【作者】 张忠品；

【导师】 孙超；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2010， 硕士

【摘要】 本研究以昆明小鼠和3T3-L1前体脂肪细胞为研究对象。普通日粮饲喂小鼠,用外源性葡萄糖酸钙和二氢吡啶钙离子通道的抑制剂Nifedipine处理小鼠30 d,并用葡萄糖酸钙注射液和Nifedipine处理培养的3T3-L1前体脂肪细胞。测定的主要指标有:称体质量,肝脏、皮下脂肪、附睾脂肪和肾周脂肪质量,测量血清中甘油三酯(TG)、总胆固醇(TC)和高密度脂蛋白(HDL-C),测量肝脏、皮下脂肪、附睾脂肪和肾周脂肪组织中的脂肪酶活性,实时定量PCR检测GPR120基因和脂代谢相关基因转录水平,SPSS软件分析小鼠附睾脂肪中GPR120与脂代谢基因的表达相关性等,旨在从体内和体外水平探讨钙信号通路对GPR120基因转录及脂肪生成的影响机制,为进一步阐明GPR120参与调控脂肪生成的作用机制提供理论依据。取得如下结果:1.日粮添加葡萄糖酸钙可延缓小鼠体质量的增加,并降低小鼠体脂率。血清中TG、TC和LDL-C水平下降(P<0.01), HDL-C水平升高(P<0.05)。此外,葡萄糖酸钙能显著抑制肝脏和附睾脂肪组织中的脂肪酶活性,下调GPR120基因和FAS基因在肝脏、皮下脂肪、附睾脂肪和肾周脂肪部位的表达,抑制PPARγ基因在附睾脂肪和肾周脂肪中的表达,C/EBPα基因在肝脏和附睾脂肪的表达也下降;钙离子通道抑制剂Nifedipine能增加体质量并提高小鼠体脂率,血清中TG和TC水平升高。Nifedipine处理组小鼠肝脏、皮下脂肪和肾周脂肪组织中的脂肪酶活性均极显著升高(P<0.01)。Nifedipine可上调GPR120基因、FAS基因、PPARγ基因和C/EBPα在肝脏、皮下脂肪、附睾脂肪和肾周脂肪部位的表达,抑制HSL基因在这些部位的表达;说明葡萄糖酸钙可降低血脂水平,抑制脂肪生成,而Nifedipine一定程度上促进体脂沉积。2.基因相关性分析结果显示,附睾脂肪中GPR120基因与脂肪生成的重要转录因子PPARγ和C/EBPα显著正相关,与脂肪生成的标志基因FAS也显著正相关,而与脂肪分解标志基因HSL显著负相关。说明GPR120可能仅参与脂肪生成过程,而与脂肪分解关系不大。3. 5 mmol/L浓度的葡萄糖酸钙注射液处理24 h可抑制3T3-L1细胞内脂质蓄积,并下调GPR120,PPARγ,C/EBPα,FAS和HSL基因的转录水平,10μmol/L Nifedipine处理24 h可促进3T3-L1细胞脂质蓄积,并上调GPR120,PPARγ,C/EBPα和FAS基因的转录水平;葡萄糖酸钙处理72 h却提高胞内脂质含量,并增加GPR120,PPARγ,C/EBPα和FAS基因的转录水平,说明钙信号通路对脂肪生成具有复杂的调控作用。更多还原

【Abstract】 The present research aimed at exploring the effects of calgium signal on the transcription of GPR120 and adipogenesis in vivo and in vitro.To provide theoretical proof for further illustrating the regulation of adipogenisis by GPR120. Kunming mice and 3T3-L1 preadipocytes were the study subjects. The mice were fed with normal rodent diets and were treated with Calglucon and Ca2+-channel antagonist Nifedipine respectively for 30 days. The cultured 3T3-L1 preadipocytes were treated with 5 mmol/L Calglucon injection and 10μmol/L Nifedipine respectively. Body weight, liver mass, subcutaneous fat pads, epididymal fat pads and perirenal fat pads were weithed. Levels of Total cholesterol(TC), triglycerides(TG) and high density lipoproteincholesterol(HDL-C) in serum were measured. Lipase activity in mice liver and adipose tissues was measured by Lipase Activity Test Kit. Real-time PCR was applied to detect the expression levels of GPR120 and adipose metabolism related genes. The expression correlations of GPR120 with adipose metabolsim related genes in mise epididymal fat tissue were analyzed by using SPSS Stastistics Analysis Software. The main results were summarized as follows:1. In calglucon treated group, dietary calglucon can attenuate the increase of body mass and decrease the ratio of body fat to body weight, the concentrations of TG, TC and LDL-C in blood serum were decreased significantly (P<0.01), and the level of HDL-C was increased(P<0.05). Lipase activities in liver and epididymal fat tissues were significantly inhibited by calglucon. The expression levels of GPR120 and FAS mRNA were reduced in mice liver tissue, subcutaneous fat tissue, epididymal fat tissue and perirenal fat tissue. Calglucon inhibited the expression of PPARγmRNA in periepodidumal fat tissue and perirenal fat tissue, and also down regulated the expression of C/EBPαin liver and epididymal fat tissues. By contrast, Nifedipine can increase the deposit of body fat mass and heighten the levels of TG and TC in blood serum. Nifedipine increased the lipase activity in liver, epididymal fat tissue and perirenal fat tissue. The transcription levels of GPR120, FAS, PPARγas well as C/EBPαwere increased in liver, subcutaneous fat, epididymal fat and perirenal fat tissues, meanwhile, in these tissues, the transcriptional levels of HSL were decreased by Nifedipine. It’s suggested that Calglucon could decrease serum blood-fat level and inhibit body fat deposit. To some extent, Nifedipine can accelerate the deposit of body fat. 2. The expression correlation analysis results in mice epididymal fat tissue showed that GPR120 correlates with PPARγ, C/EBPαand FAS but not with HSL. It indicated that GPR120 was highly associated with adipogenesis.3.Treat cultured 3T3-L1preadipocyted with 5 mmol/L calglucon injection for 24 h, the content of intracellular lipid was decreased, and the transcripional levels of GPR120, FAS, PPARγand C/EBPαwere down-regulated. When treated with 10μmol/L Nifedipine for 24 h, Nifedipine accelerated the deposition of intracellular lipid, and increased the transcriptional levels of GPR120, PPARγ, C/EBPαand FAS. After 72 h, Calglucon injection significatly increased the deposition of intracellular lipid, and upregulated the expression levels of GPR120, PPARγ, C/EBPαand FAS. It indicats that calcium signal may play a very complicated role in regulating adipogenesis.更多还原