BMP2诱导的猪前体脂肪细胞差异表达miRNA的鉴定及miR-532-5p功能研究

发布时间：2018-05-03 03:25

本文选题：BMP2 + 前体脂肪细胞　；参考：《吉林大学》2017年硕士论文

【摘要】：肌间脂肪含量是影响猪肉品质的一个重要因素,其在动物机体中具有较高的遗传变异性。因此,从基因和分子水平研究影响猪脂肪发育的生物学机制是改善猪肉品质的根本。脂肪组织由大量脂肪细胞聚集构成,脂肪细胞分化的程度是影响脂肪组织功能的关键因素。脂肪细胞主要起源于胚胎时期中胚层的间充质干细胞,由多种转录因子参与调控而形成成熟脂肪细胞。近年来,miRNA在脂肪细胞形成中的功能逐渐被人们重视,但多集中于细胞系或干细胞中,其对猪前体脂肪细胞分化影响的研究还很少。骨形态发生蛋白2(BMP2)属于BMP家族中的一员,也在近年来被发现在脂肪细胞的形成过程中发挥关键作用。因此,本研究通过体外添加BMP2刺激猪前体脂肪细胞,构建BMP2介导的差异miRNA表达谱,探讨关键miRNA对猪前体脂肪细胞分化的影响及其作用机制。本研究首先利用胶原酶法分离猪前体脂肪细胞,在诱导分化过程中添加特定浓度的BMP2,通过标志基因检测、油红O染色、甘油三酯鉴定筛法选出对猪前体脂肪细胞分化影响最为明显的BMP2浓度;随后,以此浓度的BMP2处理猪前体脂肪细胞并采用solexa深度测序技术筛选出BMP2刺激前后猪前体脂肪细胞中差异表达的miRNA,以明确受BMP2调控的差异表达miRNA。结果显示,本研究成功分离得到的猪前体脂肪细胞,经诱导剂刺激后能够成功分化为成熟脂肪细胞;BMP2处理的猪前体脂肪细胞标志基因显著上调(p0.05),细胞分泌脂滴增多且甘油三酯含量显著升高(p0.05),表明BMP2能够促进猪前体脂肪细胞分化,且当BMP2浓度为50ng/ml时作用最为明显;BMP2刺激猪前体脂肪细胞前后共有55个miRNAs存在较大的表达差异,其中30个上调表达,25个下调表达。差异表达miRNA靶基因GO功能富集结果显示,大量靶基因富集在细胞代谢、脂质结合、蛋白结合等功能;KEGG pathway富集结果显示,靶基因富集数较多的信号通路为癌症相关的通路、神经活性配体-受体相互作用通路、内吞作用通路,另外还有一些与脂肪形成关系密切的信号通路如MAPK信号通路、胰岛素信号通路、Wnt信号通路等。这些结果说明BMP2可通过介导miRNA的差异表达,调控前体脂肪细胞分化过程中靶基因富集信号通路的转导,进而影响细胞代谢、胰岛素利用、脂类合成、蛋白结合等生物学功能的发挥,最终在前体脂肪细胞的分化和脂质沉积中发挥作用。在所有差异表达的miRNA中,miR-532-5p差异较为明显;miR-532-5p随前体脂肪细胞的分化而呈下调趋势,过表达miR-532-5p的猪前体脂肪细胞脂滴聚集量和甘油三酯含量均减少,且脂肪分化标志基因mRNA水和蛋白表达水平均降低,而miR-532-5p抑制组脂肪细胞呈相反趋势,其靶基因趋化因子2(CXCL2)的表达规律与之相符。这些结果说明miR-532-5p能通过调控CXCL2影响猪前体脂肪细胞分化。综上所述,我们发现BMP2可以通过调控miR-532-5p间接影响CXCL2的表达,从而影响猪前体脂肪细胞的分化能力。研究结果为揭示BMP2对影响脂肪形成的作用机制提供新的理论依据,并可为改善猪肉品质开辟新思路。
[Abstract]:The content of intermuscular fat is an important factor affecting the quality of pork. It has high genetic variability in the animal body. Therefore, it is essential to study the biological mechanism that affects the pig fat development from the gene and molecular level. The fat tissue is made up of a large number of fat cells, and the degree of adipocyte differentiation is the shadow. The key factor in the function of fat tissue. Adipocytes are mainly derived from mesenchymal stem cells in the mesoderm of the embryonic period, which are regulated by a variety of transcription factors to form mature adipocytes. In recent years, the function of miRNA in the formation of adipocytes has gradually been paid attention to, but many of them are in cell lines or stem cells for porcine precursor fat. There are few studies on the effect of cell differentiation. Bone morphogenetic protein 2 (BMP2) is a member of the BMP family and has been found to play a key role in the formation of adipocytes in recent years. Therefore, this study was designed to stimulate porcine precursor adipocytes by adding BMP2 in vitro, to construct BMP2 mediated differential miRNA expression profiles, and to explore the key miRNA to pigs. This study first used collagenase to separate porcine precursor adipocytes, and added a specific concentration of BMP2 in the induction of differentiation. By marker gene detection, oil red O staining, and triglyceride screening method, the most obvious BMP2 concentration on porcine anterior fat cell differentiation was selected. After that, the porcine precursor adipocytes were treated with this concentration of BMP2, and the differential expression of miRNA in the porcine precursor adipocytes before and after BMP2 stimulation was screened by Solexa deep sequencing technology. The result of the differential expression of miRNA. regulated by BMP2 showed that the porcine precursor adipocytes were successfully separated by this study, and could be successfully divided by inducer stimulation. BMP2 treated porcine precursor adipocyte marker genes were significantly up-regulated (P0.05), cell secretion increased and triglyceride content increased significantly (P0.05), indicating that BMP2 could promote the differentiation of porcine preadipocytes, and when BMP2 concentration was 50ng/ml, BMP2 stimulated pig precursor adipocytes before and after 55. There were significant differences in expression of miRNAs, including 30 up-regulated and 25 down-regulated expression. Differential expression of miRNA target gene GO functional enrichment results showed that a large number of target genes were enriched in cell metabolism, lipid binding, protein binding and other functions. KEGG pathway enrichment results showed that the target based on more enrichment of the signal pathway was cancer related pathways. Neuroactive ligand receptor interaction pathway, endocytosis pathway, and other signaling pathways closely related to fat formation, such as MAPK signaling pathways, insulin signaling pathways, and Wnt signaling pathways. These results indicate that BMP2 can regulate the differential expression of miRNA and regulate the target gene enrichment in the process of preadipocyte differentiation. Transduction of number pathway, which affects cell metabolism, insulin utilization, lipid synthesis, protein binding and other biological functions, ultimately plays a role in the differentiation and lipid deposition of precursor adipocytes. In all differentially expressed miRNA, the difference in miR-532-5p is more obvious; miR-532-5p decreases with the differentiation of precursor adipocytes. The accumulation of lipid droplets and triglycerides in the porcine precursor adipocytes were decreased, and the expression level of mRNA and protein in the fat differentiation marker gene of the miR-532-5p was decreased, while the adipocytes in the miR-532-5p inhibition group were in the opposite direction, and the expression of target gene chemokine 2 (CXCL2) was consistent with that of the target gene. These results indicate that miR-532-5p can be used. In conclusion, we have found that BMP2 can indirectly influence the expression of CXCL2 by regulating miR-532-5p by regulating the effect of CXCL2 on the differentiation of porcine precursor adipocytes. The results can provide a new theoretical basis for the mechanism of BMP2 on the effect of fat formation and improve the quality of pork. Open up a new way of thinking.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

【参考文献】