本文選題：TCEA3 + 轉(zhuǎn)錄延長因子　； 參考：《東北農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：隨著人們生活水平的提高,人們對(duì)家畜肉質(zhì)品質(zhì)的需求也不斷提高,牛肉的品質(zhì)取決于牛的肌纖維數(shù)量,肌肉纖維使牛肉在品嘗時(shí)具有勁道,香嫩等口感。固牛肉成為人們?nèi)粘Ｉ钪匈徺I的肉品之一。牛肉潛在著很大商業(yè)價(jià)值與市場價(jià)值,因此,如何產(chǎn)生高產(chǎn)量,口感勁道的肌肉牛是我們的研究方向。牛骨骼肌的生長主要是通過牛骨骼肌衛(wèi)星細(xì)胞的分化形成的,本實(shí)驗(yàn)室前期通過高通量深度測序結(jié)果發(fā)現(xiàn)在牛骨骼肌衛(wèi)星細(xì)胞(Skeletal Muscle Satellite Cells,MDSCs)分化過程中有一些基因高表達(dá),他們影響牛MDSCs的分化。Zhang Wei Wei et al研究表明,早期生長反應(yīng)因子-1(early growth response-1,EGR-1)是一種轉(zhuǎn)錄因子,EGR-1會(huì)與MYOG的啟動(dòng)區(qū)結(jié)合,過表達(dá)EGR-1,會(huì)促進(jìn)MYOG的表達(dá),從而刺激牛MDSCs的分化。在同樣的高通量測序結(jié)果我們還發(fā)現(xiàn)TCEA3(transcription elongation factor A3,TCEA3)在牛骨骼肌生長發(fā)育過程中高表達(dá),本實(shí)驗(yàn)旨在研究TCEA3基因?qū)εDSCs分化的影響。研究如下:(1)用2%的馬血清培養(yǎng)液將牛MDSCs誘導(dǎo)分化成1 D,3 D,5 D和7 D這四個(gè)時(shí)期。以分化0 D的牛MDSCs作為對(duì)照,分別收集對(duì)應(yīng)時(shí)期的蛋白與RNA樣品,采用Western Blot,RT-PCR檢測分化不同時(shí)期的牛MDSCs中TCEA3蛋白表達(dá)量與RNA表達(dá)量。(2)采用免疫熒光技術(shù),細(xì)胞質(zhì)細(xì)胞核蛋白分離純化技術(shù)檢測在牛MDSCs體外分化過程中的表達(dá)規(guī)律及定位。(3)利用CRISPR/Cas9技術(shù)對(duì)TCEA3構(gòu)建激活或抑制載體,來研究TCEA3對(duì)牛MDSCs體外分化的影響。(4)轉(zhuǎn)染TCEA3激活或抑制24 h,48 h以后,采用免疫熒光的方法對(duì)肌管融合率和肌管數(shù)目進(jìn)行統(tǒng)計(jì)。采用Western Blot方法檢測肌肉分化相關(guān)基因細(xì)胞生成素(myogenin,MYOG)表達(dá)量、肌球蛋白重鏈3(myosin heavy chain 3,MYH3)的表達(dá)量。實(shí)驗(yàn)結(jié)果表明:(1)隨著牛MDSCs體外分化過程中TCEA3蛋白表達(dá)量逐漸上升,在分化第5 D時(shí),TCEA3蛋白表達(dá)量達(dá)到峰值。(2)TCEA3蛋白主要集中在牛MDSCs的胞質(zhì)中,在細(xì)胞核中未檢測到TCEA3的表達(dá),在分化程度較高的肌管中含量較多。(3)轉(zhuǎn)染TCEA3激活載體(TCEA3過表達(dá)后)24 h后,牛MDSCs體外分化過程中的肌管融合率、肌管數(shù)目、肌肉分化相關(guān)基因MYOG表達(dá)量、MYH3為對(duì)照組的1.58倍、1.76倍、1.9倍、1.89倍。轉(zhuǎn)染TCEA3激活載體(TCEA3過表達(dá)后)48 h后,肌管融合率、肌管數(shù)目、MYOG表達(dá)量、MYH3表達(dá)量為對(duì)照組的1.8倍、1.88倍、1.4倍、1.8倍。(4)轉(zhuǎn)染TCEA3抑制載體24 h后,肌管融合率、肌管數(shù)目、MYOG表達(dá)量、MYH3表達(dá)量為對(duì)照組的0.58倍、0.7倍、0.5倍、0.15倍。轉(zhuǎn)染TCEA3抑制載體48 h后,肌管融合率、肌管數(shù)目、MYOG表達(dá)量、MYH3表達(dá)量為對(duì)照組的0.6倍、0.5倍、0.66倍、0.54倍。綜上結(jié)果表明:過表達(dá)TCEA3會(huì)促進(jìn)牛MDSCs的分化,反之,抑制TCEA3的表達(dá)會(huì)抑制牛MDSCs的分化。本實(shí)驗(yàn)首次證實(shí)TCEA3基因?qū)εDSCs分化起到促進(jìn)作用�？蔀榧∪獍l(fā)育機(jī)制的研究提供理論依據(jù),同時(shí)也為在治療肌肉萎縮疾病方面的研究上提供有力的基礎(chǔ)。
[Abstract]:With the improvement of people's living standard, people's demand for meat quality of livestock is also increasing. The quality of beef depends on the quantity of muscle fiber, which makes beef taste strong and tender. Solid beef has become one of the meat products that people buy in their daily life. Beef is potentially of great commercial and market value. Therefore, how to produce high yield, strong taste muscle cattle is our research direction. The growth of bovine skeletal muscle is mainly formed by the differentiation of bovine skeletal muscle satellite cells. The results of high throughput deep sequencing in our laboratory showed that some genes were overexpressed in the differentiation process of bovine skeletal muscle satellite cells, Skeletal Muscle Satellite cells and MDSCs. Their study on the differentiation of bovine MDSCs. Zhang Wei Wei et al showed that early growth response factor-1C early growth response-1 (EGR-1) is a transcription factor that binds to the promoter region of MYOG and overexpresses EGR-1, which promotes the expression of MYOG and stimulates the differentiation of bovine MDSCs. In the same high throughput sequencing, we also found that TCEA3(transcription elongation factor A3 (TCEA3) is highly expressed during the growth and development of bovine skeletal muscle. The purpose of this study was to study the effect of TCEA3 gene on the differentiation of bovine MDSCs. The results showed that the bovine MDSCs was induced to differentiate into 1 D 3 D 5 D and 7 D with 2% equine serum culture medium. Bovine MDSCs of differentiation 0 D was used as control. The protein and RNA samples from the corresponding period were collected respectively. The expression of TCEA3 protein and RNA expression in bovine MDSCs at different stages of differentiation were detected by Western blotRT-PCR. The expression and localization of cytoplasmic nuclear protein during the differentiation of bovine MDSCs in vitro were detected by using cytoplasmic nuclear protein separation and purification technique. CRISPR/Cas9 technique was used to construct activation or inhibition vector for TCEA3. To study the effect of TCEA3 on the differentiation of bovine MDSCs in vitro. 4) after the transfection of TCEA3 was activated or inhibited for 24 h or 48 h, the myotube fusion rate and the number of myotubes were counted by immunofluorescence method. The expression of myogenin Western Blot and myosin heavy chain 3(myosin heavy chain 3 were detected by Western Blot. The results showed that the expression of TCEA3 protein increased gradually during the differentiation of bovine MDSCs in vitro, and the expression of TCEA3 protein reached a peak at 5D. The expression of TCEA3 protein was mainly concentrated in the cytoplasm of bovine MDSCs, but no expression of TCEA3 was detected in the nucleus. After transfection of TCEA3 activation vector (TCEA3), the fusion rate and the number of myotubes during the differentiation of bovine MDSCs in vitro were observed after 24 h of overexpression of TCEA3. The expression of muscle-differentiation related gene MYOG was 1.58 times, 1.76 times and 1.89 times as much as that of the control group. The myotube fusion rate and myotube number of myotube fusion were 1.8-1.88 times 1.88 times 1.8-1.88 times 1.8-1.80 times 1.8-1.8-1.8-1.8-1.8-1.8-1.8-1.8-1.80 times, 1.8 times and 1.8 times respectively, after transfection with TCEA3 activation vector, and the myotube fusion rate was 24 h after transfection into TCEA3 inhibition vector. The number of myotubes and the expression of MYOG and MYH3 were 0.58 times, 0.7 times, 0.5 times and 0.15 times of those of the control group. After 48 hours of transfection of TCEA3 inhibition vector, the myotube fusion rate and myotube number of myotubes and the expression of MYH3 were 0.66 times and 0.54 times as much as that of the control group. The results showed that overexpression of TCEA3 could promote the differentiation of bovine MDSCs, whereas inhibiting the expression of TCEA3 could inhibit the differentiation of bovine MDSCs. This study first confirmed that TCEA3 gene can promote the differentiation of bovine MDSCs. It can provide a theoretical basis for the study of the mechanism of muscle development, and also provide a strong basis for the research on the treatment of muscular atrophy.
【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】：2017
【分類號(hào)】：S823

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