【摘要】：骨骼肌的生長發(fā)育狀況直接影響到動物的產(chǎn)肉量和肉質(zhì)。作為成體中的肌源性干細胞,衛(wèi)星細胞被廣泛用于骨骼肌發(fā)育的體外研究。衛(wèi)星細胞的增殖和分化是一個復(fù)雜的生物學(xué)過程,受到多種轉(zhuǎn)錄因子和細胞信號分子的調(diào)控。近些年的研究發(fā)現(xiàn),micro RNA對衛(wèi)星細胞的增殖和分化有調(diào)控作用。在本實驗室對牛骨骼肌衛(wèi)星細胞分化過程中micro RNA的高通量測序完成之后,發(fā)現(xiàn)mi R-103在衛(wèi)星細胞分化的過程中表達量高且變化差異性顯著,但其對衛(wèi)星細胞的增殖和分化的調(diào)節(jié)機制還不明確。我們首先以牛骨骼肌衛(wèi)星細胞為試驗對象,將衛(wèi)星細胞在體外進行誘導(dǎo)分化,采用實時定量PCR技術(shù)檢測在衛(wèi)星細胞分化的過程中內(nèi)源性mi R-103的表達模式。然后又利用瞬時轉(zhuǎn)染的方法,將外源性的mi R-103過表達載體或mi R-103抑制劑轉(zhuǎn)染到衛(wèi)星細胞中,在上調(diào)或下調(diào)mi R-103的表達量之后,通過EDU功能檢測和免疫熒光實驗觀察衛(wèi)星細胞的形態(tài)變化并統(tǒng)計分析。同時又利用實時定量PCR和Western blot進行定量分析,檢測衛(wèi)星細胞分化后期的標記基因Myo G的表達水平受到的影響。最后利用生物信息學(xué)軟件預(yù)測了mi R-103可能作用于CCNE1基因的m RNA的3'UTR。通過雙熒光素酶報告基因?qū)嶒�、實時定量PCR和Western blot進行定量分析來驗證mi R-103對CCNE1的調(diào)控作用。取得的研究結(jié)果如下:1.在牛骨骼肌衛(wèi)星細胞分化的過程中,mi R-103的表達量逐漸上調(diào),且在分化的第6天達到最大值。2.將構(gòu)建成功的mi R-103過表達載體或mi R-103抑制劑轉(zhuǎn)染到牛骨骼肌衛(wèi)星細胞中,可以觀察到在mi R-103的表達量被上調(diào)后,EDU陽性細胞的數(shù)目明顯低于對照組,下降了53.2%,Myo G陽性細胞的數(shù)目明顯高于對照組,為對照組的~1.58倍(P0.05)。而當mi R-103的表達量被下調(diào)后,EDU陽性細胞數(shù)為對照組的~5.3倍,Myo G陽性細胞數(shù)與對照組相比下降了48%(P0.05)。在分析實時定量PCR和Western blot的結(jié)果中發(fā)現(xiàn)在提高mi R-103的表達量之后Myo G的表達量顯著提高,與對照組相比差異性顯著(P0.05)。相反在mi R-103的活性受到抑制之后,Myo G的表達水平也隨之下調(diào)了與對照組相比變化差異性顯著(P0.05)。這些結(jié)果表明,mi R-103能夠促進衛(wèi)星細胞的分化,抑制細胞的增殖。3.在雙熒光素酶報告基因?qū)嶒?證實了mi R-103可以通過特異性結(jié)合CCNE1的3'UTR,降低CCNE13'UTR雙熒光素酶重組質(zhì)粒的熒光素酶活性。在實時定量PCR和Western blot定量分析的結(jié)果中可以看出看出mi R-103能夠在mRNA和蛋白水平顯著下調(diào)CCNE的表達(P0.05)。CCNE1在衛(wèi)星細胞分化過程中的變化與mi R-103的變化相對應(yīng),而且CCNE1的下調(diào)可以促進衛(wèi)星細胞的分化。因此可以得出CCNE1為mi R-103的靶基因。綜上所述,在衛(wèi)星細胞分化的過程中,mi R-103可通過直接作用于CCNE1的3'UTR來影響牛骨骼肌衛(wèi)星細胞的增殖與分化。確定了mi R-103對衛(wèi)星細胞的分化作用以及其調(diào)控機制,為產(chǎn)肉性狀的分子改良提供了新的思路。
[Abstract]:The growth and development of skeletal muscle directly affect the meat yield and meat quality of animals. As adult myogenic stem cells, satellite cells are widely used in the study of skeletal muscle development in vitro. The proliferation and differentiation of satellite cells is a complex biological process, which is regulated by a variety of transcription factors and cell signal molecules. In recent years, it has been found that, micro RNA can regulate the proliferation and differentiation of satellite cells. After the high-throughput sequencing of micro RNA during the differentiation of bovine skeletal muscle satellite cells in our laboratory, it was found that the expression level of mi-Rx103 in the process of satellite cell differentiation was high and the difference was significant. However, the mechanism of its regulation on the proliferation and differentiation of satellite cells is not clear. We first used bovine skeletal muscle satellite cells to induce differentiation in vitro. Real-time quantitative PCR technique was used to detect the expression pattern of endogenous mi Rx103 in the process of satellite cell differentiation. Then, the exogenous over-expression vector or inhibitor of mi-R-mi-103 was transfected into satellite cells by transient transfection. After up-regulation or down-regulation of the expression of mi-R-103, the expression of mi-R-R was up-regulated or down-regulated. The morphological changes of satellite cells were observed by EDU function test and immunofluorescence assay. At the same time, real-time quantitative PCR and Western blot were used for quantitative analysis to detect the influence of the expression level of the marker gene Myo-G in the late stage of satellite cell differentiation. Finally, the bioinformatics software was used to predict the 3 ~ (UTR) of m-RNA which may act on the CCNE1 gene of mi R _ (R) _ (103). Double luciferase reporter gene assay, real-time quantitative PCR and Western blot quantitative analysis were used to verify the regulatory effect of mi rm 103 on CCNE1. The results obtained are as follows: 1. In the process of differentiation of bovine skeletal muscle satellite cells, the expression of mi Rx103 was up-regulated gradually, and reached the maximum on the 6th day of differentiation. It was observed that the number of EDU positive cells in bovine skeletal muscle satellite cells was significantly lower than that in the control group after up-regulation of the expression level of mi-R-mi-103 after the successful construction of the vector or mi-R-103 inhibitor was transfected into bovine skeletal muscle satellite cells. The number of Myo G positive cells was significantly higher than that of the control group, which was 1.58 times higher than that of the control group (P0.05). The number of EDU positive cells was decreased by 48% as compared with the control group (P 0.05). When the expression of mi R / R 103 was down regulated, the number of, Myo G positive cells in the control group was 5.3 times higher than that in the control group (P 0.05). The results of real-time quantitative PCR and Western blot showed that the expression of Myo-G was significantly increased after increasing the expression of mi-Rx103, which was significantly higher than that of the control group (P0.05). On the contrary, the expression level of, Myo G was down-regulated after the inhibition of the activity of mi-R-103 compared with that of the control group (P0.05). These results suggest that mi Rx103 can promote the differentiation of satellite cells and inhibit the proliferation of satellite cells. 3. In the double luciferase reporter gene assay, it was confirmed that mi Rx103 could specifically bind to 3 渭 tris of CCNE1 and decrease the luciferase activity of CCNE13'UTR double luciferase recombinant plasmid. In the quantitative analysis of real-time quantitative PCR and Western blot, it was found that the expression of CCNE was significantly down-regulated at the mRNA and protein levels by mi-Rx103 (P0.05). The changes of CCNE1 during satellite cell differentiation corresponded to the changes of mi-Rx103, and the expression of CCNE1 decreased significantly at the mRNA and protein levels (P0.05). The down-regulation of CCNE1 can promote the differentiation of satellite cells. Therefore, it can be concluded that CCNE1 is the target gene of mi RZ 103. In conclusion, in the process of satellite cell differentiation, the proliferation and differentiation of bovine skeletal muscle satellite cells can be affected by the direct action of mi Rm 103 on 3'UTR of CCNE1. The differentiation of satellite cells and its regulation mechanism were determined in this paper, which provided a new idea for molecular improvement of meat-producing traits of mi-R _ (R) _ (103).
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823

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