【作者】 周远飞；

【导师】 彭健；

【作者基本信息】 华中农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 动物脂肪组织的沉积不仅仅是增加已存在的脂肪细胞中的脂类储藏,而且始终伴随着从祖细胞生成新的脂肪细胞的过程,即“脂肪生成(adipogenesis)"。脂肪生成包括起始阶段多能干细胞向前脂肪细胞“定型”,以及随后前脂肪细胞向成熟脂肪细胞“分化”。在过去的二十年中,人们对调控脂肪生成的调网络,特别是控制前脂肪细胞向脂肪细胞“分化”的转录级联已经基本清楚。而在“定型”阶段,虽然目前已有一些转录因子和信号通路也陆续被证实,但由于缺少前脂肪细胞的标志基因,因此,多能的间充质干细胞(Mesenchymal stem cells, MSCs)向前脂肪细胞“定型”的特异性定型因子及其分子机制有待进一步研究。已有研究证实,SIRT1在前脂肪细胞分化中,不仅可以去乙酰化组蛋白,调控转录因子的转录活性；而且可以去乙酰化脂肪生成关键转录因子。此外,在MSCs细胞命运决定中,SIRT1可以促进MSCs的成骨分化,而抑制MSCs的成脂分化。但SIRT1调控MSCs成脂定向的分子机制尚不明确,有待进一步阐明。本研究由两部分组成,第一部分为体外细胞实验,研究SIRT1通过Wnt信号通路的对MSCs细胞成脂定向的影响,并阐明SIRT1通过调控Wnt信号通路,影响MSCs分化形成脂肪细胞的分子机制。第二部分为体内活体试验,以SIRT1敲除的小鼠为研究对象,通过动物活体验证SIRT1与脂肪形成的关系；同时以分离的小鼠的胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)为材料,进一步探讨了SIRT1调控MSCs成脂定向的分子机制。主要研究内容和结果如下：第一部分,体外细胞实验。在C3H10T1/2细胞中,对SIRT1用激活剂或抑制剂处理,或干涉/过表达SIRT1后,研究SIRT1对脂肪生成表型及成脂标志基因mRNA和蛋白水平的影响。在此基础上,研究干涉SIRT1对Wnt信号靶基因和关键蛋白的表达及Wnt信号通路报告系统活性的影响；用Real-time PCR芯片筛选出受SIRT1调控Wnt信号通路拮抗物；并稳定干涉这些Wnt信号通路拮抗物后,研究对成脂表型及成脂标志基因mRNA和蛋白水平的影响：通过ChIP和IP等手段,研究SIRT1调控Wnt信号通路的分子机制。主要结果如下：1.添加SIRT1的激活剂白藜芦醇抑制了C3H10T1/2细胞的成脂表型,且成脂标志基因(PPARγ, aP2和adiponectin)的mRNA和蛋白表达也显著抑制；而添加SIRT1的抑制剂烟酰胺则促进了C3H10T1/2细胞的脂滴形成,成脂标志基因(PPARyγ, aP2和adiponectin)的mRNA和蛋白表达丰度显著上调(P<0.05)。白藜芦醇处理显著增加了6h和12h的S期细胞数量(P<0.05),烟酰胺处理对细胞周期影响不显著(P>0.05)。2.在C3H10T1/2细胞中干涉SIRT1,成脂诱导后,增加了油红O染色的脂滴的数目,且显著提高了成脂标志基因(PPARy, aP2和adiponectin的mRNA和蛋白表达丰度；而过表达SIRT1组,视野中油红O染色的脂滴较少,显著抑制了成脂标志基因(PPARγ, aP2和adiponectin)的mRNA和蛋白表达。表明激活SIRT1抑制了MSCs细胞的成脂定向；而抑制SIRT1的活性,则促进了MSCs细胞的成脂定向。3.在C3H10T1/2细胞中干涉SIRT1,显著抑制了Wnt信号通路靶基因CyclinD1的mRNA和蛋白表达水平(P<0.05)以及Wnt信号通路关键分子β-catenin的蛋白水平；Wnt信号通路报告系统实验结果显示,干涉SIRT1显著抑制了Wnt信号通路报告系统的活性(P<0.05)；此外,通过转染β-catenin的突变体质粒,结果发现SIRT1对Wnt信号通路的调控是依赖β-catenin的。4.Wnt信号PCR芯片结果及对芯片的验证结果显示,激活SIRT1显著抑制了Wnt信号通路胞外拮抗物sFRPl和sFRP2以及胞内拮抗物Dactl的mRNA表达水平(P<0.05)；而抑制SIRT1显著提高了Wnt信号通路胞外拮抗物sFRP1和sFRP2以及胞内拮抗物Dactl的mRNA表达水平(P<0.05)。这表明,SIRT1对Wnt信号通路的调控可能是通过Wnt信号的胞外拮抗物sFRP1和sFRP2,以及Wnt信号的胞内拮抗物Dactl来实现的。5.分别建立稳转干涉SIRT1和干涉Wnt信号通路拮抗物的细胞材料：pLKO.1-SIRT1、pLKO.1-SIRT1+pLKO.1-sFRP1, pLKO.1-SIRT1+pLKO.1-sFRP2, pLKO.1-SIRT1+pLKO.1-Dact和pLKO.1-SIRT1+pLKO.1-sFRP1+pLKO.1-sFRP2;成脂诱导这些稳转的细胞,结果显示与pLKO.1-SIRT1组相比,这些细胞的形成的脂滴较少,且成脂标志基因(PPARγ, aP2和adiponectin)的mRNA和蛋白的表达下调；Wnt信号通路报告系统实验结果显示,与pLKO.1-SIRT1组相比,提高了Wnt信号通路的活性。以上结果表明,在MSCs细胞成脂定向过程中,SIRT1可能通过Wnt信号拮抗物sFRP1、sFRP2和Dact1来作用的。6. ChIP结果显示,干涉SIRT1显著增加了sFRP1.sFRP2和Dact1的启动子区域的组蛋白的H3K9和H4K16的乙酰化水平(P<0.05)。此外,IP结果显示,干涉SIRT1显著增加了β-catenin的乙酰化水平,且减少了β-catenin进入细胞核内的水平。这表明,SIRT1可以通过去乙酰化组蛋白和非组蛋白两种方式调控Wnt信号通路。第二部分,体内活体实验。首先,以SIRT1单敲的小鼠(SIRT1+/-)为研究对象,SIRT1野生型小鼠(SIRT1+/+)为对照,验证SIRT1与脂肪形成的关系。正常饮食饲喂12w后,对小鼠称重并取样,对各部位脂肪组织和肝脏称重,并计算脂肪组织与体重的比值；检测血液中甘油三酯的含量；通过外观和HE染色观察脂肪组织的形态；并检测了皮下和内脏脂肪组织中成脂标志基因的变化。然后,分离13.5的小鼠胚胎的MEFs细胞,经基因型鉴定后,对不同SIRT1基因型(SIRT1+/+、SIRT1+/.和SIRT1-/-)的MEFs细胞成脂诱导,研究SIRT1对脂肪生成表型及成脂标志基因mRNA和蛋白水平的影响；此外,以不同SIRT1基因型的MEFs细胞为材料,通过ChIP和IP等手段进一步探讨SIRT1调控Wnt信号通路的分子机制。主要结果如下：1.小鼠正常饮食饲喂12w后,SIRT1+/-小鼠的外观正常,但体重显著低于SIRT1+/+小鼠(P<0.05)；脂肪组织和肝脏质量没有显著差异(P>0.05),但提高了SIRT1+/.小鼠的脂肪与体重质量比(P<0.05)：血液中甘油三酯的含量也没有显著差异(P>0.05)。以上结果表明,SIRT1敲除影响了小鼠生长,但并不影响脂肪组织和肝脏组织的发育,相反可以促进脂肪的形成。2.对脂肪组织的形态研究结果显示,不同基因型小鼠的棕色脂肪组织和附睾脂肪组织外观差异不显著；HE染色结果显示,SIRT1+/.小鼠脂肪组织脂肪细胞的体积有增加的趋势。3.SIRT1+/小鼠显著提高了皮下脂肪(腹股沟脂肪)中PPARy的mRNA表达丰度(P<0.05),对内脏脂肪(附睾脂肪)中PPARγ的mRNA表达没有显著差异(P>0.05);aP2和adiponectin的mRNA在两种基因型小鼠的皮下和内脏脂肪组织中的表达也没有差异。4.成脂诱导MEFs细胞后,SIRT1+/和SIRT1-/组的MEFs细胞脂滴数目较多,而SIRT1+/组最多；SIRT1+/-组的成脂标志基因(PPARγ, aP2和adiponectin)的表达丰度极显著或显著高于SIRT1+/+和SIRT1-/-组(P<0.01；P<0.05)；而SIRT1-/.组的成脂标志基因(PPARγ, aP2和adiponectin)的表达丰度极显著高于SIRT1+/+(P<0.01)。以上结果表明,SIRT1缺失则促进了MEFs细胞的脂肪生成,SIRT1单缺失具有更强的成脂能力。5. ChIP结果显示,在SIRT1缺失的MEFs细胞中,显著增加了sFRP1、sFRP2和Dactl的启动子区域的组蛋白的H3K9和H4K16的乙酰化水平(P<0.05)。IP结果显示,干涉SIRT1缺失增加了P-catenin的乙酰化水平。这表明在SIRT1缺失的MEFs细胞中,SIRT1也可以通过去乙酰化组蛋白和非组蛋白两种方式调控Wnt信号路。本研究的主要结论：SIRT1对间充质干细胞命运选择具有决定性作用,激活SIRT1抑制了MSCs细胞的成脂定向,而抑制SIRT1则促进了MSCs细胞的成脂定向。其机制是一方面SIRT1通过去乙酰化Wnt信号的拮抗物启动子区域的组蛋白,抑制Wnt信号的拮抗物的表达,解除了对Wnt信号通路的拮抗,从而激活Wnt信号通路,抑制脂肪的生成；另一方面SIRT1通过去乙酰化Wnt信号的关键蛋白β-catenin,促进β-catenin在核内的积累,促进Wnt信号通路靶基因活性,抑制脂肪的生成。更多还原

【Abstract】 Animal fat tissue deposition is not only greater the existing lipid stored in fat cells, and always new adipocytes are growing from progenitor cells, also known as adipogenesis. Adipogenesis can be divided into two related stages:commitment and terminal differentiation. Within the last two decades it has also become clear how the activity of these transcription factors control differentiation from preadipocytes to adipocytes. During commitment, although there are some transcription factors and signaling pathways have been confirmed, due to the lack of preadipocyte marker genes. Thus, the specific factors and molecular mechanism need further research during determination process.Previous studies reported that SIRT1participate in regulation of adipocyte differentiation, and mediate transcription and post-translation modifications, can directly deacetylate histones and various transcription factors about nonhistones. In addition, SIRT1markedly inhibited adipocyte and promoted osteoblast differentiation during fate determination of MSCs. But the underlying molecular mechanism remains to be investigated.This study composes of two parts that were cell experiment and animal experiment. In vitro experiment, we studied that SIRT1regulate MSCs commitment to adipocyte lineage through Wnt signalling pathway, and elucidated the molecular mechanism about SIRT1regulation of Wnt signaling antagonists. In vivo, the heterozygous KO (SIRT1+/-) mice and wild type (WT, SIRT1+/+) littermates were used as subjects in the study. We firstly validated the relation between SIRT1and adipogenesis. The MEFs were isolated and as mainly research material. The molecular mechanism of SIRT1regulation of MSCs commitment to adipocyte lineage was made future investigated. The main contents and results are as follows:The first part:In vitro experiment.1. C3H10T1/2MSCs were treated with SIRT1activator (resveratrol)/inhibitor (nicotinamide) for2d, or were transfected RNAi/overexpression plasmid of SIRT1, then induced by adipogenic medium to study whether SIRT1regulates MSCs commitment to adipocyte lineage.2. C3H10T1/2cells were transfected RNAi plasmid of SIRT1, the cells were harvested. The Wnt signalling pathway report activity and the target genes were determined that to study whether SIRT1regulation of adipogenic commitment through Wnt signalling pathway.3. C3H10T1/2MSCs were treated with resveratrol/inhibitor; the cells were harvested and screening of Wnt antagonists by profiler PCR Array. The stable transfection of cell lines about RNAi of Wnt signaling antagonists were induced by adipogenic medium. Wnt signalling pathway were determined that to study whether SIRT1regulation of adipogenic commitment through regulating Wnt signaling antagonists.4. Using ChIP and IP, the mechanisms underlying of SIRT1regulation of Wnt signalling were analysis in infected cell. The main results are as follows:1. Activation of SIRT1by resveratrol inhibited the lipid accumulation and adipogenic gene expression, while inhibition of SIRT1by nicotinamide promoted the lipid accumulation and adipogenic gene expression of C3H10T1/2cells. Resveratrol treatment of C3H10T1/2cells markedly increased the number of cells at the S phase for6h and12h (P<0.05), whereas nicotinamide treatment failed to elicit any substantiate effect on C3H10T1/2cells.2. Overexpression of SIRT1in C3H10T1/2cells blocked lipid lipid accumulation and adipogenic gene expression, shRNA-medited knockdown of SIRTl had the opposite effect. Thus, the combined date from gain-and loss-of function consistently demonstrate that SIRT1acts as a repressor of adipogenesis in vitro.3. SIRT1RNAi significantly inhibited Cyclin D1expression of Wnt signalling target gene and β-catenin of key factor of Wnt signalling target gene. shRNA-medited knockdown of SIRT1could significantly suppressed Wnt reporter activity. The results of β-catenin reporter assay showed that a β-catenin-dependent.4. The results of Wnt signalling by profiler PCR Array showed that resveratrol treatment inhibited sFRP1and sFRP2gene expression of extracellular antagonists of the Wnt signalling pathway and Dact1mRNA expression of intracellular antagonists of the Wnt signalling pathway. In contrast, nicotinamide promoted sFRP1, sFRP2and Dact1 gene. The results of PCR Array revealed that SIRT1regulate Wnt signalling by inhibited sFRP1, sFRP2and Dactl expression.5. We used lentivirus to generate C3H10T1/2stable cell lines carrying pLKO.1-SIRT1、pLKO.1-SIRT1+pLKO.1-sFRPl、pLKO.1-SIRT1+pLKO.1-sFRP2, pLKO.1-SIRT1+pLKO.1-Dactl and pLKO.1-SIRT1+pLKO.1-sFRP1+pLKO.1-sFRP2. The stable transected cells were subjected to adipogenic differentiation protocol, and samples were collected for adipogenic analysis. Compared with cells infected with the pLKO.1-SIRT1vector, stale cells down-regulated SIRT1and Wnt signalling antagonists accumulated much less fat as determined by Oil red O staining and had a reduction in adipogenic maker gene expression. The stable cells infected with SIRT1and Wnt signalling antagonist’s vector increased Wnt reporter activity.6. We performed ChIP assay in C3H10T1/2cells and abserved robust increases in acetylation of H3K9and H4K16at the sFRPI, sFRP2and Dactl promoters with shRNA Knockdown of SIRT1. We performed IP assay in C3H10T1/2cells and abserved that SIRT1deacetylaes β-catenin to promote its accumulation in the nucleus.The second part: In vivo experiment. The heterozygous KO (SIRT1+/-) mice and wild type (WT, SIRT1+/+) littermates were used as subjects in the study. The SIRT1+/+mice and SIRT1+/-mice maintained on the regular chow diet for12w, blood was collected first, and then liver, intrescapular brown adipose tissue, intrescapular adipose tissue, inginal fat, epididymal fat and perirenal fatwere taken and weighed for the tissues to body weight ratio. Histology study on adipose tissue by HE staining.2. The MEFs were isolated and as mainly research material. The molecular mechanism of SIRT1regulation of MSCs commitment to adipocyte lineage was made future investigated. The main contents and results are as follows:1. The appearance was no difference in SIRT1+/-mice compare with SIRT1+/+mice, but the body weigh had a reduction. Although SIRT1+/-mice exhibited no different in fat weigh and liver weigh, the ratio of fat mass to body weigh was significantly higher compare with SIRT1+/+mice, suggesting that the fat content is increased in the null mice. 2. The appearance and HE staining showed that SIRT1+/+mice display moderately enlarged adipose depots relative to SIRT1+/+littermates when assessed by histology.3. Expression of PPARy mRNA was much higher in inginal fat of SIRT+/-mice and was not change in epididymal fat of SIRT1+/-mice. Expression of aP2and adiponectin mRNA was not change in epididymal fat and inginal fat of SIRT1+/-mice.4. MEF cells were prepared from13.5d embryo and induced for adipogenesis in the culture medium. Compared with WT cells lipid accumulation in differentiated cells from SIRT+/-and SIRT1-/-MEF were enhanced as observed by the red color from the Oil red-O staining. SIRT1+/-MEFs exhibited significant elevation in mRNA and protein for PPARγ, aP2and adiponectin, followed by the SIRT1-/-MEFs. The results suggest that SIRT1null cell promote adipogenesis, Adipogenic maker genes, while heterozygous SIRT1have greater ability of adipogenic commitment.5. We performed ChIP assay in MEFs and abserved robust increases in acetylation of H3K9and H4K16at the sFRPl, sFRP2and Dactl promoters in null cells of SIRT1. IP assay showed that in null cells of SIRT1increases acetylation of (3-catenin.In summary, the conclusions of this study are:1. SIRT1is a negative regulator of MSCs commitment to adipocyte lineage.2. SIRT1may affect MSC fate by regulating Wnt signalling pathway. On the one hand, SIRT1deacetylate hisone of sFRP1, sFRP2and Dactl promoters, inhibit the sFRPl, sFRP2and Dactl mRNA expression, and remove the inhibition for Wnt signaling, then activate Wnt signaling pathway. On the other hand, SIRT1deacetylates β-catenin to promote its accumulation in the nucleus leading to transcription of genes for blocking adipogenesis of MSCs.更多还原