本文關(guān)鍵詞： LYRM1 脂肪細(xì)胞 RNA干擾 線粒體 UCP2　出處：《南京醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來(lái)，肥胖發(fā)生率逐漸增高，兒童肥胖發(fā)生率的升高也非常顯著。肥胖是一種具有遺傳性、多因子參與的代謝性疾病，是能量攝入與能量消耗失衡的結(jié)果。肥胖是引起糖尿病、高血壓等多種疾病的高危因素。肥胖病因復(fù)雜，主要致病因素包括：機(jī)體遺傳易感性、環(huán)境因素、運(yùn)動(dòng)和營(yíng)養(yǎng)，其中遺傳因素在肥胖的發(fā)生發(fā)展中起重要作用。 LYRM1基因是本研究小組應(yīng)用抑制性差減雜交技術(shù)（suppression subtractivehybridization，SSH）篩選肥胖與正常人網(wǎng)膜脂肪組織中差異表達(dá)基因時(shí)獲得并克隆到的一條肥胖相關(guān)全長(zhǎng)新基因。前期研究發(fā)現(xiàn)：LYRM1基因高表達(dá)于肥胖者網(wǎng)膜脂肪組織，定位于染色體16P11.2，包括4個(gè)外顯子和3個(gè)內(nèi)含子，開(kāi)放閱讀框369bp，mRNA全長(zhǎng)1589bp，編碼蛋白質(zhì)產(chǎn)物122aa，蛋白N端附近有一高度保守的三肽結(jié)構(gòu)（LYR-motif containing，，LYR），提示該基因與線粒體能量代謝可能相關(guān)。 已知線粒體是供能物質(zhì)ATP的主要合成場(chǎng)所，是能量代謝的重要細(xì)胞器。線粒體功能障礙所引起的細(xì)胞活性氧（Reactive oxygen species，ROS）增高，可通過(guò)活化各種絲氨酸激酶（serine kinases）的機(jī)制，磷酸化胰島素受體底物蛋白（insulin receptor substrate，IRS）的絲氨酸殘基，而阻礙胰島素受體（insulinreceptor，IR）自身酪氨酸激酶（Intrinsic tyrosine kinase）對(duì)IRS的磷酸化作用，最終導(dǎo)致胰島素抵抗。由于前期研究顯示，LYRM1基因在3T3-L1脂肪細(xì)胞中高表達(dá)（overexpression），能夠?qū)е戮�粒體功能障礙、顯著降低胰島素刺激下的葡萄糖攝取率，提示該基因可能通過(guò)改變線粒體功能影響胰島素敏感性，因此本研究通過(guò)小干擾RNA（small interference RNA，siRNA）技術(shù)，進(jìn)一步觀察LYRM1基因表達(dá)沉默后成熟脂肪細(xì)胞線粒體功能是否發(fā)生改變，以論證該基因?qū)�粒體功能的影響。 目的：觀察LYRM1基因表達(dá)沉默對(duì)成熟脂肪細(xì)胞線粒體功能的影響，并初步分析其機(jī)制。 方法：體外培養(yǎng)穩(wěn)定轉(zhuǎn)染LYRM1干擾慢病毒（Lentiviral-Lyrm1-shRNA）的3T3-L1前體脂肪細(xì)胞，以僅轉(zhuǎn)染陰性慢病毒（Lentiviral-NC-shRNA）的3T3-L1前體脂肪細(xì)胞為對(duì)照，RT-PCR驗(yàn)證LYRM1基因的干擾效率。胰島素、地塞米松、1-甲基-3-異丁基黃嘌呤（MDI）方案誘導(dǎo)脂肪細(xì)胞分化成熟。透射電鏡下觀察成熟脂肪細(xì)胞的線粒體形態(tài)和超微結(jié)構(gòu)。采用RT-PCR法檢測(cè)線粒體DNA拷貝數(shù)（mtDNA）及相關(guān)基因Mfn1、Mfn2、Drp1、Fis1、UCP2、UCP4的表達(dá)�；瘜W(xué)發(fā)光酶標(biāo)儀檢測(cè)細(xì)胞ATP含量。應(yīng)用線粒體熒光探針（Mito-Tracker Red）、2,7二氯熒光素探針（2’,7’-Dichlorofluorescein diacetate，DCFDA）預(yù)染成熟脂肪細(xì)胞，激光共聚焦顯微鏡及流式細(xì)胞儀觀測(cè)線粒體膜電位及細(xì)胞活性氧簇（reactiveoxygen species，ROS）的變化。 結(jié)果：（1）沉默組脂肪細(xì)胞的LYRM1基因表達(dá)水平明顯低于陰性對(duì)照組，Lentiviral-LYRM1-shRNA的干擾效率達(dá)40%；（2）LYRM1基因沉默組成熟脂肪細(xì)胞線粒體形態(tài)、超微結(jié)構(gòu)以及mtDNA與陰性對(duì)照組之間無(wú)明顯差異；（3）LYRM1基因沉默組成熟脂肪細(xì)胞的線粒體膜電位和細(xì)胞ATP含量較陰性對(duì)照組顯著升高；（4）LYRM1基因沉默組細(xì)胞活性氧（ROS）水平明顯降低；（5）LYRM1基因沉默組成熟脂肪細(xì)胞中的Mfn2、Ucp2mRNA表達(dá)明顯上調(diào)，Ucp4、Mfn1、Drp1、Fis1等mRNA表達(dá)在兩組間無(wú)明顯差異。 結(jié)論：LYRM1基因表達(dá)沉默可以影響成熟脂肪細(xì)胞線粒體功能狀態(tài)，一定程度上改善線粒體代謝的功能。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R723.14

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