睾酮调节巨噬细胞极化介导脂肪细胞分化与小鼠附睾脂肪积累

发布时间：2018-05-02 07:58

本文选题：睾酮 + 巨噬细胞极化　；参考：《华中农业大学》2017年博士论文

【摘要】：随着社会的进步与生活条件的改善,肥胖已经成为全球化健康问题。已有研究证明,睾酮作为体内重要的雄性激素广泛参与多种生物学过程,男性血清睾酮水平与脂代谢密切相关,血清睾酮偏低是导致男性肥胖的一大诱因。巨噬细胞是机体内重要的免疫细胞,它可在局部微环境的诱导下活化形成具有不同表型和功能的亚细胞型,这被称为巨噬细胞极化。经典的M1型极化以高表达促炎因子为特征,而M2型极化与过敏、免疫调节等作用相关,M2型极化分为3种亚型,本文主要以典型的M2a型极化为研究对象。已有研究证明,脂肪组织中的巨噬细胞极化表型的平衡与肥胖有着紧密的联系,但其中机制尚不明确。因此,本课题旨在探究睾酮与脂肪细胞分化和巨噬细胞极化之间的关联,为雄性激素与肥胖的研究提供理论基础。本课题首先在体外以小鼠3T3-L1前脂肪细胞为研究对象,在诱导分化过程中给予不同浓度的睾酮(0-10-5 mol/L)处理或含有睾酮(10-7 mol/L)的诱导后M1/M2极化条件培养基处理,通过油红O染色、RT-qPCR检测脂肪细胞标志基因脂联素,瘦素,Cebpb,Ppara,Il6,Tnfa表达水平以测定前脂细胞的分化程度。以小鼠RAW264.7巨噬细胞为研究对象,以LPS(1mg/mL)或IL-4(20 ng/mL)诱导其分别向M1与M2a方向极化,同时给予不同浓度的睾酮刺激,通过RT-qPCR检测M1型极化标志基因Nos2,Tnfa,Il6,Il1b与M2a型极化标志基因Arg1,Il10,Mgl2,Mrc1的mRNA相对表达水平,从而检测睾酮对巨噬细胞极化的作用。通过Western blot方法检测Akt蛋白S~(473)位点磷酸化水平,从而探究睾酮在巨噬细胞内活化的信号通路。本研究发现,睾酮并不直接影响3T3-L1前脂肪细胞分化,而是可通过诱导极化后的M1型巨噬细胞条件培养基显著抑制前脂细胞分化,而含有或不含有睾酮的M2a极化条件培养基对3T3-L1细胞分化的影响并不显著。RAW264.7细胞处理结果显示,睾酮并不直接引起RAW264.7巨噬细胞极化,但是睾酮可抑制LPS诱导的M1极化和促进IL-4诱导的M2a极化,并呈浓度依赖性。通过使用雄激素拮抗剂(HF)、雄激素受体抑制剂(ASC-J9)、Gai蛋白抑制剂(PTX)、Akt1/2-siRNA进行细胞信号转导的研究,结果表明,睾酮对巨噬细胞极化的调节作用是通过Gai蛋白和Akt蛋白的磷酸化产生的,而不是通过经典的雄激素受体通路。在体内,本课题通过外源注射黄体生成素受体(LHR)多肽降低小鼠血清睾酮水平。结果显示,低血清睾酮小鼠附睾白色脂肪组织增大。石蜡切片结果显示,LHR多肽组脂肪细胞较对照组相比显著增大。免疫组化结果显示,肥胖的脂肪组织中有较多的M1型巨噬细胞,而其他对照组的脂肪组织中则是以M2a型巨噬细胞为主,且数量较少。而注射睾酮增补剂丙酸睾酮注射液(TPI)能逆转这一结果。本研究首次在体外证明了睾酮对巨噬细胞极化有显著的调节作用。这种作用主要是通过Gai蛋白与Akt蛋白通路产生,而不是经典的雄激素受体。睾酮对脂肪细胞分化的影响是通过调节巨噬细胞极化而间接产生的。本研究成功建立LHR多肽降低小鼠血清睾酮模型,从此模型中证明低血清睾酮可导致小鼠附睾白色脂肪组织增大,并伴有M1型巨噬细胞极化。
[Abstract]:With the progress of society and the improvement of living conditions, obesity has become a global health problem. It has been proved that testosterone is widely involved in many biological processes as an important androgen in the body. The serum testosterone level of men is closely related to lipid metabolism. Low serum testosterone is a major cause of obesity in men. Macrophages are the main cause of obesity. The important immune cells in the body can be activated by local microenvironment to form subcellular types with different phenotypes and functions. This is called macrophage polarization. The classic M1 polarization is characterized by high expression of proinflammatory factors, and M2 polarization is related to allergy and immunoregulation. The M2 type polarization is divided into 3 subtypes. This paper is the main article. With typical M2 A-type polarization, studies have shown that the balance of the polarization phenotype of macrophages in adipose tissue is closely related to obesity, but the mechanism is not yet clear. Therefore, the aim of this study is to explore the relationship between testosterone and adipocyte differentiation and macrophage polarization, and the study of androgens and obesity. For the theoretical basis. First of all, in vitro, the mouse 3T3-L1 preadipocytes were taken as the research object. In the induction of differentiation, different concentrations of testosterone (0-10-5 mol/L) were treated with or containing testosterone (10-7 mol/L) induced M1/M2 polarization condition culture medium treatment. The fat cell marker gene adiponectin was detected by RT-qPCR, and the fat cell marker gene adiponectin was detected by RT-qPCR. The expression level of Cebpb, Ppara, Il6, and Tnfa was used to determine the degree of differentiation of the anterior fat cells. The mice RAW264.7 macrophages were used as the research object, and they were induced by LPS (1mg/mL) or IL-4 (20 ng/mL) to polarize them to M1 and M2a direction respectively, and were given different concentrations of testosterone. Polarization marker gene Arg1, Il10, Mgl2, Mrc1 mRNA relative expression level, thus detecting the effect of testosterone on macrophage polarization. The Western blot method was used to detect the phosphorylation level of S~ (473) site of Akt protein, thus exploring the signaling pathway of testosterone activation in macrophages. This study found that testosterone does not directly affect the preadipocytes before 3T3-L1. Differentiation, but can significantly inhibit the differentiation of preadipocyte by induced polarization M1 macrophage conditioned medium, and the effect of M2a polarization conditioned medium containing or without testosterone on 3T3-L1 cell differentiation is not significant.RAW264.7 cell processing results show that testosterone does not directly cause RAW264.7 macrophage polarization, but testosterone is available Inhibiting the M1 polarization induced by LPS and promoting IL-4 induced M2a polarization are concentration dependent. By using the androgen receptor inhibitor (HF), the androgen receptor inhibitor (ASC-J9), the Gai protein inhibitor (PTX), and Akt1/2-siRNA to carry out the cell signal transduction. The results show that the regulation of testosterone on the polarization of macrophages is through Gai protein and Akt. The phosphorylation of the protein was produced rather than through the classical androgenic receptor pathway. In the body, the serum testosterone level of mice was reduced by exogenous injection of the luteinizing hormone receptor (LHR) peptide. The results showed that the white adipose tissue in the epididymis of the low serum testosterone mice increased. The results of paraffin section showed that the LHR polypeptide group of adipocytes was compared to the control. The results of the group were significantly increased. The results of immunohistochemical staining showed that there were more M1 type macrophages in fat tissue, while the other control groups were mainly M2a type macrophages in the adipose tissue, and the number was small. And the testosterone supplementing agent Testosterone Propionate Injection (TPI) could reverse this result. It has a significant regulatory effect on the polarization of macrophages. This effect is produced mainly through the Gai protein and the Akt protein pathway, rather than the classical androgen receptor. The effect of testosterone on the differentiation of adipocytes is produced indirectly by regulating the polarization of macrophages. This study successfully established the LHR polypeptide to reduce the serum testosterone model in mice. It is shown that low serum testosterone can increase the white adipose tissue of epididymis in mice and increase the polarization of M1 type macrophages.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R589.2

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