRNA is increased at 2,3,4,6,9,13d after tauma, IGF-II mRNA is increased at 3,4,6,9d after tauma, especially at 4,6d. (I) IGF-I mRNA is increased at 1,2,3,4,6,9d after tauma, IGF-II mRNA is also increased at 1,2,3,4,6,9,13d after tauma. (II) IGF-I mRNA is increased at 1,2,3,4d after tauma, IGF-II mRNA is increased at 1,2,3,4,6,9,13d after tauma. (2)Histological changes: the gastrocnemius had congestion，edema and rupture after trauma. The myoblasts and myotubes grew more and faster in groups I and II than group S, the myotubes fused to myofibres earlier in I and II. (3) Ultrastructural changes: groups I and II formed basement membrane "borders"myofibrils and myotubes earlier than group S. Conclusion: We tested the expression of the endogenic IGF mRNA with RT-PCR methods for the first time. We found that Local injection of IGF could promote earlier secretion of endogenic IGF mRNA; could stimulate the proliferation of myoblasts, to hasten the formation and fusion of myotubes, to accelerate the healing of skeletal muscular injury. Key words: Insulin-like growth factor,skeletal muscle,injury,heal更多还原