Sidt2介导的自噬紊乱及其相关骨骼肌病

发布时间：2018-06-20 12:48

  本文选题：Sidt2 + 骨骼肌肌病　； 参考：《皖南医学院》2017年硕士论文

【摘要】：目的:通过构建Sidt2基因剔除小鼠模型,观察Sidt2剔除后对小鼠骨骼肌的影响,探讨其所致骨骼肌肌病的具体发病机制。方法:运用Cre-loxp系统建立Sidt2基因条件性剔除小鼠模型,通过HE染色、PAS染色、COX染色、免疫荧光检测Laminin等技术观察Sidt2剔除后骨骼肌的病理学改变,此外通过电镜观察Sidt2剔除后骨骼肌组织超微结构的变化。接着运用Western blot、免疫荧光技术检测自噬过程中关键蛋白以及自噬调控途径关键蛋白的变化,然后通过饥饿诱导自噬发生后,再次检测自噬过程中的关键蛋白及自噬调控途径关键蛋白的变化,观察Sidt2缺失对骨骼肌自噬的影响。同时运用Western blot检测凋亡的三种途径过程中的关键蛋白表达水平的变化,以及通过TUNEL法检测凋亡,观察Sidt2剔除后对凋亡的影响。结果:首先我们成功获得了骨骼肌Sidt2剔除的小鼠模型,并通过DNA、RNA、蛋白水平鉴定造模成功。其次我们通过光镜及电镜下观察到,骨骼肌Sidt2剔除的小鼠模型存在严重的骨骼肌病变,通过HE染色后在光镜下可以观察到Sidt2剔除组骨骼肌的变性、炎性浸润及肌纤维尺寸的缩小甚至肌纤维的萎缩;通过PAS染色后在光镜下可以观察到Sidt2剔除组骨骼肌存在着大量的糖原积累;通过COX染色后在光镜下可以观察到Sidt2剔除组骨骼肌细胞色素氧化酶明显减少;通过免疫荧光检测Laminin在Sidt2剔除组可观察到肌纤维尺寸的缩小,肌膜的异常;在电镜下可以看到Sidt2剔除组骨骼肌细胞的结构紊乱,线粒体的肿胀与堆积,大量的自体吞噬泡的堆积以及细胞核染色质的凝集与边集等大量病理改变。接着Western blot检测自噬关键蛋白发现Sidt2剔除组的LC3II、P62、Beclin1、Atg12等表达量增加,自噬调控途径的关键蛋白mTOR、S6K1、Akt的磷酸化是减少的,而Foxo3的表达含量无明显变化。通过骨骼肌冰冻切片的免疫荧光进行Laminin与P62免疫双标,可以更加直接的观察到Sidt2剔除后导致了P62的堆积。紧接着通过饥饿诱导自噬发生后,在正常对照组中可以发现饥饿诱导自噬发生后会导致LC3II的增加,P62含量减少及磷酸化的Akt、磷酸化的mTOR及磷酸化的S6K1减少,而Sidt2剔除组中饥饿诱导并没有出现上述的变化。同时通过Western blot检测凋亡途径的关键蛋白发现Sidt2剔除组的Caspase3、Caspase9、Caspase12、Chop、Bip、P-eIF2a、P-perk、ATF6、Bax、Bad等关键蛋白的表达含量增加,此外通过TUNEL试剂盒检测凋亡,发现Sidt2剔除组的骨骼肌凋亡明显增加。结论:Sidt2基因缺失后会导致严重的骨骼肌肌病,一方面是由于Sidt2缺失后导致了骨骼肌的自噬紊乱,包括自噬降解阶段受阻及自噬诱导阶段受损;另一方面是由于Sidt2缺失后导致了骨骼肌的凋亡的激活,主要是线粒体途径和内质网应激途径介导的凋亡。
[Abstract]:Objective: to study the effect of Sidt2 gene knockout on skeletal muscle of mice, and to explore the mechanism of the skeletal muscle myopathy induced by Sidt2 gene knockout. Methods: Sidt2 gene conditioned knockout mice model was established by Cre-loxp system. The pathological changes of skeletal muscle after Sidt2 deletion were observed by HE staining, pas staining and Cox staining. In addition, the ultrastructural changes of skeletal muscle after Sidt 2 removal were observed by electron microscope. Then Western blot and immunofluorescence technique were used to detect the changes of key proteins and key proteins of autophagy regulation pathway during autophagy, and then induced autophagy by starvation. The changes of key proteins in autophagy and autophagy regulation pathway were detected again to observe the effect of Sidt2 deletion on skeletal muscle autophagy. At the same time, Western blot was used to detect the expression of key proteins in the three pathways of apoptosis, and Tunel was used to detect the effect of Sidt2 knockout on apoptosis. Results: first of all, we successfully obtained the mouse model of skeletal muscle Sidt2 knockout, and successfully identified the model by DNA-RNA and protein level. Secondly, we observed that there was serious skeletal muscle lesion in Sidt2 knockout mice model by light and electron microscope, and the degeneration of skeletal muscle in Sidt2 knockout group could be observed by HE staining under light microscope. After pas staining, a large amount of glycogen accumulation was observed in the skeletal muscle of Sidt2 knockout group. The cytochrome oxidase of skeletal muscle in Sidt2 knockout group was obviously decreased after Cox staining, and the size of muscle fiber and the abnormality of myomembrane could be observed in Sidt2 knockout group by immunofluorescence detection. The structural disorder of skeletal muscle cells, the swelling and accumulation of mitochondria, the accumulation of autologous phagocytosis and nuclear chromatin agglutination and edge agglutination were observed under electron microscope in Sidt2 knockout group. Then Western blot analysis showed that the expression of LC3IIP62P / Beclin1 / Atg12 in Sidt2 knockout group was increased, and the phosphorylation of mTORS6K1 / Akt, the key protein of autophagy regulation pathway, was decreased, but the expression of Foxo3 was not changed significantly. The double labeling of laminin and P62 by immunofluorescence of frozen sections of skeletal muscle can be observed more directly after Sidt2 is removed which leads to the accumulation of P62. Then, after induced autophagy by starvation, it was found that starvation induced autophagy could increase the content of LC3II and decrease the content of phosphorylated Akt, phosphorylated mTOR and phosphorylated S6K1 in normal control group. These changes were not observed in the Sidt2 knockout group. At the same time, the expression of Caspase3 (Caspase3) and Caspase9 (Caspase9) in Sidt2 knockout group was found to be increased by Western blot. The expression of the key proteins such as P-eIF2aHATF6Bax-Bad in Sidt2 knockout group was also found to be increased, and the apoptosis of skeletal muscle in Sidt2 knockout group was significantly increased by Tunel assay. Conclusion the deletion of the 1: Sidt2 gene can lead to serious skeletal myopathy. On the one hand, the loss of Sidt2 gene leads to the dysfunction of skeletal muscle autophagy, including the inhibition of autophagy degradation stage and the damage of autophagy induction stage. On the other hand, the activation of skeletal muscle apoptosis was caused by Sidt2 deletion, which was mainly mediated by mitochondrial pathway and endoplasmic reticulum stress pathway.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R685

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