miR-103对牛骨骼肌卫星细胞分化的影响研究

发布时间：2019-04-08 20:07

【摘要】：骨骼肌的生长发育状况直接影响到动物的产肉量和肉质。作为成体中的肌源性干细胞,卫星细胞被广泛用于骨骼肌发育的体外研究。卫星细胞的增殖和分化是一个复杂的生物学过程,受到多种转录因子和细胞信号分子的调控。近些年的研究发现,micro RNA对卫星细胞的增殖和分化有调控作用。在本实验室对牛骨骼肌卫星细胞分化过程中micro RNA的高通量测序完成之后,发现mi R-103在卫星细胞分化的过程中表达量高且变化差异性显著,但其对卫星细胞的增殖和分化的调节机制还不明确。我们首先以牛骨骼肌卫星细胞为试验对象,将卫星细胞在体外进行诱导分化,采用实时定量PCR技术检测在卫星细胞分化的过程中内源性mi R-103的表达模式。然后又利用瞬时转染的方法,将外源性的mi R-103过表达载体或mi R-103抑制剂转染到卫星细胞中,在上调或下调mi R-103的表达量之后,通过EDU功能检测和免疫荧光实验观察卫星细胞的形态变化并统计分析。同时又利用实时定量PCR和Western blot进行定量分析,检测卫星细胞分化后期的标记基因Myo G的表达水平受到的影响。最后利用生物信息学软件预测了mi R-103可能作用于CCNE1基因的m RNA的3'UTR。通过双荧光素酶报告基因实验、实时定量PCR和Western blot进行定量分析来验证mi R-103对CCNE1的调控作用。取得的研究结果如下:1.在牛骨骼肌卫星细胞分化的过程中,mi R-103的表达量逐渐上调,且在分化的第6天达到最大值。2.将构建成功的mi R-103过表达载体或mi R-103抑制剂转染到牛骨骼肌卫星细胞中,可以观察到在mi R-103的表达量被上调后,EDU阳性细胞的数目明显低于对照组,下降了53.2%,Myo G阳性细胞的数目明显高于对照组,为对照组的~1.58倍(P0.05)。而当mi R-103的表达量被下调后,EDU阳性细胞数为对照组的~5.3倍,Myo G阳性细胞数与对照组相比下降了48%(P0.05)。在分析实时定量PCR和Western blot的结果中发现在提高mi R-103的表达量之后Myo G的表达量显著提高,与对照组相比差异性显著(P0.05)。相反在mi R-103的活性受到抑制之后,Myo G的表达水平也随之下调了与对照组相比变化差异性显著(P0.05)。这些结果表明,mi R-103能够促进卫星细胞的分化,抑制细胞的增殖。3.在双荧光素酶报告基因实验,证实了mi R-103可以通过特异性结合CCNE1的3'UTR,降低CCNE13'UTR双荧光素酶重组质粒的荧光素酶活性。在实时定量PCR和Western blot定量分析的结果中可以看出看出mi R-103能够在mRNA和蛋白水平显著下调CCNE的表达(P0.05)。CCNE1在卫星细胞分化过程中的变化与mi R-103的变化相对应,而且CCNE1的下调可以促进卫星细胞的分化。因此可以得出CCNE1为mi R-103的靶基因。综上所述,在卫星细胞分化的过程中,mi R-103可通过直接作用于CCNE1的3'UTR来影响牛骨骼肌卫星细胞的增殖与分化。确定了mi R-103对卫星细胞的分化作用以及其调控机制,为产肉性状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).
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

【共引文献】