本文選題：Sidt2 + 骨骼肌肌病　； 參考：《皖南醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:通過構(gòu)建Sidt2基因剔除小鼠模型,觀察Sidt2剔除后對(duì)小鼠骨骼肌的影響,探討其所致骨骼肌肌病的具體發(fā)病機(jī)制。方法:運(yùn)用Cre-loxp系統(tǒng)建立Sidt2基因條件性剔除小鼠模型,通過HE染色、PAS染色、COX染色、免疫熒光檢測(cè)Laminin等技術(shù)觀察Sidt2剔除后骨骼肌的病理學(xué)改變,此外通過電鏡觀察Sidt2剔除后骨骼肌組織超微結(jié)構(gòu)的變化。接著運(yùn)用Western blot、免疫熒光技術(shù)檢測(cè)自噬過程中關(guān)鍵蛋白以及自噬調(diào)控途徑關(guān)鍵蛋白的變化,然后通過饑餓誘導(dǎo)自噬發(fā)生后,再次檢測(cè)自噬過程中的關(guān)鍵蛋白及自噬調(diào)控途徑關(guān)鍵蛋白的變化,觀察Sidt2缺失對(duì)骨骼肌自噬的影響。同時(shí)運(yùn)用Western blot檢測(cè)凋亡的三種途徑過程中的關(guān)鍵蛋白表達(dá)水平的變化,以及通過TUNEL法檢測(cè)凋亡,觀察Sidt2剔除后對(duì)凋亡的影響。結(jié)果:首先我們成功獲得了骨骼肌Sidt2剔除的小鼠模型,并通過DNA、RNA、蛋白水平鑒定造模成功。其次我們通過光鏡及電鏡下觀察到,骨骼肌Sidt2剔除的小鼠模型存在嚴(yán)重的骨骼肌病變,通過HE染色后在光鏡下可以觀察到Sidt2剔除組骨骼肌的變性、炎性浸潤及肌纖維尺寸的縮小甚至肌纖維的萎縮;通過PAS染色后在光鏡下可以觀察到Sidt2剔除組骨骼肌存在著大量的糖原積累;通過COX染色后在光鏡下可以觀察到Sidt2剔除組骨骼肌細(xì)胞色素氧化酶明顯減少;通過免疫熒光檢測(cè)Laminin在Sidt2剔除組可觀察到肌纖維尺寸的縮小,肌膜的異常;在電鏡下可以看到Sidt2剔除組骨骼肌細(xì)胞的結(jié)構(gòu)紊亂,線粒體的腫脹與堆積,大量的自體吞噬泡的堆積以及細(xì)胞核染色質(zhì)的凝集與邊集等大量病理改變。接著Western blot檢測(cè)自噬關(guān)鍵蛋白發(fā)現(xiàn)Sidt2剔除組的LC3II、P62、Beclin1、Atg12等表達(dá)量增加,自噬調(diào)控途徑的關(guān)鍵蛋白mTOR、S6K1、Akt的磷酸化是減少的,而Foxo3的表達(dá)含量無明顯變化。通過骨骼肌冰凍切片的免疫熒光進(jìn)行Laminin與P62免疫雙標(biāo),可以更加直接的觀察到Sidt2剔除后導(dǎo)致了P62的堆積。緊接著通過饑餓誘導(dǎo)自噬發(fā)生后,在正常對(duì)照組中可以發(fā)現(xiàn)饑餓誘導(dǎo)自噬發(fā)生后會(huì)導(dǎo)致LC3II的增加,P62含量減少及磷酸化的Akt、磷酸化的mTOR及磷酸化的S6K1減少,而Sidt2剔除組中饑餓誘導(dǎo)并沒有出現(xiàn)上述的變化。同時(shí)通過Western blot檢測(cè)凋亡途徑的關(guān)鍵蛋白發(fā)現(xiàn)Sidt2剔除組的Caspase3、Caspase9、Caspase12、Chop、Bip、P-eIF2a、P-perk、ATF6、Bax、Bad等關(guān)鍵蛋白的表達(dá)含量增加,此外通過TUNEL試劑盒檢測(cè)凋亡,發(fā)現(xiàn)Sidt2剔除組的骨骼肌凋亡明顯增加。結(jié)論:Sidt2基因缺失后會(huì)導(dǎo)致嚴(yán)重的骨骼肌肌病,一方面是由于Sidt2缺失后導(dǎo)致了骨骼肌的自噬紊亂,包括自噬降解階段受阻及自噬誘導(dǎo)階段受損;另一方面是由于Sidt2缺失后導(dǎo)致了骨骼肌的凋亡的激活,主要是線粒體途徑和內(nèi)質(zhì)網(wǎng)應(yīng)激途徑介導(dǎo)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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R685

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