本文選題：BMP2 + 前體脂肪細胞　； 參考：《吉林大學》2017年碩士論文

【摘要】：肌間脂肪含量是影響豬肉品質的一個重要因素,其在動物機體中具有較高的遺傳變異性。因此,從基因和分子水平研究影響豬脂肪發(fā)育的生物學機制是改善豬肉品質的根本。脂肪組織由大量脂肪細胞聚集構成,脂肪細胞分化的程度是影響脂肪組織功能的關鍵因素。脂肪細胞主要起源于胚胎時期中胚層的間充質干細胞,由多種轉錄因子參與調控而形成成熟脂肪細胞。近年來,miRNA在脂肪細胞形成中的功能逐漸被人們重視,但多集中于細胞系或干細胞中,其對豬前體脂肪細胞分化影響的研究還很少。骨形態(tài)發(fā)生蛋白2(BMP2)屬于BMP家族中的一員,也在近年來被發(fā)現(xiàn)在脂肪細胞的形成過程中發(fā)揮關鍵作用。因此,本研究通過體外添加BMP2刺激豬前體脂肪細胞,構建BMP2介導的差異miRNA表達譜,探討關鍵miRNA對豬前體脂肪細胞分化的影響及其作用機制。本研究首先利用膠原酶法分離豬前體脂肪細胞,在誘導分化過程中添加特定濃度的BMP2,通過標志基因檢測、油紅O染色、甘油三酯鑒定篩法選出對豬前體脂肪細胞分化影響最為明顯的BMP2濃度;隨后,以此濃度的BMP2處理豬前體脂肪細胞并采用solexa深度測序技術篩選出BMP2刺激前后豬前體脂肪細胞中差異表達的miRNA,以明確受BMP2調控的差異表達miRNA。結果顯示,本研究成功分離得到的豬前體脂肪細胞,經誘導劑刺激后能夠成功分化為成熟脂肪細胞;BMP2處理的豬前體脂肪細胞標志基因顯著上調(p0.05),細胞分泌脂滴增多且甘油三酯含量顯著升高(p0.05),表明BMP2能夠促進豬前體脂肪細胞分化,且當BMP2濃度為50ng/ml時作用最為明顯;BMP2刺激豬前體脂肪細胞前后共有55個miRNAs存在較大的表達差異,其中30個上調表達,25個下調表達。差異表達miRNA靶基因GO功能富集結果顯示,大量靶基因富集在細胞代謝、脂質結合、蛋白結合等功能;KEGG pathway富集結果顯示,靶基因富集數(shù)較多的信號通路為癌癥相關的通路、神經活性配體-受體相互作用通路、內吞作用通路,另外還有一些與脂肪形成關系密切的信號通路如MAPK信號通路、胰島素信號通路、Wnt信號通路等。這些結果說明BMP2可通過介導miRNA的差異表達,調控前體脂肪細胞分化過程中靶基因富集信號通路的轉導,進而影響細胞代謝、胰島素利用、脂類合成、蛋白結合等生物學功能的發(fā)揮,最終在前體脂肪細胞的分化和脂質沉積中發(fā)揮作用。在所有差異表達的miRNA中,miR-532-5p差異較為明顯;miR-532-5p隨前體脂肪細胞的分化而呈下調趨勢,過表達miR-532-5p的豬前體脂肪細胞脂滴聚集量和甘油三酯含量均減少,且脂肪分化標志基因mRNA水和蛋白表達水平均降低,而miR-532-5p抑制組脂肪細胞呈相反趨勢,其靶基因趨化因子2(CXCL2)的表達規(guī)律與之相符。這些結果說明miR-532-5p能通過調控CXCL2影響豬前體脂肪細胞分化。綜上所述,我們發(fā)現(xiàn)BMP2可以通過調控miR-532-5p間接影響CXCL2的表達,從而影響豬前體脂肪細胞的分化能力。研究結果為揭示BMP2對影響脂肪形成的作用機制提供新的理論依據(jù),并可為改善豬肉品質開辟新思路。
[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.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S828

【參考文獻】

相關期刊論文 前1條

1 邢雪琨;武紅艷;林俊堂;豐慧根;原志慶;;趨化因子2促進肝再生中脂肪的形成[J];解剖學報;2016年05期

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本文編號：1836776