本文選題：BMP2 + 前體脂肪細(xì)胞��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：肌間脂肪含量是影響豬肉品質(zhì)的一個(gè)重要因素,其在動(dòng)物機(jī)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(xué)機(jī)制是改善豬肉品質(zhì)的根本。脂肪組織由大量脂肪細(xì)胞聚集構(gòu)成,脂肪細(xì)胞分化的程度是影響脂肪組織功能的關(guān)鍵因素。脂肪細(xì)胞主要起源于胚胎時(shí)期中胚層的間充質(zhì)干細(xì)胞,由多種轉(zhuǎn)錄因子參與調(diào)控而形成成熟脂肪細(xì)胞。近年來(lái),miRNA在脂肪細(xì)胞形成中的功能逐漸被人們重視,但多集中于細(xì)胞系或干細(xì)胞中,其對(duì)豬前體脂肪細(xì)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(lái)被發(fā)現(xiàn)在脂肪細(xì)胞的形成過(guò)程中發(fā)揮關(guān)鍵作用。因此,本研究通過(guò)體外添加BMP2刺激豬前體脂肪細(xì)胞,構(gòu)建BMP2介導(dǎo)的差異miRNA表達(dá)譜,探討關(guān)鍵miRNA對(duì)豬前體脂肪細(xì)胞分化的影響及其作用機(jī)制。本研究首先利用膠原酶法分離豬前體脂肪細(xì)胞,在誘導(dǎo)分化過(guò)程中添加特定濃度的BMP2,通過(guò)標(biāo)志基因檢測(cè)、油紅O染色、甘油三酯鑒定篩法選出對(duì)豬前體脂肪細(xì)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(xì)胞并采用solexa深度測(cè)序技術(shù)篩選出BMP2刺激前后豬前體脂肪細(xì)胞中差異表達(dá)的miRNA,以明確受BMP2調(diào)控的差異表達(dá)miRNA。結(jié)果顯示,本研究成功分離得到的豬前體脂肪細(xì)胞,經(jīng)誘導(dǎo)劑刺激后能夠成功分化為成熟脂肪細(xì)胞;BMP2處理的豬前體脂肪細(xì)胞標(biāo)志基因顯著上調(diào)(p0.05),細(xì)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(jìn)豬前體脂肪細(xì)胞分化,且當(dāng)BMP2濃度為50ng/ml時(shí)作用最為明顯;BMP2刺激豬前體脂肪細(xì)胞前后共有55個(gè)miRNAs存在較大的表達(dá)差異,其中30個(gè)上調(diào)表達(dá),25個(gè)下調(diào)表達(dá)。差異表達(dá)miRNA靶基因GO功能富集結(jié)果顯示,大量靶基因富集在細(xì)胞代謝、脂質(zhì)結(jié)合、蛋白結(jié)合等功能;KEGG pathway富集結(jié)果顯示,靶基因富集數(shù)較多的信號(hào)通路為癌癥相關(guān)的通路、神經(jīng)活性配體-受體相互作用通路、內(nèi)吞作用通路,另外還有一些與脂肪形成關(guān)系密切的信號(hào)通路如MAPK信號(hào)通路、胰島素信號(hào)通路、Wnt信號(hào)通路等。這些結(jié)果說(shuō)明BMP2可通過(guò)介導(dǎo)miRNA的差異表達(dá),調(diào)控前體脂肪細(xì)胞分化過(guò)程中靶基因富集信號(hào)通路的轉(zhuǎn)導(dǎo),進(jìn)而影響細(xì)胞代謝、胰島素利用、脂類(lèi)合成、蛋白結(jié)合等生物學(xué)功能的發(fā)揮,最終在前體脂肪細(xì)胞的分化和脂質(zhì)沉積中發(fā)揮作用。在所有差異表達(dá)的miRNA中,miR-532-5p差異較為明顯;miR-532-5p隨前體脂肪細(xì)胞的分化而呈下調(diào)趨勢(shì),過(guò)表達(dá)miR-532-5p的豬前體脂肪細(xì)胞脂滴聚集量和甘油三酯含量均減少,且脂肪分化標(biāo)志基因mRNA水和蛋白表達(dá)水平均降低,而miR-532-5p抑制組脂肪細(xì)胞呈相反趨勢(shì),其靶基因趨化因子2(CXCL2)的表達(dá)規(guī)律與之相符。這些結(jié)果說(shuō)明miR-532-5p能通過(guò)調(diào)控CXCL2影響豬前體脂肪細(xì)胞分化。綜上所述,我們發(fā)現(xiàn)BMP2可以通過(guò)調(diào)控miR-532-5p間接影響CXCL2的表達(dá),從而影響豬前體脂肪細(xì)胞的分化能力。研究結(jié)果為揭示BMP2對(duì)影響脂肪形成的作用機(jī)制提供新的理論依據(jù),并可為改善豬肉品質(zhì)開(kāi)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S828

【參考文獻(xiàn)】

相關(guān)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(jìn)肝再生中脂肪的形成[J];解剖學(xué)報(bào);2016年05期

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本文編號(hào)：1836776