【摘要】：骨骼肌的生長(zhǎng)發(fā)育狀況直接影響到動(dòng)物的產(chǎn)肉量和肉質(zhì)。作為成體中的肌源性干細(xì)胞,衛(wèi)星細(xì)胞被廣泛用于骨骼肌發(fā)育的體外研究。衛(wèi)星細(xì)胞的增殖和分化是一個(gè)復(fù)雜的生物學(xué)過程,受到多種轉(zhuǎn)錄因子和細(xì)胞信號(hào)分子的調(diào)控。近些年的研究發(fā)現(xiàn),micro RNA對(duì)衛(wèi)星細(xì)胞的增殖和分化有調(diào)控作用。在本實(shí)驗(yàn)室對(duì)牛骨骼肌衛(wèi)星細(xì)胞分化過程中micro RNA的高通量測(cè)序完成之后,發(fā)現(xiàn)mi R-103在衛(wèi)星細(xì)胞分化的過程中表達(dá)量高且變化差異性顯著,但其對(duì)衛(wèi)星細(xì)胞的增殖和分化的調(diào)節(jié)機(jī)制還不明確。我們首先以牛骨骼肌衛(wèi)星細(xì)胞為試驗(yàn)對(duì)象,將衛(wèi)星細(xì)胞在體外進(jìn)行誘導(dǎo)分化,采用實(shí)時(shí)定量PCR技術(shù)檢測(cè)在衛(wèi)星細(xì)胞分化的過程中內(nèi)源性mi R-103的表達(dá)模式。然后又利用瞬時(shí)轉(zhuǎn)染的方法,將外源性的mi R-103過表達(dá)載體或mi R-103抑制劑轉(zhuǎn)染到衛(wèi)星細(xì)胞中,在上調(diào)或下調(diào)mi R-103的表達(dá)量之后,通過EDU功能檢測(cè)和免疫熒光實(shí)驗(yàn)觀察衛(wèi)星細(xì)胞的形態(tài)變化并統(tǒng)計(jì)分析。同時(shí)又利用實(shí)時(shí)定量PCR和Western blot進(jìn)行定量分析,檢測(cè)衛(wèi)星細(xì)胞分化后期的標(biāo)記基因Myo G的表達(dá)水平受到的影響。最后利用生物信息學(xué)軟件預(yù)測(cè)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(bào)告基因?qū)嶒?yàn)、實(shí)時(shí)定量PCR和Western blot進(jìn)行定量分析來驗(yàn)證mi R-103對(duì)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(xì)胞分化的過程中,mi R-103的表達(dá)量逐漸上調(diào),且在分化的第6天達(dá)到最大值。2.將構(gòu)建成功的mi R-103過表達(dá)載體或mi R-103抑制劑轉(zhuǎn)染到牛骨骼肌衛(wèi)星細(xì)胞中,可以觀察到在mi R-103的表達(dá)量被上調(diào)后,EDU陽性細(xì)胞的數(shù)目明顯低于對(duì)照組,下降了53.2%,Myo G陽性細(xì)胞的數(shù)目明顯高于對(duì)照組,為對(duì)照組的~1.58倍(P0.05)。而當(dāng)mi R-103的表達(dá)量被下調(diào)后,EDU陽性細(xì)胞數(shù)為對(duì)照組的~5.3倍,Myo G陽性細(xì)胞數(shù)與對(duì)照組相比下降了48%(P0.05)。在分析實(shí)時(shí)定量PCR和Western blot的結(jié)果中發(fā)現(xiàn)在提高mi R-103的表達(dá)量之后Myo G的表達(dá)量顯著提高,與對(duì)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(dá)水平也隨之下調(diào)了與對(duì)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(jìn)衛(wèi)星細(xì)胞的分化,抑制細(xì)胞的增殖。3.在雙熒光素酶報(bào)告基因?qū)嶒?yàn),證實(shí)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(shí)時(shí)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(dá)(P0.05)。CCNE1在衛(wèi)星細(xì)胞分化過程中的變化與mi R-103的變化相對(duì)應(yīng),而且CCNE1的下調(diào)可以促進(jìn)衛(wèi)星細(xì)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(xì)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(xì)胞的增殖與分化。確定了mi R-103對(duì)衛(wèi)星細(xì)胞的分化作用以及其調(diào)控機(jī)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(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐心浩;力量和耐力訓(xùn)練對(duì)肌球蛋白重鏈及肌纖維轉(zhuǎn)型的影響[J];北京體育大學(xué)學(xué)報(bào);2004年11期

2 駱泉豐,祁佐良,王煒,王興;三叉神經(jīng)支配對(duì)面癱后肌球蛋白重鏈亞型的影響[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年05期

3 郭云雁;王立賢;程篤學(xué);顏華;;豬肌纖維組織學(xué)特性研究進(jìn)展[J];中國畜牧獸醫(yī);2007年08期

4 王來娣;鄭云;蔣拾貝;王星果;張軍;龔道清;;miRNA在脂代謝中的研究進(jìn)展[J];動(dòng)物營養(yǎng)學(xué)報(bào);2013年07期

5 張燕軍;韓志玲;張文廣;李金泉;;骨骼肌發(fā)育及其相關(guān)miRNA表達(dá)機(jī)制的研究進(jìn)展[J];中國畜牧獸醫(yī);2014年03期

6 陳歡;彭博;李富運(yùn);于雪;盧虎英;衛(wèi)肖艷;彭圓圓;范伊凡;張莉;;基于U0126阻斷ERK信號(hào)通路的電針阿是穴對(duì)兔鈍挫傷后肌肉再生影響的研究[J];中華中醫(yī)藥雜志;2014年07期

7 王月超;蔡輝益;閆海潔;陳曉琳;張姝;張愛華;楊斌;郭軍蕊;;家禽骨骼肌的生長(zhǎng)發(fā)育規(guī)律及調(diào)控[J];飼料工業(yè);2014年17期

8 王辰;張晶;謝炳坤;周榮;李奎;唐中林;;miR-143在肌細(xì)胞中的表達(dá)及其影響[J];中國畜牧獸醫(yī);2014年10期

9 習(xí)欠云;張永亮;;簡(jiǎn)論動(dòng)物生物化學(xué)對(duì)營養(yǎng)與飼料科學(xué)的科研教學(xué)啟示[J];飼料工業(yè);2015年06期

10 王子帥;化朝舉;王辰;李奎;唐中林;;長(zhǎng)鏈非編碼RNA(H19)在小鼠骨骼肌生長(zhǎng)發(fā)育過程中的表達(dá)[J];中國畜牧獸醫(yī);2015年10期

相關(guān)會(huì)議論文 前2條

1 徐子偉;門小明;齊珂珂;;豬肌肉纖維類型及其代謝特征與肉質(zhì)形成的關(guān)系及機(jī)理探討[A];動(dòng)物營養(yǎng)研究進(jìn)展（2012年版）[C];2012年

2 彭嶺;李杰;唐梅森;向忠軍;黃海波;郭宗耀;;循環(huán)miRNA與冠心病及其中醫(yī)證候研究相關(guān)進(jìn)展[A];第九次全國中西醫(yī)結(jié)合診斷學(xué)術(shù)研討會(huì)論文集[C];2015年

相關(guān)博士學(xué)位論文 前10條

1 史新娥;FoxO1調(diào)控豬骨骼肌纖維類型分化的作用及分子機(jī)理[D];西北農(nóng)林科技大學(xué);2011年

2 王恒;肌肉中calsarcin基因的差異表達(dá)和轉(zhuǎn)錄調(diào)控研究[D];華中農(nóng)業(yè)大學(xué);2007年

3 李曉箐;雌激素對(duì)大鼠咬肌ER和MHC mRNA表達(dá)的影響[D];四川大學(xué);2007年

4 門小明;肌肉纖維類型組成對(duì)豬肉品質(zhì)的影響及其機(jī)理研究[D];江南大學(xué);2012年

5 李曉;PRDM16對(duì)C2C12細(xì)胞分化的影響及其PR結(jié)構(gòu)域的功能分析[D];南京農(nóng)業(yè)大學(xué);2011年

6 陳晨;miR-135a和miR-183對(duì)3T3-L1前脂肪細(xì)胞分化及脂肪形成的調(diào)控作用研究[D];華中農(nóng)業(yè)大學(xué);2013年

7 崔麗娜;間充質(zhì)干細(xì)胞肝向分化特異性microRNA表達(dá)譜的鑒定及功能學(xué)研究[D];第四軍醫(yī)大學(xué);2013年

8 任偉剛;microRNA-34基因抑制膀胱癌細(xì)胞生長(zhǎng)以及順鉑抗腫瘤效應(yīng)的機(jī)制研究[D];中南大學(xué);2012年

9 韓宏曉;日本血吸蟲感染不同適宜性嚙齒類動(dòng)物后宿主及蟲體miRNA表達(dá)分析[D];揚(yáng)州大學(xué);2013年

10 龔家驥;小鼠骨折愈合中mmu-miR-142-5p表達(dá)激活支持成骨細(xì)胞功能的研究[D];中南大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 李沐;MicroRNA介導(dǎo)的IGF-1基因沉默與端粒酶對(duì)鹿茸細(xì)胞增殖抑制的影響[D];吉林農(nóng)業(yè)大學(xué);2013年

2 尹靈芝;穴位電針對(duì)前導(dǎo)下頜大鼠咀嚼肌MMP-2和MyHC-Ⅰ表達(dá)的影響[D];重慶醫(yī)科大學(xué);2013年

3 王樂樂;雞let-7a的組織表達(dá)、功能預(yù)測(cè)及其靶基因ADIPOR2遺傳效應(yīng)分析[D];河南農(nóng)業(yè)大學(xué);2013年

4 李挺;利用表達(dá)譜芯片技術(shù)篩選家兔肌肉發(fā)育相關(guān)基因[D];揚(yáng)州大學(xué);2013年

5 汪芳杰;四個(gè)候選基因在雞成肌細(xì)胞和肌肉組織的mRNA表達(dá)特性分析[D];安徽農(nóng)業(yè)大學(xué);2013年

6 潘霄;豬偽狂犬超強(qiáng)毒株的分離鑒定及非編碼短片段DNA文庫的構(gòu)建[D];河南科技大學(xué);2013年

7 劉佩佩;Arx的表達(dá)與C2C12細(xì)胞分化的關(guān)系[D];廈門大學(xué);2014年

8 王月超;L-肉堿和賴氨酸對(duì)愛拔益加(AA)肉公雞骨骼肌生長(zhǎng)發(fā)育及蛋白質(zhì)表達(dá)的影響[D];中國農(nóng)業(yè)科學(xué)院;2014年

9 翟焱盼;高脂飲食誘導(dǎo)的C57BL/6J肥胖小鼠脂肪組織RIP140基因表達(dá)水平的研究[D];青島大學(xué);2014年

10 王亞輝;miR-133b對(duì)牛骨骼肌衛(wèi)星細(xì)胞增殖的影響[D];東北農(nóng)業(yè)大學(xué);2014年

，

本文編號(hào)：2454874