植物提取物Embelin对小鼠肥胖的影响及机制研究

发布时间：2018-06-30 05:16

本文选题：恩贝酸 + 肥胖　；参考：《天津医科大学》2016年硕士论文

【摘要】：背景:肥胖是一种代谢性疾病,由多种因素引起。其特点是体内脂肪细胞体积的增大和细胞数量的增加,由此导致脂肪质量占身体总质量的百分比异常增高,并且脂肪过多沉积在附睾周围、肠系膜和肾周等部位[1]。肥胖会使多种疾病的患病风险明显增加,如心脑血管疾病、脂肪肝、糖尿病等[2]。恩贝酸(embelin)是从植物中提取的一种醌类化合物。已有文献研究表明对于高脂饮食诱导的大鼠肥胖模型,Embelin能够抑制肥胖和由于肥胖导致的高血脂和氧化应激[3]。本研究主要探讨了Embelin是否通过抑制骨髓间充质干细胞向脂肪细胞分化分化来抑制肥胖,并初步探究其机制。方法:1、骨髓基质细胞系C3H10T1/2和间充质干细胞系ST2分别用二甲基亚砜(DMSO)和不同浓度的Embelin处理后,用成脂诱导培养基培养,诱导细胞成脂分化,通过油红O染色检测分化程度,通过q RT-PCR和Western blotting从m RNA丰度和蛋白表达水平检测成脂分化标记基因的水平,从而确定Embelin对骨髓基质细胞向脂肪细胞分化过程的影响;2、骨髓基质细胞C3H10T1/2分别用DMSO和Embelin处理,通过细胞免疫荧光检测β-连环蛋白(β-catenin)入核的改变,从而确定Embelin对wnt通路的作用;3、C3H10T1/2细胞分别用二甲基亚砜(DMSO)和不同浓度的Embelin处理后,成脂诱导培养基诱导成脂,提取核蛋白,通过Western blotting检测细胞核中β-catenin和T细胞因子(T cell factor,TCF)-4蛋白水平,确定Embelin对wnt通路的作用;4、C3H10T1/2细胞分别用二甲基亚砜(DMSO)或10μM的Embelin处理后,q RT-PCR检测Wnt通路配体Wnt10b、共受体Lrp5、抑制因子Sfrp1和Dkk1的m RNA水平,以此确定Embelin影响Wnt通路的机制;5、雄性C57BL/6小鼠,4周龄,用高脂饲料喂养构建小鼠肥胖模型,同时皮下注射不同剂量的Embelin,造模过程中记录各组小鼠体重,成模后用骨密度仪测量体脂率,取血清检测葡萄糖、甘油三酯、游离脂肪酸和总胆固醇水平,以此确定Embelin对肥胖的影响;6、实验组与对照组小鼠处死后,分离各个部位脂肪组织,提取RNA,逆转录,通过q RT-PCR检测不同部位脂肪组织中成脂分化标记基因和关键转录因子的表达水平;7、取实验组与对照组小鼠的脂肪组织,制作石蜡切片,HE染色,观察脂肪组织中脂肪细胞的体积。结果:1、油红O染色表明Embelin对骨髓基质细胞系C3H10T1/2和ST2成脂分化有抑制作用,q RT-PCR和Western blotting结果表明Embelin能抑制成脂分化标记基因过氧化物酶体增殖物激活受体γ(Peroxisome proliferator-activated receptorγ,PPARγ)、CCATT增强结合蛋白(CCAAT enhancer binding proteinα,C/EBP-α)、脂肪细胞表征因子a P2(Adipocyte fatty acid-binding Protein 2)和Adipsin的表达,且有剂量依赖效应;2、细胞免疫荧光表明在C3H10T1/2中Embelin可以促进β-catenin入核,提取核蛋白,Western blotting结果表明核内β-catenin和TCF-4水平上升,且有剂量依赖效应,说明Embelin可以激活wnt通路。C3H10T1/2细胞经Embelin处理后,q RT-PCR检测Wnt通路相关基因的表达,包括Wnt通路的配体wnt10b、共受体Lrp5、Wnt通路抑制因子sfrp1和Dkk1,发现Dkk1的m RNA水平明显下降,说明Embelin下调Wnt通路抑制因子Dkk1的表达,进而激活Wnt通路;3、饲喂高脂饲料组小鼠较饲喂普通饲料组小鼠体重明显上升,造模成功,而高脂饲料喂养同时皮下注射Embelin组的体重较高脂同时注射溶剂组明显下降,骨密度仪检测体脂率也表明Embelin能够明显降低脂肪占体重的百分比,且有剂量依赖效应,表明Embelin能够抑制小鼠肥胖;测定血清中葡萄糖、游离脂肪酸、总胆固醇和甘油三酯水平,结果表明Embelin组均比溶剂组低,进一步说明Embelin能够抑制小鼠肥胖;4、取各组小鼠脂肪组织制作石蜡切片,HE染色表明,注射Embelin后,小鼠的脂肪细胞体积变小,说明Embelin能够在体内抑制脂肪组织中脂肪细胞的肥大;5、提取各组小鼠脂肪组织总RNA,用q RT-PCR检测成脂标记基因和关键转录因子的表达,注射Embelin组a P2、C/EBP-α、PPARγ和Adipsin表达水平均低于模型组,也说明Embelin能够在体内抑制成脂分化。结论:1、在体外,Embelin能够通过下调Wnt通路抑制因子Dkk1的表达从而激活wnt通路,抑制骨髓基质细胞成脂分化;2、在体内,Embelin能够通过抑制间充质干细胞成脂分化来抑制小鼠肥胖。
[Abstract]:Background : Obesity is a metabolic disease caused by a variety of factors . It is characterized by an increase in the volume of fat cells in the body and an increase in the number of cells , thereby causing an abnormal increase in the percentage of fat mass in the total mass of the body , and excessive deposition of fat around the cryptorchidism , mesenteric and perirenal parts . Obesity can increase the risk of multiple diseases , such as cardiovascular and cerebrovascular diseases , fatty liver , diabetes , etc . embelin is a kind of quinone compound extracted from plants . It has been found that Embelin can inhibit obesity and high blood lipid and oxidative stress caused by obesity . The effects of Embelin on the differentiation of bone marrow stromal cells into adipocytes were determined by using DMSO and Embelin treatment . The expression of adipogenic differentiation marker gene and key transcription factor in adipose tissue of bone marrow stromal cell line C3H10T1 / 2 was detected by means of q RT - PCR . The results showed that Embelin could inhibit the expression of 尾 - catenin in bone marrow stromal cell line C3H10T1 / 2 and ST2 . The results showed that Embelin could inhibit the expression of 尾 - catenin in bone marrow stromal cell line C3H10T1 / 2 and ST2 . Conclusion : In vitro , Embelin can inhibit the fat cells in adipose tissue by downregulating the expression of fat cells in adipose tissue . Conclusion : In vitro , Embelin can inhibit the proliferation of fat cells in adipose tissue . In vivo , Embelin can inhibit the differentiation of adipose cells in bone marrow stromal cells .
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R589.2

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