SIRT1敲除对C57BL／6J小鼠脂肪细胞分化的影响和机制探讨  

SIRT1 knockout impairs the differentiation of adipocyte in C57BL/6J mice and its underlying mechanism

作　　者：徐芬[1] 严晋华[1] 梁华[1] 许雯[1] 叶建平[1] 翁建平[1] 

机构地区：[1]中山大学附属第三医院内分泌与代谢病学科广东省糖尿病防治重点实验室,广州510630

出　　处：《中华医学杂志》2013年第36期2857-2860,共4页National Medical Journal of China

基　　金：国家重点基础研究发展计划（973计划）（2012CB517506）;国家自然科学基金（30900506）;国家自然青年科学基金（81100556）;国家杰出青年科学基金（81025005）;广东省自然科学基金（$2012010009323）

摘　　要：目的探讨沉默信息调节因子1（SIRT1）在脂肪分化和发育中的作用及相关机制。方法建立以C57BL／6J为基因背景的SIRTl全敲除小鼠（简称SIRT1^-/-小鼠），以同窝的野生型小鼠作为正常对照。28周龄小鼠测体重和体内脂肪含量，留取附睾脂肪垫。用BODIPY558／568和isolectinAlexaFluor488分别对离体后新鲜的附睾脂肪尖端的脂肪细胞和毛细血管进行荧光染色，在共聚焦显微镜下成像后用Imafis图像分析处理软件重建为三维图像。将固定后的附睾脂肪进行组织切片，行HE染色；用免疫组化的方法测血管内皮细胞标志物CD31蛋白的表达。体外实验取怀孕小鼠第13～14天的胚胎，制备小鼠胚胎成纤维（MEF）细胞，诱导分化为成熟的脂肪细胞，进行油红O染色。结果与野生型小鼠相比，SIRT1^-/-小鼠的体重显著低[（42．1±1．6）比（25．4±1．0）g，P〈0．05]，体内脂肪含量明显少[（13．4±1．0）比（7．8±0．5）g，P〈0．05]，附睾脂肪组织明显发育不良，组织切片HE染色显示SIRT1^-/-小鼠的脂肪细胞显著小、细胞外基质少；而体外实验中SIRT1^-/-小鼠MEF细胞的成脂能力反而强于野生型小鼠[油红O定量测吸光度（A）值：1．93±0．09比0．71±0．04，P〈0．05]。进一步实验发现，与野生型小鼠相比，SIRTl1^-/-小鼠附睾脂肪组织中血管生成显著少（毛细血管所占体积百分比：2．92％±0．03％比1．34％±0．02％，P〈0．05），表现为三维成像中毛细血管网较野生型对照组少近1／2，且SIRT1^-/-小鼠附睾脂肪组织中血管内皮细胞标志物CD31的表达受损。结论SIRT1全敲除导致C57BL／6J基因背景的SIRT1^-/-小鼠脂肪组织发育不良，其机制并非由于成脂能力减弱，而可能与脂肪组织中血管生成受损使得脂肪细胞的分化成熟受阻有关。Objective To explore the role and mechanism of SIRT1 （Sirtuinl） in the differentiation of adipocyte. Methods SIRTI -/- mice with C57BL/6J gene background were generated and litter-mate wild-type （WT） mice were used as controls. Body weight and fat content were detected and their epididymal fat pads were collected at 28 weeks old of age. The tip part of epididymal fat tissue was incubated in phosphate buffered saline （PBS） containing both BODIPY 558/568 （5 txmol/L in PBS） for adipoeytes and isoleetin Alexa Fluor 488 （40 μg/ml in PBS） for endothelial cells overnight. The stained cells were then visualized under confocal microscope. Reconstruction of 3D data sets was accomplished with image processing software Imaris. Immunohistoehemistry was used to detect the protein level of endothelial cell marker CD31. Hematoxylin and eosin staining was performed for fixed epididymal fat tissue. Mouse embryonic fibroblast （MEF） cells were prepared from 13 - 14 days embryos of SIRT1 -/- or WT mice and differentiated into adipocytes. Then oil-red 0 staining was performed. Results Compared with wild-type controls, both body weight （ WT 42. 1 g ± 1.6 g vs SIRT1 -/- 25.4 g± 1.0 g, P 〈 0. 05 ） and epididymal fat mass （ WT 13.4 g 1.0 g vs SIRT1-/- 7.8 g ±0.5 g, P 〈0.05）were much smaller in the SIRT1-/- mice. HE staining of fat tissue exhibited a significant reduction in adipocyte size and extracellular matrix in SIRTI -/- mice. However, the adipogenesis ability of MEF ceils was significantly enhanced in vitro in SIRT1 -/- MEF cells. Further study found that the density of vascular network decreased by 50% in the tip portion of epididymal fat pads of SIRT1-/- mice （capillary density：WT 2.92% ±0.03% vs SIRT1 / 1.34% ± 0. 02%, P 〈 0. 05 ）. CD31, a cellular marker of decreased angiogenesis, decreased significantly in epididymal fat pads of SIRT1 -/ - mice. Conclusion SIRT1 knockout impairs adipocyte differentiation in SIRT1 -/ mice with C57BL/6J ene backzround through reduced angiozenesis

关 键 词：抗衰老酶 脂肪细胞 细胞分化 血管生成 

分 类 号：R589.2[医药卫生—内分泌]