TCEA3对牛骨骼肌卫星细胞分化的影响

发布时间：2018-06-05 05:10

本文选题：TCEA3 + 转录延长因子　；参考：《东北农业大学》2017年硕士论文

【摘要】：随着人们生活水平的提高,人们对家畜肉质品质的需求也不断提高,牛肉的品质取决于牛的肌纤维数量,肌肉纤维使牛肉在品尝时具有劲道,香嫩等口感。固牛肉成为人们日常生活中购买的肉品之一。牛肉潜在着很大商业价值与市场价值,因此,如何产生高产量,口感劲道的肌肉牛是我们的研究方向。牛骨骼肌的生长主要是通过牛骨骼肌卫星细胞的分化形成的,本实验室前期通过高通量深度测序结果发现在牛骨骼肌卫星细胞(Skeletal Muscle Satellite Cells,MDSCs)分化过程中有一些基因高表达,他们影响牛MDSCs的分化。Zhang Wei Wei et al研究表明,早期生长反应因子-1(early growth response-1,EGR-1)是一种转录因子,EGR-1会与MYOG的启动区结合,过表达EGR-1,会促进MYOG的表达,从而刺激牛MDSCs的分化。在同样的高通量测序结果我们还发现TCEA3(transcription elongation factor A3,TCEA3)在牛骨骼肌生长发育过程中高表达,本实验旨在研究TCEA3基因对牛MDSCs分化的影响。研究如下:(1)用2%的马血清培养液将牛MDSCs诱导分化成1 D,3 D,5 D和7 D这四个时期。以分化0 D的牛MDSCs作为对照,分别收集对应时期的蛋白与RNA样品,采用Western Blot,RT-PCR检测分化不同时期的牛MDSCs中TCEA3蛋白表达量与RNA表达量。(2)采用免疫荧光技术,细胞质细胞核蛋白分离纯化技术检测在牛MDSCs体外分化过程中的表达规律及定位。(3)利用CRISPR/Cas9技术对TCEA3构建激活或抑制载体,来研究TCEA3对牛MDSCs体外分化的影响。(4)转染TCEA3激活或抑制24 h,48 h以后,采用免疫荧光的方法对肌管融合率和肌管数目进行统计。采用Western Blot方法检测肌肉分化相关基因细胞生成素(myogenin,MYOG)表达量、肌球蛋白重链3(myosin heavy chain 3,MYH3)的表达量。实验结果表明:(1)随着牛MDSCs体外分化过程中TCEA3蛋白表达量逐渐上升,在分化第5 D时,TCEA3蛋白表达量达到峰值。(2)TCEA3蛋白主要集中在牛MDSCs的胞质中,在细胞核中未检测到TCEA3的表达,在分化程度较高的肌管中含量较多。(3)转染TCEA3激活载体(TCEA3过表达后)24 h后,牛MDSCs体外分化过程中的肌管融合率、肌管数目、肌肉分化相关基因MYOG表达量、MYH3为对照组的1.58倍、1.76倍、1.9倍、1.89倍。转染TCEA3激活载体(TCEA3过表达后)48 h后,肌管融合率、肌管数目、MYOG表达量、MYH3表达量为对照组的1.8倍、1.88倍、1.4倍、1.8倍。(4)转染TCEA3抑制载体24 h后,肌管融合率、肌管数目、MYOG表达量、MYH3表达量为对照组的0.58倍、0.7倍、0.5倍、0.15倍。转染TCEA3抑制载体48 h后,肌管融合率、肌管数目、MYOG表达量、MYH3表达量为对照组的0.6倍、0.5倍、0.66倍、0.54倍。综上结果表明:过表达TCEA3会促进牛MDSCs的分化,反之,抑制TCEA3的表达会抑制牛MDSCs的分化。本实验首次证实TCEA3基因对牛MDSCs分化起到促进作用。可为肌肉发育机制的研究提供理论依据,同时也为在治疗肌肉萎缩疾病方面的研究上提供有力的基础。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

【参考文献】