无机焦磷酸酶（PPA1）在脂肪细胞分化过程中的功能研究

发布时间：2018-12-12 02:07

【摘要】：脂肪组织是调节机体能量平衡的一个重要器官,成人脂肪细胞存在每年约10%的更新,且BMI越高对脂肪细胞更新的速率需求越高,脂肪细胞分化异常与机体胰岛素抵抗及2型糖尿病的发生密切相关。线粒体是细胞内进行氧化磷酸化和能量供应的主要场所,脂肪细胞线粒体的功能也与脂肪细胞的分化及更新有着紧密的联系。无机焦磷酸酶(Inorganic Pyrophosphatase,PPA1)是一种催化一分子无机焦磷酸转化为两分子磷酸盐离子的酶,近年来诸多研究表明PPA1与细胞的高能量代谢状况相关,也与细胞的活化关系密切。实验室前期研究发现PPA1在脂肪组织中表达丰度较其他组织要高,且在高脂喂养的2型糖尿病小鼠中发现PPA1表达的改变与文献报道中脂肪细胞数目的变化有着相同的趋势,因此本研究对PPA1在脂肪细胞分化的作用进行了初步的探讨。在细胞水平,我们在3T3-L1前体脂肪细胞中考察了PPA1对脂肪细胞分化的影响。结果显示,PPA1在3T3-L1脂肪细胞分化过程中表达量逐渐升高,并且其表达量随着细胞分化效率的提高进一步升高;干扰PPA1可以明显抑制3T3-L1脂肪细胞的成脂能力、分化后期的胰岛素敏感性和细胞因子的表达,且这种影响与细胞的增殖状况无关。我们通过透射电镜对细胞的形态进行观察,发现在分化前期,干扰PPA1使细胞线粒体出现嵴溶解和肿胀的现象,而在分化后期,PPA1干扰组细胞的线粒体数目与对照组相比较多但是形态短小。通过进一步对调控线粒体分裂融合的基因进行检测,我们发现调控线粒体融合相关基因Mfn1、Mfn2以及调控线粒体分裂相关基因Drp1的表达均下降,说明干扰PPA1打乱了线粒体分裂融合的平衡。在此基础上我们检测了线粒体功能,发现与线粒体呼吸链、线粒体转运、脂肪酸代谢以及三羧酸循环相关基因的表达也有不同程度的下降。在动物水平,我们对干扰了PPA1同源基因Nurf38的果蝇模型进行研究发现,干扰Nurf38使果蝇幼虫体长缩短,脂肪体细胞脂滴减小、核浆比例增大,体内甘油三酯含量降低。在对成蝇进行的考察中发现,干扰Nurf38使成蝇产率降低,其中雄性成蝇的产率只有对照组的1/2。对这些雄性成蝇进行低糖饥饿实验,干扰Nurf38组的果蝇较早出现死亡并且死亡率高,抗饥饿能力大大降低。综上所述,我们在细胞层面与动物水平对PPA1基因在脂肪分化过程中的作用进行了研究,并对其可能的机制进行了初步探讨,为PPA1基因功能的研究及应用提供了参考。
[Abstract]:Adipose tissue is an important organ to regulate the energy balance of the body. Adult adipocytes have about 10% renewal every year, and the higher the BMI is, the higher the rate of adipocyte renewal is required. Abnormal adipocyte differentiation is closely related to insulin resistance and type 2 diabetes. Mitochondria are the main sites for oxidative phosphorylation and energy supply in cells. The function of mitochondria in adipocytes is closely related to the differentiation and regeneration of adipocytes. Inorganic pyrophosphatase (Inorganic Pyrophosphatase,PPA1) is an enzyme that catalyzes the conversion of one molecule of inorganic pyrophosphoric acid to two molecules of phosphate ions. In recent years, many studies have shown that PPA1 is related to the high energy metabolism of cells and to the activation of cells. Previous laboratory studies found that PPA1 expression was more abundant in adipose tissue than in other tissues, and the change of PPA1 expression in type 2 diabetic mice fed with high fat had the same trend as that reported in the literature. Therefore, the effect of PPA1 on adipocyte differentiation was studied. At the cellular level, we investigated the effects of PPA1 on adipocyte differentiation in 3T3-L1 precursor adipocytes. The results showed that the expression of PPA1 increased gradually during the differentiation of 3T3-L1 adipocytes, and its expression increased with the increase of cell differentiation efficiency. Interfering with PPA1 could significantly inhibit the adipogenic ability of 3T3-L1 adipocytes, insulin sensitivity and cytokine expression in the late stage of differentiation, and this effect was not related to the proliferation of 3T3-L1 adipocytes. We observed the morphology of the cells by transmission electron microscope and found that interfering with PPA1 made the mitochondria appear cristolysis and swelling in the early stage of differentiation, but in the late stage of differentiation, The number of mitochondria in PPA1 interference group was more than that in control group, but its morphology was short. By further detecting the genes regulating mitochondrial fission fusion, we found that the expression of mitochondrial fusion related gene Mfn1,Mfn2 and mitochondrial mitotic related gene Drp1 decreased. It was suggested that interfering with PPA1 disturbed the balance of mitochondrial fission and fusion. On this basis, we detected mitochondrial function and found that the expression of genes related to mitochondrial respiratory chain, mitochondrial transport, fatty acid metabolism and tricarboxylic acid cycle were also decreased in varying degrees. At the animal level, we studied the Drosophila melanogaster model which interfered with the PPA1 homologous gene Nurf38. It was found that interfering with Nurf38 shortened the larva length, decreased lipid droplets in fat body cells, increased the ratio of nucleus and plasma, and decreased the content of triglyceride in the body of Drosophila melanogaster. In the investigation of adult fly, it was found that interference with Nurf38 reduced the yield of adult fly, in which male adult fly yield was only 1 / 2 of that of control group. The male adult flies were tested with low sugar starvation. The fruit flies in the Nurf38 group died earlier and the mortality rate was high. The ability to resist hunger was greatly reduced. In conclusion, we studied the role of PPA1 gene in adipose differentiation at the cellular level and animal level, and preliminarily discussed its possible mechanism, which provides a reference for the study and application of PPA1 gene function.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R58

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