【摘要】：第一部分Nesprin-1在C2C12肌原細(xì)胞系分化過程中的作用 目的探討Nesprin-1,一個(gè)新發(fā)現(xiàn)的含多個(gè)血影蛋白重復(fù)序列的細(xì)胞骨架蛋白家族成員,在肌細(xì)胞分化過程中的作用。 方法 1.將C2C12肌原細(xì)胞細(xì)胞分化成肌小管。 2.采用Western-blotting和免疫熒光技術(shù)檢測(cè)Nesprin-1蛋白表達(dá)水平和亞細(xì)胞定位在肌小管分化成熟過程中的改變。 3.利用RNA干擾技術(shù)抑制Nesprin-1蛋白的表達(dá),觀察對(duì)C2C12肌原細(xì)胞分化的影響。 結(jié)果 1.Western-blotting結(jié)果顯示nesprin-1各亞型在C2C12肌原細(xì)胞系分化過程中表達(dá)水平有所改變。 2.免疫熒光結(jié)果顯示nesprin-1各亞型在C2C12肌原細(xì)胞系分化過程中細(xì)胞中定位有明顯改變。 3. Nesprin-1 siRNA組與對(duì)照組相比,分化前后C2C12細(xì)胞中nesprin-1蛋白表達(dá)明顯降低。 4. Nesprin-1 siRNA組與對(duì)照組相比,分化后肌組織分化成熟的蛋白標(biāo)志肌球蛋白(myosin)的表達(dá)量以及肌小節(jié)的數(shù)量比對(duì)照組均明顯減少(P0.001)。 結(jié)論nesprin-1對(duì)骨胳肌細(xì)胞分化至關(guān)重要,不同分子大小nesprin-1亞型在C2C12肌原細(xì)胞分化過程中發(fā)揮各自特殊作用,在該細(xì)胞質(zhì)與細(xì)胞核中作用不同。 第二部分Nesprin-1在小鼠胚胎干細(xì)胞分化為心肌細(xì)胞過程中的作用 目的探討nesprin-1在胚胎干細(xì)胞分化為心肌細(xì)胞過程中的作用。 方法 1.采用懸滴-懸浮-貼壁三步法,5-氮雜胞苷(5-azacytidine ,5-aza)與丹參共同誘導(dǎo)胚胎干細(xì)胞分化為心肌細(xì)胞。 2.采用western-blotting與免疫熒光技術(shù)檢測(cè)nesprin-1蛋白表達(dá)水平和在分化成熟過程中的改變。 3.同時(shí)利用RNA干擾(RNAi)技術(shù)抑制nesprin-1蛋白的表達(dá)。Ⅰ組(RNA干擾目的序列AAAGCCAAGCACGCAACTA),Ⅱ組(RNA干擾目的序列GGGAACCAACAGTGAGATT),Ⅲ組(RNA干擾目的序列ACCAGGACATTGCGTACTA),Ⅳ組對(duì)照組,觀察對(duì)胚胎干細(xì)胞分化的影響。 結(jié)果 1.Nesprin-1各亞型在胚胎干細(xì)胞分化為心肌細(xì)胞過程中的表達(dá)水平有所變化。 2.各干擾組分化后心肌組織分化成熟的蛋白標(biāo)志肌球蛋白(myosin)的表達(dá)量減少。 3.各組心肌細(xì)胞分化率分別為(17.78±1.92)%,(36.67±3.34)%, (44.42±5.08)%,(77.78±1.92)%,各干擾組與對(duì)照組相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 本文首次證實(shí)nesprin-1亞型在胚胎干細(xì)胞分化為心肌細(xì)胞過程中至關(guān)重要,不同分子大小的nesprin-1亞型在此分化過程中發(fā)揮各自特殊的作用,某些亞型可能有更加重要的作用。
[Abstract]:Part I the role of Nesprin-1 in the differentiation of C2C12 myogenic cell lines objective to investigate the role of a newly discovered member of Nesprin-1, cytoskeleton family containing multiple repeat sequences of hematocapsid proteins in the process of myocyte differentiation. Method 1. Differentiation of C2C12 myogenic cells into myotubules. 2. Western-blotting and immunofluorescence techniques were used to detect the changes of Nesprin-1 protein expression and subcellular localization during the differentiation and maturation of myotubule. RNA interference technique was used to inhibit the expression of Nesprin-1 protein and to observe the effect on the differentiation of C2C12 myogenic cells. Results 1.Western-blotting results showed that the expression level of nesprin-1 subtypes changed during the differentiation of C2C12 myogenic cell lines. 2. The results of immunofluorescence showed that the localization of nesprin-1 subtypes in C2C12 myogenic cell line was significantly changed. 3. 3. Compared with the control group, the expression of nesprin-1 protein in C2C12 cells decreased significantly in Nesprin-1 siRNA group before and after differentiation. 4. 4. Compared with the control group, the expression of myosin (myosin) and the number of muscle sections in the Nesprin-1 siRNA group were significantly lower than those in the control group (P0.001). Conclusion nesprin-1 plays an important role in the differentiation of skeletal muscle cells. Nesprin-1 subtypes of different molecular sizes play a special role in the differentiation of C2C12 myogenic cells and play different roles in the cytoplasm and nucleus. The role of Nesprin-1 in the differentiation of murine embryonic stem cells into cardiomyocytes objective to investigate the role of nesprin-1 in the differentiation of embryonic stem cells into cardiomyocytes. Method 1. 5-azacytidine 5-azacytidine (5-azacytidine 5-aza) and salvia miltiorrhiza (Salvia miltiorrhiza) were used to induce embryonic stem cells to differentiate into cardiomyocytes. 2. Western-blotting and immunofluorescence techniques were used to detect the expression level of nesprin-1 protein and its changes during differentiation and maturation. At the same time, RNA interference (RNAi) technique was used to inhibit the expression of nesprin-1 protein. Group 鈪，

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