Nesprin-1在胚胎干细胞分化为心肌细胞过程中的作用
发布时间：2018-08-26 07:32
【摘要】：第一部分Nesprin-1在C2C12肌原细胞系分化过程中的作用 目的探讨Nesprin-1,一个新发现的含多个血影蛋白重复序列的细胞骨架蛋白家族成员,在肌细胞分化过程中的作用。 方法 1.将C2C12肌原细胞细胞分化成肌小管。 2.采用Western-blotting和免疫荧光技术检测Nesprin-1蛋白表达水平和亚细胞定位在肌小管分化成熟过程中的改变。 3.利用RNA干扰技术抑制Nesprin-1蛋白的表达,观察对C2C12肌原细胞分化的影响。 结果 1.Western-blotting结果显示nesprin-1各亚型在C2C12肌原细胞系分化过程中表达水平有所改变。 2.免疫荧光结果显示nesprin-1各亚型在C2C12肌原细胞系分化过程中细胞中定位有明显改变。 3. Nesprin-1 siRNA组与对照组相比,分化前后C2C12细胞中nesprin-1蛋白表达明显降低。 4. Nesprin-1 siRNA组与对照组相比,分化后肌组织分化成熟的蛋白标志肌球蛋白(myosin)的表达量以及肌小节的数量比对照组均明显减少(P0.001)。 结论nesprin-1对骨胳肌细胞分化至关重要,不同分子大小nesprin-1亚型在C2C12肌原细胞分化过程中发挥各自特殊作用,在该细胞质与细胞核中作用不同。 第二部分Nesprin-1在小鼠胚胎干细胞分化为心肌细胞过程中的作用 目的探讨nesprin-1在胚胎干细胞分化为心肌细胞过程中的作用。 方法 1.采用悬滴-悬浮-贴壁三步法,5-氮杂胞苷(5-azacytidine ,5-aza)与丹参共同诱导胚胎干细胞分化为心肌细胞。 2.采用western-blotting与免疫荧光技术检测nesprin-1蛋白表达水平和在分化成熟过程中的改变。 3.同时利用RNA干扰(RNAi)技术抑制nesprin-1蛋白的表达。Ⅰ组(RNA干扰目的序列AAAGCCAAGCACGCAACTA),Ⅱ组(RNA干扰目的序列GGGAACCAACAGTGAGATT),Ⅲ组(RNA干扰目的序列ACCAGGACATTGCGTACTA),Ⅳ组对照组,观察对胚胎干细胞分化的影响。 结果 1.Nesprin-1各亚型在胚胎干细胞分化为心肌细胞过程中的表达水平有所变化。 2.各干扰组分化后心肌组织分化成熟的蛋白标志肌球蛋白(myosin)的表达量减少。 3.各组心肌细胞分化率分别为(17.78±1.92)%,(36.67±3.34)%, (44.42±5.08)%,(77.78±1.92)%,各干扰组与对照组相比,差异具有统计学意义(P0.05)。 结论 本文首次证实nesprin-1亚型在胚胎干细胞分化为心肌细胞过程中至关重要,不同分子大小的nesprin-1亚型在此分化过程中发挥各自特殊的作用,某些亚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，
本文编号：2204165
[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，
本文编号：2204165
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