Follistatin对鸭骨骼肌卫星细胞增殖的影响及其作用机制的初步研究
发布时间：2019-05-30 02:06
【摘要】：Follistatin作为TGF-β超家族的一个成员,在哺乳动物胚胎期骨骼肌及其生长发育的过程中发挥着广泛的生理作用,在骨骼肌细胞增殖、调节肝功能、诱导红细胞再生、神经细胞分化等多种信号调节通路中也发挥着重要作用。因此,探索Follistatin对骨骼肌卫星细胞增殖的影响,同时期望探索Follistatin对鸭骨骼肌卫星细胞增殖的作用机制,希望为深入研究Follistatin调控鸭骨骼肌生长发育的作用机理奠定基础。本实验以孵化14d的鸭胚为试验材料,采用差速贴壁的方法分离培养骨骼肌卫星细胞,使用浓度分别为0、1、10、100ng/ml的Follistatin处理鸭骨骼肌卫星细胞,在培养箱中培养36h后,采用CCK-8检测骨骼肌卫星细胞增殖情况,使用抗pax7抗体染色,DAPI染核,鉴定骨骼肌卫星细胞;使用流式细胞仪检测骨骼肌卫星细胞周期变化情况;采用real-time qPCR方法检测Follistatin对骨骼肌卫星细胞增殖过程中的标记基因PCNA,基因MyoD以及TGF-P信号通路中TGF-β、Smad2和Smad3的表达的影响;Western blot检测Smad2和Smad3蛋白表达及其磷酸化水平,确定Follistatin对鸭骨骼肌卫星细胞的增殖影响及其作用机制。主要结果如下:1.通过使用Pax7抗体进行免疫荧光染色,结果显示有95%以上的Pax7呈阳性表达,说明培养的细胞为骨骼肌卫星细胞。2.通过CCK-8检测不同浓度Follistatin(0、1、10、100ng/ml)对骨骼肌卫星细胞增殖的影响,结果表明10ng/ml的Follistatin对骨骼肌卫星细胞增殖效果最明显(P0.01)。3.采用Pax7进行免疫荧光染色显示,与对照组相比,Pax7阳性表达显著升高(P0.05)。流式检测骨骼肌卫星细胞周期变化结果显示,与对照组相比,细胞G1期显著降低(P0.05),而G2期和S期显著升高(P0.05)。real-time qPCR方法检测PCNA、MyoD基因的表达情况,与对照组相比,结果显示PCNA基因表达量显著升高(P0.01),MyoD基因表达量显著降低(P0.05)。通过使用real-time qPCR方法检测TGF-β、Smad2和Smad3基因的表达量,与对照组相比,结果表明TGF-β、Smad2和Smad3基因的表达量都显著升高(P0.01)。Western blot检测结果表明Smad2和Smad3的蛋白表达及其磷酸化水平显著升高(P0.05)。4.使用LY2109761抑制剂处理鸭骨骼肌卫星细胞后,采用流式细胞仪检测骨骼肌卫星细胞周期的变化情况,结果表明,与对照组相比,LY(LY2109761)抑制剂处理组中,细胞G1期显著升高(P0.05),细胞S期和G2期显著降低(P0.05)。Realtime qPCR 检测 MyoD、PCNA、TGF-β、Smad2 和 Smad3基因的表达情况,结果发现MyoD基因和PCNA基因分别呈现显著上升和下降(P0.05),TGF-β基因表达量没有显著变化,Smad2、Smad3基因的表达量显著降低(P0.05),LY2109761抑制剂和Follistatin共同处理鸭骨骼肌卫星细胞后,细胞G1期显著升高(P0.05),细胞S期和G2期显著降低(P0.05)。MyoD基因和PCNA基因分别呈现显著上升和下降(P0.05),TGF-β基因表达量显著升高(P0.05),Smad2、Smad3基因的表达量显著降低(P0.05),Western blot检测Smad2、Smad3蛋白的表达和磷酸化水平情况,结果表明Smad2和Smad3蛋白的表达和磷酸化水平显著降低(P0.05)。
[Abstract]:Folistatin, as a member of the TGF-1 superfamily, plays a broad physiological role in the skeletal muscle of the mammalian embryo and its growth and development, and plays an important role in the proliferation of skeletal muscle cells, the regulation of liver function, and the induction of red blood cell regeneration. It also plays an important role in various signal conditioning pathways, such as the differentiation of nerve cells. Therefore, the effect of Folistatin on the proliferation of skeletal muscle satellite cells is explored, and the mechanism of Folistatin on the proliferation of skeletal muscle satellite cells is also expected to lay a foundation for studying the mechanism of Folistatin to regulate the growth and development of duck skeletal muscle. In this experiment, a 14-day hatching duck embryo was used as the test material, and the satellite cells of the skeletal muscle were isolated and cultured by using the method of differential apposition. The satellite cells of the duck skeletal muscle were treated with Folistatin in the concentration of 0,1,10 and 100 ng/ ml, respectively. After the incubation for 36 h in the incubator, the proliferation of skeletal muscle satellite cells was detected by CCK-8. the cell cycle change of the skeletal muscle satellite is detected by using the anti-pax7 antibody staining and the DAPI staining core; the cell cycle change of the skeletal muscle satellite is detected by the flow cytometry; the marker gene PCNA, the gene MyoD and the TGF-2 in the TGF-P signal path in the proliferation process of the skeletal muscle satellite cells are detected by using a real-time qPCR method, The effects of the expression of Smad2 and Smad3 on the expression of Smad2 and Smad3 were detected by Western blot. The main results are as follows:1. The results showed that more than 95% of Pax7 was expressed as a positive expression, indicating that the cultured cells were skeletal muscle satellite cells. The effect of different concentrations of Folistatin (0,1,10,100 ng/ ml) on the proliferation of skeletal muscle satellite cells was detected by CCK-8. The results showed that the effect of 10 ng/ ml of Folistatin on the proliferation of skeletal muscle satellite cells was most significant (P0.01). The expression of Pax7 was significantly higher than that in the control group (P0.05). The results showed that the cell cycle of the skeletal muscle satellite was significantly lower than that in the control group (P0.05). The expression of PCNA and MyoD was detected by the real-time qPCR method, and compared with the control group. The results showed that the expression of PCNA was significantly higher (P0.01), and the expression of MyoD gene was significantly lower (P0.05). The expression of TGF-1, Smad2 and Smad3 gene was detected by using the real-time qPCR method. The results showed that the expression of TGF-1, Smad2 and Smad3 was significantly higher than that in the control group (P0.01). Western blot showed that the expression of Smad2 and Smad3 and the level of phosphorylation of Smad3 were significantly higher (P0.05). After treatment of duck skeletal muscle satellite cells with LY2109761 inhibitor, the changes of cell cycle of skeletal muscle satellite were detected by flow cytometry. The results showed that in the treatment group of LY (LY2109761) inhibitor, the G1 phase of the cells increased significantly (P0.05). The expression of MyoD, PCNA, TGF-1, Smad2 and Smad3 was detected by Realtime qPCR. The expression of Smad3 gene was significantly lower (P0.05). After LY2109761 and Folistatin co-treated duck skeletal muscle satellite cells, the G1 phase of the cells increased significantly (P0.05), and the S and G2 phases of the cells decreased significantly (P0.05). The expression of Smad2 and Smad3 was significantly lower than that of Smad2 and Smad3 (P0.05). The results showed that the expression of Smad2 and Smad3 was significantly lower than that of Smad2 and Smad3 (P0.05).
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S834
本文编号：2488423
[Abstract]:Folistatin, as a member of the TGF-1 superfamily, plays a broad physiological role in the skeletal muscle of the mammalian embryo and its growth and development, and plays an important role in the proliferation of skeletal muscle cells, the regulation of liver function, and the induction of red blood cell regeneration. It also plays an important role in various signal conditioning pathways, such as the differentiation of nerve cells. Therefore, the effect of Folistatin on the proliferation of skeletal muscle satellite cells is explored, and the mechanism of Folistatin on the proliferation of skeletal muscle satellite cells is also expected to lay a foundation for studying the mechanism of Folistatin to regulate the growth and development of duck skeletal muscle. In this experiment, a 14-day hatching duck embryo was used as the test material, and the satellite cells of the skeletal muscle were isolated and cultured by using the method of differential apposition. The satellite cells of the duck skeletal muscle were treated with Folistatin in the concentration of 0,1,10 and 100 ng/ ml, respectively. After the incubation for 36 h in the incubator, the proliferation of skeletal muscle satellite cells was detected by CCK-8. the cell cycle change of the skeletal muscle satellite is detected by using the anti-pax7 antibody staining and the DAPI staining core; the cell cycle change of the skeletal muscle satellite is detected by the flow cytometry; the marker gene PCNA, the gene MyoD and the TGF-2 in the TGF-P signal path in the proliferation process of the skeletal muscle satellite cells are detected by using a real-time qPCR method, The effects of the expression of Smad2 and Smad3 on the expression of Smad2 and Smad3 were detected by Western blot. The main results are as follows:1. The results showed that more than 95% of Pax7 was expressed as a positive expression, indicating that the cultured cells were skeletal muscle satellite cells. The effect of different concentrations of Folistatin (0,1,10,100 ng/ ml) on the proliferation of skeletal muscle satellite cells was detected by CCK-8. The results showed that the effect of 10 ng/ ml of Folistatin on the proliferation of skeletal muscle satellite cells was most significant (P0.01). The expression of Pax7 was significantly higher than that in the control group (P0.05). The results showed that the cell cycle of the skeletal muscle satellite was significantly lower than that in the control group (P0.05). The expression of PCNA and MyoD was detected by the real-time qPCR method, and compared with the control group. The results showed that the expression of PCNA was significantly higher (P0.01), and the expression of MyoD gene was significantly lower (P0.05). The expression of TGF-1, Smad2 and Smad3 gene was detected by using the real-time qPCR method. The results showed that the expression of TGF-1, Smad2 and Smad3 was significantly higher than that in the control group (P0.01). Western blot showed that the expression of Smad2 and Smad3 and the level of phosphorylation of Smad3 were significantly higher (P0.05). After treatment of duck skeletal muscle satellite cells with LY2109761 inhibitor, the changes of cell cycle of skeletal muscle satellite were detected by flow cytometry. The results showed that in the treatment group of LY (LY2109761) inhibitor, the G1 phase of the cells increased significantly (P0.05). The expression of MyoD, PCNA, TGF-1, Smad2 and Smad3 was detected by Realtime qPCR. The expression of Smad3 gene was significantly lower (P0.05). After LY2109761 and Folistatin co-treated duck skeletal muscle satellite cells, the G1 phase of the cells increased significantly (P0.05), and the S and G2 phases of the cells decreased significantly (P0.05). The expression of Smad2 and Smad3 was significantly lower than that of Smad2 and Smad3 (P0.05). The results showed that the expression of Smad2 and Smad3 was significantly lower than that of Smad2 and Smad3 (P0.05).
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S834
【参考文献】
相关期刊论文 前1条
1 单艳菊;束婧婷;宋迟;胡艳;朱春红;李慧芳;;鸭骨骼肌卫星细胞的分离培养与鉴定[J];江苏农业科学;2012年12期
，本文编号：2488423
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