IGF-1通过线粒体信号途径对感觉神经再支配骨骼肌结构和功能维持的作用
发布时间：2018-09-10 12:41
【摘要】：骨骼肌(skeletal muscle,SKM)正常结构和功能的维持是保证人体正常运动状态的最基本条件,蛋白合成和降解平衡的稳定维持着SKM的正常结构和功能,而这一平衡状态的维持有赖于正常的神经支配。当失神经支配时SKM蛋白合成与降解的平衡被打破,导致肌萎缩,并伴有一系列严重的临床症状,如运动功能的丧失。在运动神经损毁严重等特殊情况下,SKM的神经支配无法及时恢复,长时间的运动神经支配缺失会导致SKM收缩功能的丧失,即使SKM再次得到运动神经支配,其运动功能也无法恢复。因此,尽快恢复失神经SKM的神经支配至关重要。将感觉神经近侧端与运动神经远侧残端连接的感觉保护成为保护无法及时恢复运动神经支配SKM的一种方案,但感觉保护的作用较弱,其治疗效果有待进一步加强。体内和体外的实验均证实胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)作为一种多功能的多肽类生长因子,可通过激活磷脂酸肌醇3激酶(phosphatidylinositol3-kinase,PI3K)/Akt信号通路延缓多种病理性肌萎缩并维持和促进神经系统的生长及存活,但IGF-1对SKM感觉保护是否有增强作用尚有待证实。已有研究表明,Akt激活能够影响线粒体的功能状态,而线粒体的功能与其形态密切相关,线粒体在分裂和融合状态之间持续转换,线粒体的异常形态会导致多种疾病。但IGF-1对SKM感觉保护模型中线粒体的影响尚无相关研究。本课题通过建立在体感觉神经支配动物模型和具有感觉神经支配的SKM细胞体外联合培养模型,研究IGF-1对SKM感觉保护的作用及相关机制,本课题的研究结果将会为延缓失神经肌萎缩的治疗提供新的理论基础和实验依据。第一部分IGF-1增强在体感觉神经再支配对骨骼肌的保护作用如果SKM失去运动神经支配的时间过长,即使SKM再次获得神经支配,其运动功能也将无法恢复。在运动神经损伤严重,无法及时恢复SKM神经支配时,可将感觉神经与运动神经残端连接进行修复,使失神经支配SKM得到临时的感觉神经支配,这种技术手段被称为感觉保护。虽然运动功能仍无法恢复,但感觉保护可保护SKM的收缩功能及重要结构,待运动神经恢复至能够正常修复后,再将运动神经近侧与远侧残端连接,使SKM运动功能逐步恢复。但感觉保护对失神经SKM的作用程度有限,效果有待加强。IGF-1作为一种多功能多肽类因子,对SKM和神经系统均具有保护作用,但IGF-1能否增强SKM感觉保护的作用尚有待于进一步研究。mtDNA数量的变化与线粒体正常功能的维持密切相关,而这种变化与SKM的功能有密切的关系,而这些变化过程中IGF-1所扮演的角色,目前也不清楚。基于以上研究背景,本课题设计如下实验进行研究。取25只260 ± 10 g的Wistar雄性大鼠,采用随机分组的方法分为:(1)假手术组:在大鼠后肢作纵行切口,显露坐骨神经分叉部后缝合;(2)失神经组:做同样切口后离断胫神经,近侧残端结扎并缝入塑料帽后缝合;(3)IGF-1组:手术操作同失神经组,胫神经离断后缝合刀口,术后8 w时间内每3d向腓肠肌多点注射10 μgIGF-1;(4)感觉保护组:作纵行切口后离断胫神经和腓肠神经,将腓肠神经的近侧残端和胫神经的远侧残端缝合,胫神经的近侧残端同样缝合到塑料帽中,缝合刀口;(5)IGF-1+感觉保护组,手术操作同感觉保护组,IGF-1应用时间、部位及剂量同IGF-1组。术后8 w收集腓肠肌组织样本进行相关检测。结果表明,对肌萎缩最为直观反映为肌湿重和肌横截面积(cross-sectional area,CSA),在失神经组出现显著下降,IGF-1组和感觉保护组增加,而IGF-1 +感觉保护组增加的幅度最大。SKM关键收缩蛋白肌球蛋白重链1(myosin heavy chain 1,MyHC1)变化趋势与腓肠肌肌湿重和肌横截面积相一致,失神经组下降最明显,而IGF-1增强了 SKM感觉保护的作用,使IGF-1 +感觉保护组的MyHC1表达量较感觉保护组进一步提高。同时,IGF-1不仅能够直接提升失神经SKM线粒体含量,而且IGF-1还可进一步增强感觉神经再支配对线粒体含量的上调作用。以上结果表明,感觉神经保护可延缓大鼠失神经肌萎缩和线粒体数量的减少,在感觉神经保护的基础上应用IGF-1后,可使感觉神经保护延缓肌萎缩和线粒体数量减少的作用得到明显增强。上述实验结果为进一步探讨IGF-1对SKM感觉保护的增强作用提供了实验依据和新的思路。第二部分IGF-1对具有感觉神经支配的骨骼肌细胞保护作用的体外培养研究维持SKM正常运动功能的神经支配的发生、发展和作用过程是极其复杂的。失神经支配的SKM会即刻丧失其应有的运动功能,而且还由于神经营养作用的缺失而导致SKM很快就会出现萎缩。为了研究延缓失神经支配SKM的策略,本课题利用具有背根神经节(dorsal root ganglion,DRG)神经元感觉神经支配的SKM细胞模型,研究外源性IGF-1对SKM细胞直接作用或通过作用于支配SKM细胞的感觉神经而改善SKM细胞状态的新的治疗策略。在Wistar新生鼠后肢SKM细胞培养2 d后,取15 d胎鼠器官型DRG来建立DRG组织块和分散SKM细胞的联合培养体系,此培养体系将继续培养4 d。本课题的实验分组为:(1)对照组(SKM组),SKM细胞在SKM培养液中培养2d后更换为共培养体系培养液继续培养4d;(2)联合培养组,单纯SKM细胞培养2 d后加入DRG组织块继续共培养4d;(3)SKM + IGF-1组,SKM细胞在培养2d后,更换为共培养体系使用的培养液培养2d,然后在共培养培养液中加入IGF-1(20nmol/L)继续培养2d;(4)联合培养+IGF-1组,单纯SKM细胞培养2 d后,与DRG组织块共培养2 d,此后在联合培养培养液中加入IGF-1继续培养2 d。通过免疫荧光标记检测SKM细胞的长度和表面积以衡量细胞生长状态,通过检测SKM关键收缩蛋白MyHC1的表达量以间接反映SKM细胞的收缩潜能,检测mtDNA/nuDNA比值代表线粒体数量的变化,用MitoTracker Red活体染料标定线粒体的形态,检测胞浆细胞色素C(Cytochrome C,CytC)表达量衡量线粒体完整性。结果显示,IGF-1可直接作用于SKM细胞或通过作用于DRG感觉神经元而间接影响SKM细胞的生长状态;IGF-1可通过影响SKM细胞收缩性蛋白MyHC1的表达来改善SKM细胞的功能状态;IGF-1对SKM细胞线粒体数量、线粒体分裂/融合的动态平衡以及线粒体完整性的维持等多个方面具有重要的促进作用。以上结果表明,作为单因素作用的感觉神经支配或IGF-1可对培养的SKM细胞状态具有一定的影响作用,联合应用感觉神经保护和IGF-1对SKM细胞的效果更为明显。本课题的研究结果为IGF-1对感觉保护的作用提供新的资料和观点,为SKM细胞状态和线粒体之间的关系补充了新的实验资料和依据。第三部分IGF-1保护具有感觉神经支配的骨骼肌细胞的线粒体信号途径IGF-1和感觉神经支配对失神经SKM具有确定的保护作用,对于长时间失神经支配SKM运动功能的恢复具有重要的指导意义,尤其是IGF-1通过增强感觉神经支配信号而对SKM细胞的间接作用研究策略是本研究领域的一个重要发展方向,但IGF-1的这种保护作用机制仍不清楚。但由于失神经支配SKM状态变化和功能丧失的严重性表现在多个方面,又由于IGF-1信号通路对SKM细胞作用靶点的不确定性,致使IGF-1通过增强感觉神经支配信号改善SKM细胞状态的机制探索带来了一定的难度。本课题利用具有DRG神经元感觉神经支配的SKM细胞模型,通过检测IGF-1激活的Akt通路及下游的相关靶点,并运用相应的激活/抑制剂和过表达载体研究相应的机制。结果表明,感觉神经支配并没有激活SKM细胞的Akt通路,感觉神经保护对SKM细胞的改善可能是通过其他途径来实现的。但IGF-1对感觉神经-肌联合培养的SKM细胞Akt磷酸化水平则有显著的上调作用。通过用LY294002抑制Akt磷酸化,进一步确认Akt磷酸化水平的上调是IGF-1诱导的线粒体数量增加、线粒体融合增多、线粒体完整性改善、蛋白水解泛素蛋白酶体途径(ubiquitin-proteasome system,UPS)中泛素连接酶 atrogin-1 和肌环指蛋白 1(muscle RING finger 1,MuRF1)抑制、MyHC1蛋白表达量上升的关键步骤。与未给予IGF-1的感觉保护组相比,应用IGF-1的感觉保护组SKM的Akt磷酸化水平的上调抑制了线粒体外膜蛋白线粒体E3泛素蛋白连接酶1(mitochondrial E3 ubiquitin protein ligase 1,Mull)的表达。为进一步确认Mull在Akt对线粒体及下游改变中发挥的作用,通过构建Mull过表达载体,应用于IGF-1孵育的联合培养体系内,证实了 Mull过表达可抑制SKM细胞的Akt磷酸化水平升高诱导的线粒体状态的改善、蛋白降解途径抑制和SKM收缩蛋白表达的上调,确认了 Mull可能为IGF-1下游作用于线粒体的重要靶点。IGF-1抑制Mull,使SKM细胞线粒体状态改善,能量产生增多,从而抑制了线粒体下游能量感受器5-磷酸腺苷依赖的蛋白激酶 α(adenosine 5'-monophosphate-activated protein kinase α,AMPKΩ)的活化。通过应用1 mmol/LAMPKα激活剂5-氨基咪唑-4-甲酰胺-1-D-核糖核苷酸(5-aminoimidazole-4-carbox-amide-1-D-ribonucleoside,AICAR),进一步确认IGF-1改善线粒体能量产生后,抑制AMPKα活化,进而减少atrogin-1 mRNA、MuRF1 mRNA表达和促进MyHCl蛋白表达。本课题的研究结果明确了在神经-肌联合培养体系中IGF-1对线粒体功能形态和SKM细胞状态的进一步促进作用的机制。IGF-1通过对其下游靶点Akt、Mull以及AMPKα的调控来改善感觉神经再支配SKM细胞的状态。该途径的主要调控机制为IGF-1诱导Akt磷酸化和抑制Mull的表达,从而改善线粒体形态及功能状态,最终通过抑制UPS关键分子atrogin-1和MuRF1的表达来增加收缩性蛋白MyHC1的合成。这些数据为通过调控IGF-1信号通路及线粒体改善长期失神经SKM细胞的功能状态提供了全新的实验依据,同时也为该途径的关键分子的研究开拓了全新的领域。
[Abstract]:The maintenance of normal structure and function of skeletal muscle (SKM) is the basic condition to ensure the normal movement of human body. The stability of protein synthesis and degradation balance maintains the normal structure and function of SKM. The maintenance of this balance depends on the normal innervation. When denervation occurs, the synthesis and degradation of SKM protein are smooth. The balance is broken, resulting in muscular atrophy, and accompanied by a series of serious clinical symptoms, such as loss of motor function. In special cases such as severe motor nerve damage, the nerve innervation of SKM can not be restored in time. Long-term loss of motor innervation can lead to the loss of contraction function of SKM, even if SKM is reinnervated by motor nerve, its motor function will be lost. Therefore, it is very important to restore the innervation of the denervated SKM as soon as possible. The sensory protection of the proximal end of the sensory nerve connected with the distal stump of the motor nerve is a scheme that can not restore the innervation of the motor nerve in time. Insulin-like growth factor-1 (IGF-1), as a multifunctional polypeptide growth factor, has been demonstrated to delay various pathological muscular atrophy and maintain and promote the growth and survival of the nervous system by activating phosphatidylinositol 3-kinase (PI3K) / Akt signaling pathway. Whether IGF-1 enhances the sensory protection of SKM remains to be confirmed. Studies have shown that Akt activation can affect the functional state of mitochondria, and the function of mitochondria is closely related to its morphology. Mitochondria continue to change between splitting and fusion states. Abnormal morphology of mitochondria can lead to many diseases. However, IGF-1 can affect the sensory protection mode of SKM. In this study, the effects of IGF-1 on sensory protection of SKM and its related mechanisms were studied by establishing the animal model of somatosensory innervation and the SKM cell model with sensory innervation in vitro. The results of this study will provide a new theory for delaying the treatment of denervated muscular atrophy. The first part is about the protective effect of IGF-1 on skeletal muscle by sensory nerve reinnervation in vivo. If SKM loses its motor nerve reinnervation for too long, its motor function will not be restored even if SKM is reinnervation again. This technique is called sensory protection. Although motor function can not be restored, sensory protection can protect the contractile function and important structure of the motor nerve. After the motor nerve can be restored to normal, the motor nerve can be closed to the motor nerve. However, the effect of sensory protection on denervated SKM is limited and needs to be strengthened. As a multifunctional polypeptide factor, IGF-1 has protective effects on both SKM and nervous system, but whether IGF-1 can enhance the sensory protection of SKM remains to be further studied. Changes in mitochondrial function are closely related to the maintenance of normal mitochondrial function, and this change is closely related to the function of SKM. The role of IGF-1 in these changes is not clear at present. Based on the above research background, the following experiments were designed to study. 25 Wistar male rats of 260 + 10 g were randomly divided into two groups. Methods: (1) Sham operation group: longitudinal incision was made in the hind limbs of rats, sciatic nerve bifurcation was exposed and sutured; (2) denervation group: tibial nerve was severed after the same incision, proximal stump ligation and suture into plastic cap after suture; (3) IGF-1 group: the operation was the same as denervation group, tibial nerve was severed after suture knife mouth, 8 weeks after the operation more gastrocnemius muscle every 3 days. (4) Sensory protection group: the tibial nerve and sural nerve were severed after longitudinal incision, the proximal stump of the sural nerve and the distal stump of the tibial nerve were sutured, and the proximal stump of the tibial nerve was also sutured into the plastic cap to suture the knife edge; (5) IGF-1 + sensory protection group, the operation was the same as sensory protection group, the application time and location of IGF-1. The results showed that muscle atrophy was most intuitively reflected as muscle wet weight and cross-sectional area (CSA). In denervated group, there was a significant decrease, while in IGF-1 group and sensory protection group increased, while in IGF-1 + sensory protection group the increase was the greatest. The change trend of myosin heavy chain 1 (MyHC1) was consistent with the wet weight and cross-sectional area of gastrocnemius muscle. In denervated group, the decrease was most obvious. IGF-1 enhanced the sensory protective effect of SKM, and further increased the expression of MyHC1 in the sensory protective group compared with the sensory protective group. These results suggest that sensory nerve protection can delay denervated muscle atrophy and mitochondrial decrease in rats, and sensory nerve protection can be delayed by the application of IGF-1 on the basis of sensory nerve protection. Muscle atrophy and mitochondrial decrease were significantly enhanced. These results provide experimental evidence and new ideas for further exploring the sensory protective effect of IGF-1 on SKM. Part II In vitro study of the protective effect of IGF-1 on skeletal muscle cells with sensory innervation to maintain normal motor function of SKM The occurrence, development and action of denervation are extremely complex. The denervated SKM immediately loses its motor function and soon atrophies due to the absence of neurotrophic function. In order to study the strategy of delaying denervation of SKM, dorsal root ganglion (DRG) is used in this study. NGlion, DRG) neuron sensory innervation of SKM cell model, to study exogenous IGF-1 on SKM cells directly or through the role of sensory nerve innervation of SKM cells to improve the state of SKM cells new treatment strategies. Wistar newborn rat hindlimb SKM cells cultured for 2 days, take 15 days fetal rat organotype DRG to establish DRG tissue blocks and disperse SKM cells. The experimental groups of this subject are: (1) control group (SKM group), SKM cells were cultured in SKM medium for 2 days and then replaced by co-culture medium for 4 days; (2) co-culture group, SKM cells were cultured in DRG tissue block for 4 days after 2 days; (3) SKM + IGF-1 group, SKM + IGF-1 group M cells were cultured in co-culture medium for 2 days, then added with IGF-1 (20 nmol/L) for 2 days; (4) co-culture + IGF-1 group, SKM cells were co-cultured with DRG tissue block for 2 days after 2 days, and then co-cultured with IGF-1 in co-culture medium for 2 days. The length and surface area of SKM cells were measured by labeling. The contractile potential of SKM cells was indirectly reflected by detecting the expression of MyHC1, the ratio of mtDNA to nuDNA, the number of mitochondria, the morphology of mitochondria and cytochrome C (Cytochrome C) were measured by MitoTracker Red dye in vivo. The results showed that IGF-1 could directly affect SKM cells or indirectly affect the growth of SKM cells by acting on DRG sensory neurons; IGF-1 could improve the functional status of SKM cells by affecting the expression of contractile protein MyHC 1; and IGF-1 could improve the mitochondrial number and mitochondria of SKM cells. These results suggest that sensory innervation or IGF-1, as a single factor, can affect the state of cultured SKM cells to some extent, and the combination of sensory nerve protection and IGF-1 is more effective in SKM cells. The results of this study provide new data and viewpoints for the role of IGF-1 in sensory protection, and provide new experimental data and evidence for the relationship between SKM cell state and mitochondria. Part III The protective effect of IGF-1 on the mitochondrial signaling pathway of skeletal muscle cells with sensory innervation and the loss of sensory innervation. Neural SKM has a definite protective effect, which is of great significance to the recovery of motor function of long-term denervated SKM. In particular, the indirect effect of IGF-1 on SKM cells by enhancing sensory innervation signal is an important research direction in this field, but the protective mechanism of IGF-1 is still not clear. However, the severity of the changes of denervated SKM state and the loss of function is manifested in many aspects, and the uncertainty of the target of IGF-1 signaling pathway on SKM cells leads to the difficulty of exploring the mechanism of IGF-1 improving SKM cell state by enhancing sensory innervation signals. The results showed that sensory innervation did not activate the Akt pathway of SKM cells, and the improvement of sensory nerve protection on SKM cells might be through the detection of the Akt pathway activated by IGF-1 and related downstream targets, and the corresponding activation/inhibitor and overexpression vector. However, IGF-1 can significantly up-regulate the phosphorylation of Akt in SKM cells co-cultured with sensory nerves and muscles. By inhibiting Akt phosphorylation with LY294002, it is further confirmed that the up-regulation of Akt phosphorylation is due to the increase of mitochondrial number, mitochondrial fusion, mitochondrial integrity and ubiquitous protein hydrolysis induced by IGF-1. Inhibition of ubiquitin-proteasome system (UPS) ubiquitin-ligase Atrogin-1 and myRING finger-1 (MuRF1) is a key step in the increase of MyHC1 protein expression. The up-regulation of Akt phosphorylation in SKM treated with IGF-1 inhibited mitochondria compared with the sensory protection group without IGF-1. Expression of mitochondrial E3 ubiquitin protein ligase 1 (Mull) in vitro. To further confirm the role of Mull in mitochondrial and downstream alterations induced by Akt, Mull overexpression vector was constructed and applied to the co-culture system incubated with IGF-1. It was confirmed that Mull overexpression could inhibit SKM fineness. Mull may be an important target for downstream action of IGF-1 on mitochondria. IGF-1 inhibits Mull, improves mitochondrial state and increases energy production in SKM cells, thus inhibiting downstream energy perception in mitochondria. Activation of adenosine 5'-monophosphate-activated protein kinase alpha (AMPK_) by receptor 5-adenosine 5'-monophosphate-activated protein kinase alpha (AMPK_). Improvement of mitochondrial energy by using 1 mmol/LAMPK alpha activator 5-aminoimidazole-4-formamide-1-D-ribonucleoside (AICAR) was further confirmed. After production, it inhibits the activation of AMPK alpha, thereby reducing the expression of Atrogin-1 mRNA, MuRF1 mRNA and promoting the expression of MyHCl protein.The results of this study clarify the further mechanism of IGF-1 in promoting the functional morphology of mitochondria and the state of SKM cells in the neuromuscular co-culture system. The main regulatory mechanism of this pathway is that IGF-1 induces Akt phosphorylation and inhibits Mull expression, thereby improving mitochondrial morphology and functional status, and ultimately increasing the synthesis of contractile protein MyHC 1 by inhibiting the expression of the key UPS molecules Atrogin-1 and MuRF 1. Controlling the IGF-1 signaling pathway and mitochondria to improve the functional status of long-term denervated SKM cells provides a new experimental basis, but also for this
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R685
本文编号：2234476
[Abstract]:The maintenance of normal structure and function of skeletal muscle (SKM) is the basic condition to ensure the normal movement of human body. The stability of protein synthesis and degradation balance maintains the normal structure and function of SKM. The maintenance of this balance depends on the normal innervation. When denervation occurs, the synthesis and degradation of SKM protein are smooth. The balance is broken, resulting in muscular atrophy, and accompanied by a series of serious clinical symptoms, such as loss of motor function. In special cases such as severe motor nerve damage, the nerve innervation of SKM can not be restored in time. Long-term loss of motor innervation can lead to the loss of contraction function of SKM, even if SKM is reinnervated by motor nerve, its motor function will be lost. Therefore, it is very important to restore the innervation of the denervated SKM as soon as possible. The sensory protection of the proximal end of the sensory nerve connected with the distal stump of the motor nerve is a scheme that can not restore the innervation of the motor nerve in time. Insulin-like growth factor-1 (IGF-1), as a multifunctional polypeptide growth factor, has been demonstrated to delay various pathological muscular atrophy and maintain and promote the growth and survival of the nervous system by activating phosphatidylinositol 3-kinase (PI3K) / Akt signaling pathway. Whether IGF-1 enhances the sensory protection of SKM remains to be confirmed. Studies have shown that Akt activation can affect the functional state of mitochondria, and the function of mitochondria is closely related to its morphology. Mitochondria continue to change between splitting and fusion states. Abnormal morphology of mitochondria can lead to many diseases. However, IGF-1 can affect the sensory protection mode of SKM. In this study, the effects of IGF-1 on sensory protection of SKM and its related mechanisms were studied by establishing the animal model of somatosensory innervation and the SKM cell model with sensory innervation in vitro. The results of this study will provide a new theory for delaying the treatment of denervated muscular atrophy. The first part is about the protective effect of IGF-1 on skeletal muscle by sensory nerve reinnervation in vivo. If SKM loses its motor nerve reinnervation for too long, its motor function will not be restored even if SKM is reinnervation again. This technique is called sensory protection. Although motor function can not be restored, sensory protection can protect the contractile function and important structure of the motor nerve. After the motor nerve can be restored to normal, the motor nerve can be closed to the motor nerve. However, the effect of sensory protection on denervated SKM is limited and needs to be strengthened. As a multifunctional polypeptide factor, IGF-1 has protective effects on both SKM and nervous system, but whether IGF-1 can enhance the sensory protection of SKM remains to be further studied. Changes in mitochondrial function are closely related to the maintenance of normal mitochondrial function, and this change is closely related to the function of SKM. The role of IGF-1 in these changes is not clear at present. Based on the above research background, the following experiments were designed to study. 25 Wistar male rats of 260 + 10 g were randomly divided into two groups. Methods: (1) Sham operation group: longitudinal incision was made in the hind limbs of rats, sciatic nerve bifurcation was exposed and sutured; (2) denervation group: tibial nerve was severed after the same incision, proximal stump ligation and suture into plastic cap after suture; (3) IGF-1 group: the operation was the same as denervation group, tibial nerve was severed after suture knife mouth, 8 weeks after the operation more gastrocnemius muscle every 3 days. (4) Sensory protection group: the tibial nerve and sural nerve were severed after longitudinal incision, the proximal stump of the sural nerve and the distal stump of the tibial nerve were sutured, and the proximal stump of the tibial nerve was also sutured into the plastic cap to suture the knife edge; (5) IGF-1 + sensory protection group, the operation was the same as sensory protection group, the application time and location of IGF-1. The results showed that muscle atrophy was most intuitively reflected as muscle wet weight and cross-sectional area (CSA). In denervated group, there was a significant decrease, while in IGF-1 group and sensory protection group increased, while in IGF-1 + sensory protection group the increase was the greatest. The change trend of myosin heavy chain 1 (MyHC1) was consistent with the wet weight and cross-sectional area of gastrocnemius muscle. In denervated group, the decrease was most obvious. IGF-1 enhanced the sensory protective effect of SKM, and further increased the expression of MyHC1 in the sensory protective group compared with the sensory protective group. These results suggest that sensory nerve protection can delay denervated muscle atrophy and mitochondrial decrease in rats, and sensory nerve protection can be delayed by the application of IGF-1 on the basis of sensory nerve protection. Muscle atrophy and mitochondrial decrease were significantly enhanced. These results provide experimental evidence and new ideas for further exploring the sensory protective effect of IGF-1 on SKM. Part II In vitro study of the protective effect of IGF-1 on skeletal muscle cells with sensory innervation to maintain normal motor function of SKM The occurrence, development and action of denervation are extremely complex. The denervated SKM immediately loses its motor function and soon atrophies due to the absence of neurotrophic function. In order to study the strategy of delaying denervation of SKM, dorsal root ganglion (DRG) is used in this study. NGlion, DRG) neuron sensory innervation of SKM cell model, to study exogenous IGF-1 on SKM cells directly or through the role of sensory nerve innervation of SKM cells to improve the state of SKM cells new treatment strategies. Wistar newborn rat hindlimb SKM cells cultured for 2 days, take 15 days fetal rat organotype DRG to establish DRG tissue blocks and disperse SKM cells. The experimental groups of this subject are: (1) control group (SKM group), SKM cells were cultured in SKM medium for 2 days and then replaced by co-culture medium for 4 days; (2) co-culture group, SKM cells were cultured in DRG tissue block for 4 days after 2 days; (3) SKM + IGF-1 group, SKM + IGF-1 group M cells were cultured in co-culture medium for 2 days, then added with IGF-1 (20 nmol/L) for 2 days; (4) co-culture + IGF-1 group, SKM cells were co-cultured with DRG tissue block for 2 days after 2 days, and then co-cultured with IGF-1 in co-culture medium for 2 days. The length and surface area of SKM cells were measured by labeling. The contractile potential of SKM cells was indirectly reflected by detecting the expression of MyHC1, the ratio of mtDNA to nuDNA, the number of mitochondria, the morphology of mitochondria and cytochrome C (Cytochrome C) were measured by MitoTracker Red dye in vivo. The results showed that IGF-1 could directly affect SKM cells or indirectly affect the growth of SKM cells by acting on DRG sensory neurons; IGF-1 could improve the functional status of SKM cells by affecting the expression of contractile protein MyHC 1; and IGF-1 could improve the mitochondrial number and mitochondria of SKM cells. These results suggest that sensory innervation or IGF-1, as a single factor, can affect the state of cultured SKM cells to some extent, and the combination of sensory nerve protection and IGF-1 is more effective in SKM cells. The results of this study provide new data and viewpoints for the role of IGF-1 in sensory protection, and provide new experimental data and evidence for the relationship between SKM cell state and mitochondria. Part III The protective effect of IGF-1 on the mitochondrial signaling pathway of skeletal muscle cells with sensory innervation and the loss of sensory innervation. Neural SKM has a definite protective effect, which is of great significance to the recovery of motor function of long-term denervated SKM. In particular, the indirect effect of IGF-1 on SKM cells by enhancing sensory innervation signal is an important research direction in this field, but the protective mechanism of IGF-1 is still not clear. However, the severity of the changes of denervated SKM state and the loss of function is manifested in many aspects, and the uncertainty of the target of IGF-1 signaling pathway on SKM cells leads to the difficulty of exploring the mechanism of IGF-1 improving SKM cell state by enhancing sensory innervation signals. The results showed that sensory innervation did not activate the Akt pathway of SKM cells, and the improvement of sensory nerve protection on SKM cells might be through the detection of the Akt pathway activated by IGF-1 and related downstream targets, and the corresponding activation/inhibitor and overexpression vector. However, IGF-1 can significantly up-regulate the phosphorylation of Akt in SKM cells co-cultured with sensory nerves and muscles. By inhibiting Akt phosphorylation with LY294002, it is further confirmed that the up-regulation of Akt phosphorylation is due to the increase of mitochondrial number, mitochondrial fusion, mitochondrial integrity and ubiquitous protein hydrolysis induced by IGF-1. Inhibition of ubiquitin-proteasome system (UPS) ubiquitin-ligase Atrogin-1 and myRING finger-1 (MuRF1) is a key step in the increase of MyHC1 protein expression. The up-regulation of Akt phosphorylation in SKM treated with IGF-1 inhibited mitochondria compared with the sensory protection group without IGF-1. Expression of mitochondrial E3 ubiquitin protein ligase 1 (Mull) in vitro. To further confirm the role of Mull in mitochondrial and downstream alterations induced by Akt, Mull overexpression vector was constructed and applied to the co-culture system incubated with IGF-1. It was confirmed that Mull overexpression could inhibit SKM fineness. Mull may be an important target for downstream action of IGF-1 on mitochondria. IGF-1 inhibits Mull, improves mitochondrial state and increases energy production in SKM cells, thus inhibiting downstream energy perception in mitochondria. Activation of adenosine 5'-monophosphate-activated protein kinase alpha (AMPK_) by receptor 5-adenosine 5'-monophosphate-activated protein kinase alpha (AMPK_). Improvement of mitochondrial energy by using 1 mmol/LAMPK alpha activator 5-aminoimidazole-4-formamide-1-D-ribonucleoside (AICAR) was further confirmed. After production, it inhibits the activation of AMPK alpha, thereby reducing the expression of Atrogin-1 mRNA, MuRF1 mRNA and promoting the expression of MyHCl protein.The results of this study clarify the further mechanism of IGF-1 in promoting the functional morphology of mitochondria and the state of SKM cells in the neuromuscular co-culture system. The main regulatory mechanism of this pathway is that IGF-1 induces Akt phosphorylation and inhibits Mull expression, thereby improving mitochondrial morphology and functional status, and ultimately increasing the synthesis of contractile protein MyHC 1 by inhibiting the expression of the key UPS molecules Atrogin-1 and MuRF 1. Controlling the IGF-1 signaling pathway and mitochondria to improve the functional status of long-term denervated SKM cells provides a new experimental basis, but also for this
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R685
【参考文献】
相关期刊论文 前1条
1 Gang Li;Qing-shan Li;Wen-bin Li;Jian Wei;Wen-kai Chang;Zhi Chen;Hu-yun Qiao;Ying-wei Jia;Jiang-hua Tian;Bing-sheng Liang;;miRNA targeted signaling pathway in the early stage of denervated fast and slow muscle atrophy[J];Neural Regeneration Research;2016年08期
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