Follistatin對(duì)鴨骨骼肌衛(wèi)星細(xì)胞增殖的影響及其作用機(jī)制的初步研究
發(fā)布時(shí)間：2019-05-30 02:06
【摘要】：Follistatin作為T(mén)GF-β超家族的一個(gè)成員,在哺乳動(dòng)物胚胎期骨骼肌及其生長(zhǎng)發(fā)育的過(guò)程中發(fā)揮著廣泛的生理作用,在骨骼肌細(xì)胞增殖、調(diào)節(jié)肝功能、誘導(dǎo)紅細(xì)胞再生、神經(jīng)細(xì)胞分化等多種信號(hào)調(diào)節(jié)通路中也發(fā)揮著重要作用。因此,探索Follistatin對(duì)骨骼肌衛(wèi)星細(xì)胞增殖的影響,同時(shí)期望探索Follistatin對(duì)鴨骨骼肌衛(wèi)星細(xì)胞增殖的作用機(jī)制,希望為深入研究Follistatin調(diào)控鴨骨骼肌生長(zhǎng)發(fā)育的作用機(jī)理奠定基礎(chǔ)。本實(shí)驗(yàn)以孵化14d的鴨胚為試驗(yàn)材料,采用差速貼壁的方法分離培養(yǎng)骨骼肌衛(wèi)星細(xì)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(xì)胞,在培養(yǎng)箱中培養(yǎng)36h后,采用CCK-8檢測(cè)骨骼肌衛(wèi)星細(xì)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(xì)胞;使用流式細(xì)胞儀檢測(cè)骨骼肌衛(wèi)星細(xì)胞周期變化情況;采用real-time qPCR方法檢測(cè)Follistatin對(duì)骨骼肌衛(wèi)星細(xì)胞增殖過(guò)程中的標(biāo)記基因PCNA,基因MyoD以及TGF-P信號(hào)通路中TGF-β、Smad2和Smad3的表達(dá)的影響;Western blot檢測(cè)Smad2和Smad3蛋白表達(dá)及其磷酸化水平,確定Follistatin對(duì)鴨骨骼肌衛(wèi)星細(xì)胞的增殖影響及其作用機(jī)制。主要結(jié)果如下:1.通過(guò)使用Pax7抗體進(jìn)行免疫熒光染色,結(jié)果顯示有95%以上的Pax7呈陽(yáng)性表達(dá),說(shuō)明培養(yǎng)的細(xì)胞為骨骼肌衛(wèi)星細(xì)胞。2.通過(guò)CCK-8檢測(cè)不同濃度Follistatin(0、1、10、100ng/ml)對(duì)骨骼肌衛(wèi)星細(xì)胞增殖的影響,結(jié)果表明10ng/ml的Follistatin對(duì)骨骼肌衛(wèi)星細(xì)胞增殖效果最明顯(P0.01)。3.采用Pax7進(jìn)行免疫熒光染色顯示,與對(duì)照組相比,Pax7陽(yáng)性表達(dá)顯著升高(P0.05)。流式檢測(cè)骨骼肌衛(wèi)星細(xì)胞周期變化結(jié)果顯示,與對(duì)照組相比,細(xì)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(cè)PCNA、MyoD基因的表達(dá)情況,與對(duì)照組相比,結(jié)果顯示PCNA基因表達(dá)量顯著升高(P0.01),MyoD基因表達(dá)量顯著降低(P0.05)。通過(guò)使用real-time qPCR方法檢測(cè)TGF-β、Smad2和Smad3基因的表達(dá)量,與對(duì)照組相比,結(jié)果表明TGF-β、Smad2和Smad3基因的表達(dá)量都顯著升高(P0.01)。Western blot檢測(cè)結(jié)果表明Smad2和Smad3的蛋白表達(dá)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(xì)胞后,采用流式細(xì)胞儀檢測(cè)骨骼肌衛(wèi)星細(xì)胞周期的變化情況,結(jié)果表明,與對(duì)照組相比,LY(LY2109761)抑制劑處理組中,細(xì)胞G1期顯著升高(P0.05),細(xì)胞S期和G2期顯著降低(P0.05)。Realtime qPCR 檢測(cè) MyoD、PCNA、TGF-β、Smad2 和 Smad3基因的表達(dá)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(dá)量沒(méi)有顯著變化,Smad2、Smad3基因的表達(dá)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(xì)胞后,細(xì)胞G1期顯著升高(P0.05),細(xì)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(dá)量顯著升高(P0.05),Smad2、Smad3基因的表達(dá)量顯著降低(P0.05),Western blot檢測(cè)Smad2、Smad3蛋白的表達(dá)和磷酸化水平情況,結(jié)果表明Smad2和Smad3蛋白的表達(dá)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(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S834
本文編號(hào)：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(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S834
【參考文獻(xiàn)】
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(xì)胞的分離培養(yǎng)與鑒定[J];江蘇農(nóng)業(yè)科學(xué);2012年12期
，本文編號(hào)：2488423
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