Follistatin對鴨骨骼肌衛(wèi)星細胞增殖的影響及其作用機制的初步研究
發(fā)布時間：2019-05-30 02:06
【摘要】：Follistatin作為TGF-β超家族的一個成員,在哺乳動物胚胎期骨骼肌及其生長發(fā)育的過程中發(fā)揮著廣泛的生理作用,在骨骼肌細胞增殖、調(diào)節(jié)肝功能、誘導紅細胞再生、神經(jīng)細胞分化等多種信號調(diào)節(jié)通路中也發(fā)揮著重要作用。因此,探索Follistatin對骨骼肌衛(wèi)星細胞增殖的影響,同時期望探索Follistatin對鴨骨骼肌衛(wèi)星細胞增殖的作用機制,希望為深入研究Follistatin調(diào)控鴨骨骼肌生長發(fā)育的作用機理奠定基礎(chǔ)。本實驗以孵化14d的鴨胚為試驗材料,采用差速貼壁的方法分離培養(yǎng)骨骼肌衛(wèi)星細胞,使用濃度分別為0、1、10、100ng/ml的Follistatin處理鴨骨骼肌衛(wèi)星細胞,在培養(yǎng)箱中培養(yǎng)36h后,采用CCK-8檢測骨骼肌衛(wèi)星細胞增殖情況,使用抗pax7抗體染色,DAPI染核,鑒定骨骼肌衛(wèi)星細胞;使用流式細胞儀檢測骨骼肌衛(wèi)星細胞周期變化情況;采用real-time qPCR方法檢測Follistatin對骨骼肌衛(wèi)星細胞增殖過程中的標記基因PCNA,基因MyoD以及TGF-P信號通路中TGF-β、Smad2和Smad3的表達的影響;Western blot檢測Smad2和Smad3蛋白表達及其磷酸化水平,確定Follistatin對鴨骨骼肌衛(wèi)星細胞的增殖影響及其作用機制。主要結(jié)果如下:1.通過使用Pax7抗體進行免疫熒光染色,結(jié)果顯示有95%以上的Pax7呈陽性表達,說明培養(yǎng)的細胞為骨骼肌衛(wèi)星細胞。2.通過CCK-8檢測不同濃度Follistatin(0、1、10、100ng/ml)對骨骼肌衛(wèi)星細胞增殖的影響,結(jié)果表明10ng/ml的Follistatin對骨骼肌衛(wèi)星細胞增殖效果最明顯(P0.01)。3.采用Pax7進行免疫熒光染色顯示,與對照組相比,Pax7陽性表達顯著升高(P0.05)。流式檢測骨骼肌衛(wèi)星細胞周期變化結(jié)果顯示,與對照組相比,細胞G1期顯著降低(P0.05),而G2期和S期顯著升高(P0.05)。real-time qPCR方法檢測PCNA、MyoD基因的表達情況,與對照組相比,結(jié)果顯示PCNA基因表達量顯著升高(P0.01),MyoD基因表達量顯著降低(P0.05)。通過使用real-time qPCR方法檢測TGF-β、Smad2和Smad3基因的表達量,與對照組相比,結(jié)果表明TGF-β、Smad2和Smad3基因的表達量都顯著升高(P0.01)。Western blot檢測結(jié)果表明Smad2和Smad3的蛋白表達及其磷酸化水平顯著升高(P0.05)。4.使用LY2109761抑制劑處理鴨骨骼肌衛(wèi)星細胞后,采用流式細胞儀檢測骨骼肌衛(wèi)星細胞周期的變化情況,結(jié)果表明,與對照組相比,LY(LY2109761)抑制劑處理組中,細胞G1期顯著升高(P0.05),細胞S期和G2期顯著降低(P0.05)。Realtime qPCR 檢測 MyoD、PCNA、TGF-β、Smad2 和 Smad3基因的表達情況,結(jié)果發(fā)現(xiàn)MyoD基因和PCNA基因分別呈現(xiàn)顯著上升和下降(P0.05),TGF-β基因表達量沒有顯著變化,Smad2、Smad3基因的表達量顯著降低(P0.05),LY2109761抑制劑和Follistatin共同處理鴨骨骼肌衛(wèi)星細胞后,細胞G1期顯著升高(P0.05),細胞S期和G2期顯著降低(P0.05)。MyoD基因和PCNA基因分別呈現(xiàn)顯著上升和下降(P0.05),TGF-β基因表達量顯著升高(P0.05),Smad2、Smad3基因的表達量顯著降低(P0.05),Western blot檢測Smad2、Smad3蛋白的表達和磷酸化水平情況,結(jié)果表明Smad2和Smad3蛋白的表達和磷酸化水平顯著降低(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).
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S834
本文編號：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).
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S834
【參考文獻】
相關(guān)期刊論文 前1條
1 單艷菊;束婧婷;宋遲;胡艷;朱春紅;李慧芳;;鴨骨骼肌衛(wèi)星細胞的分離培養(yǎng)與鑒定[J];江蘇農(nóng)業(yè)科學;2012年12期
，本文編號：2488423
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