LYRM1基因表达沉默对成熟脂肪细胞线粒体功能的影响

发布时间：2018-02-27 19:38

本文关键词： LYRM1 脂肪细胞 RNA干扰线粒体 UCP2　出处：《南京医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：近年来，肥胖发生率逐渐增高，儿童肥胖发生率的升高也非常显著。肥胖是一种具有遗传性、多因子参与的代谢性疾病，是能量摄入与能量消耗失衡的结果。肥胖是引起糖尿病、高血压等多种疾病的高危因素。肥胖病因复杂，主要致病因素包括：机体遗传易感性、环境因素、运动和营养，其中遗传因素在肥胖的发生发展中起重要作用。 LYRM1基因是本研究小组应用抑制性差减杂交技术（suppression subtractivehybridization，SSH）筛选肥胖与正常人网膜脂肪组织中差异表达基因时获得并克隆到的一条肥胖相关全长新基因。前期研究发现：LYRM1基因高表达于肥胖者网膜脂肪组织，定位于染色体16P11.2，包括4个外显子和3个内含子，开放阅读框369bp，mRNA全长1589bp，编码蛋白质产物122aa，蛋白N端附近有一高度保守的三肽结构（LYR-motif containing，，LYR），提示该基因与线粒体能量代谢可能相关。已知线粒体是供能物质ATP的主要合成场所，是能量代谢的重要细胞器。线粒体功能障碍所引起的细胞活性氧（Reactive oxygen species，ROS）增高，可通过活化各种丝氨酸激酶（serine kinases）的机制，磷酸化胰岛素受体底物蛋白（insulin receptor substrate，IRS）的丝氨酸残基，而阻碍胰岛素受体（insulinreceptor，IR）自身酪氨酸激酶（Intrinsic tyrosine kinase）对IRS的磷酸化作用，最终导致胰岛素抵抗。由于前期研究显示，LYRM1基因在3T3-L1脂肪细胞中高表达（overexpression），能够导致线粒体功能障碍、显著降低胰岛素刺激下的葡萄糖摄取率，提示该基因可能通过改变线粒体功能影响胰岛素敏感性，因此本研究通过小干扰RNA（small interference RNA，siRNA）技术，进一步观察LYRM1基因表达沉默后成熟脂肪细胞线粒体功能是否发生改变，以论证该基因对线粒体功能的影响。目的：观察LYRM1基因表达沉默对成熟脂肪细胞线粒体功能的影响，并初步分析其机制。方法：体外培养稳定转染LYRM1干扰慢病毒（Lentiviral-Lyrm1-shRNA）的3T3-L1前体脂肪细胞，以仅转染阴性慢病毒（Lentiviral-NC-shRNA）的3T3-L1前体脂肪细胞为对照，RT-PCR验证LYRM1基因的干扰效率。胰岛素、地塞米松、1-甲基-3-异丁基黄嘌呤（MDI）方案诱导脂肪细胞分化成熟。透射电镜下观察成熟脂肪细胞的线粒体形态和超微结构。采用RT-PCR法检测线粒体DNA拷贝数（mtDNA）及相关基因Mfn1、Mfn2、Drp1、Fis1、UCP2、UCP4的表达。化学发光酶标仪检测细胞ATP含量。应用线粒体荧光探针（Mito-Tracker Red）、2,7二氯荧光素探针（2’,7’-Dichlorofluorescein diacetate，DCFDA）预染成熟脂肪细胞，激光共聚焦显微镜及流式细胞仪观测线粒体膜电位及细胞活性氧簇（reactiveoxygen species，ROS）的变化。结果：（1）沉默组脂肪细胞的LYRM1基因表达水平明显低于阴性对照组，Lentiviral-LYRM1-shRNA的干扰效率达40%；（2）LYRM1基因沉默组成熟脂肪细胞线粒体形态、超微结构以及mtDNA与阴性对照组之间无明显差异；（3）LYRM1基因沉默组成熟脂肪细胞的线粒体膜电位和细胞ATP含量较阴性对照组显著升高；（4）LYRM1基因沉默组细胞活性氧（ROS）水平明显降低；（5）LYRM1基因沉默组成熟脂肪细胞中的Mfn2、Ucp2mRNA表达明显上调，Ucp4、Mfn1、Drp1、Fis1等mRNA表达在两组间无明显差异。结论：LYRM1基因表达沉默可以影响成熟脂肪细胞线粒体功能状态，一定程度上改善线粒体代谢的功能。
[Abstract]:In recent years, the incidence of obesity increased gradually, higher incidence of childhood obesity is significant. Obesity is a hereditary metabolic disease, many factors involved in, is the result of the imbalance of energy intake and energy expenditure. Obesity is caused by diabetes, hypertension and other risk factors of many diseases. The obesity etiology is complex, mainly including pathogenic factors: genetic predisposition, body movement and environmental factors, nutrition, and genetic factors in the occurrence of obesity plays an important role in the development.
The LYRM1 gene is the research group using suppression subtractive hybridization (suppression subtractivehybridization SSH) and cloned a full-length gene related to obesity screening differentially expressed genes between obese and normal human omental adipose tissue. Previous studies showed that the expression of LYRM1 gene in omental adipose tissue of obese subjects, located on chromosome 16P11.2, including 4 exons and 3 introns, the open reading frame of 369bp, mRNA is 1589bp, encoding protein 122aa, a highly conserved three peptide structure near the N terminus of the protein (LYR-motif containing, LYR), suggesting that this gene may be associated with the mitochondrial energy metabolism.
Mitochondria are the main synthesis for known places of ATP, is an important organelle for energy metabolism. Cell mitochondrial dysfunction caused by reactive oxygen species (Reactive oxygen, species, ROS) increased through activation of various serine kinase (serine kinases) the mechanism of phosphorylation of insulin receptor substrate protein (insulin receptor, substrate, IRS). Serine residues, blocking insulin receptors (insulinreceptor, IR) (Intrinsic tyrosine kinase its tyrosine kinase) phosphorylation of IRS, eventually leading to insulin resistance. The preliminary study shows that the high expression of LYRM1 gene in 3T3-L1 fat cells (overexpression), can lead to mitochondrial dysfunction, decreased insulin stimulated glucose uptake. The rate, suggesting that the gene may influence insulin sensitivity through altered mitochondrial function, so this study by small interfering RNA (SMA Ll interference RNA (siRNA) technology was used to further observe whether mitochondrial function changes in mature adipocytes after silencing of LYRM1 gene expression, so as to demonstrate the effect of the gene on mitochondrial function.
Objective: To observe the effect of LYRM1 gene expression silencing on mitochondrial function of mature adipocytes and to analyze its mechanism.
Methods: the stable transfection of LYRM1 interference lentivirus in vitro (Lentiviral-Lyrm1-shRNA) 3T3-L1 preadipocytes, which only transfected with lentivirus (Lentiviral-NC-shRNA) 3T3-L1 preadipocytes as control, the interference efficiency RT-PCR validation of LYRM1 gene. Insulin, dexamethasone, 1- (MDI) -3- methyl isobutyl xanthine induced adipocyte differentiation scheme under transmission electron microscope. The mature fat cells mitochondrial morphology and ultrastructure. Analysis of mitochondrial DNA copy number by RT-PCR method (mtDNA) and the related gene Mfn1, Mfn2, Drp1, Fis1, UCP2, UCP4. The expression of chemiluminescent microplate cell ATP content. The application of mitochondrial fluorescence probe (Mito-Tracker Red), 2,7 two dichlorofluorescein probe (2 ', 7' -Dichlorofluorescein, diacetate, DCFDA) with pre mature fat cells, laser confocal microscopy and flow cytometry observation of mitochondrial membrane Changes in the reactiveoxygen species (ROS) of the cell and the cell active oxygen cluster.
Results: (1) LYRM1 gene silencing group of fat cells expression levels were significantly lower than the negative control group, the interference efficiency of Lentiviral-LYRM1-shRNA was 40%; (2) LYRM1 gene silencing group mature fat cells mitochondrial morphology and ultrastructure between mtDNA and the negative control group and no significant difference; (3) LYRM1 gene silencing and mitochondrial membrane potential the cell content of ATP group mature fat cells compared with the negative control group were significantly increased; (4) LYRM1 gene silencing group of cellular reactive oxygen species (ROS) significantly decreased; (5) LYRM1 gene silencing group mature fat cells in Mfn2, Ucp2mRNA expression was significantly increased, Ucp4, Mfn1, Drp1, Fis1 and mRNA showed no significant difference between the two groups.
Conclusion: the silence of LYRM1 gene expression can affect the mitochondrial function of mature adipocytes and improve the function of mitochondrial metabolism to some extent.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R723.14

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