【作者】 王明明；

【导师】 秦正红； 程钢；

【作者基本信息】 苏州大学 ， 药学（专业学位）， 2020， 硕士

【摘要】 目的:采用TIGAR转基因小鼠,观察TIGAR基因对小鼠脂质代谢的影响,同时在3T3-L1脂肪细胞中进行验证,进一步对其机制进行探究。方法:体内构建TIGAR转基因小鼠模型,5周龄TIGAR转基因小鼠以及同窝出生的野生型小鼠在同等条件下喂养16周,同一时间内测量每组小鼠体重、摄食量和饮水量;采用血糖测定方法测量8周龄小鼠葡萄糖耐量及胰岛素抵抗;采用间接量热法测定8周龄小鼠的能量指标(氧气消耗量,二氧化碳的产出量,呼吸率和能量消耗);用TC,TG,LDL-C和HDL-C试剂盒检测TIGAR对血脂的影响;称量小鼠不同部位的脂肪量(附睾,皮下,腹膜后,棕色脂肪)以及肝脏重量,通过HE和油红O染色观察脂肪的大小,数量以及损伤情况;采用RT-PCR,Western blot技术观察过表达TIGAR对脂肪合成和脂肪分解相关蛋白mRNA及蛋白的表达。在体外按照通常的方法对3T3-L1前脂肪细胞培养和诱导分化,采用脂质体法将小RNA干扰链转入到细胞,通过油红O染色观察敲低TIGAR对脂滴数量和大小的影响;采用RT-PCR,Western blot技术检测敲低TIGAR对脂肪合成和脂肪分解相关蛋白mRNA及蛋白的表达。结果:体内实验中,与野生型小鼠相比,TIGAR转基因小鼠能使小鼠体重明显增加(P<0.05),糖耐量和对胰岛素敏感性降低(P<0.01),具有统计学意义;TIGAR转基因小鼠耗氧量以及二氧化碳产出量较高(P<0.05),具有统计学意义,但能量消耗和呼吸率没有显著差异(P>0.05);TIGAR转基因小鼠脂肪组织以及肝脏重量增加,脂滴增加,肝脏严重损伤,具有明显差异(P<0.01);TIGAR转基因小鼠能够使血糖血脂升高,具有统计学意义(P<0.01);TIGAR转基因小鼠脂肪的合成相关因子mRNA水平和蛋白表达量增加(FAS,ACC,PPAR-γ),脂肪的分解相关因子mRNA水平和蛋白表达量降低(HSL,ACOX),具有统计学意义(P<0.05)。体外试验中,敲低TIGAR基因能够使脂滴减小,脂肪的合成相关因子mRNA水平和蛋白表达量下降,但脂肪的分解相关因子mRNA水平和蛋白表达量增加,具有统计学意义(P<0.05)。结论:与野生型小鼠相比,TIGAR转基因小鼠体重增高,摄食量和摄水量增加,脂肪组织和肝脏的含量增加并有不同程度损伤;TIGAR转基因小鼠血脂、血糖升高;TIGAR转基因小鼠的糖耐量以及胰岛素敏感性下降,但是耗氧量和二氧化碳产出量均有增加;过表达TIGAR有利于脂肪的合成抑制脂肪的分解。本课题着重探讨了TIGAR对脂肪合成酶和分解酶的直接调节作用,发现TIGAR的新功能,为肥胖的基因治疗提供了新思路。更多还原

【Abstract】 Aim:TIGAR transgenic mice were used to observe the effects of TIGAR on the lipid metabolism in mice.In vitro,3T3-L1 adipocytes were used to further explore the mechanisms by which TIGAR affects lipid metabolism.Methods:The model of TIGAR transgenic mice was constructed in vivo.The 5 weeks old wild-type mice and TIGAR transgenic mice born in the same nest were fed for 16 weeks under the same condition.The weight,diet and water consumption of each group of mice were measured at the same time.The glucose tolerance and insulin resistance of 8-week-old mice were measured by blood glucose measurement.Indirect calorimetry was used to measure the energy indexes(oxygen consumption,carbon dioxide production,respiration rate and energy consumption)of 8-week-old mice;TC,TG,LDL-C and HDL-C kits were applied to detect the influence of TIGAR on blood lipid;The weight of fat in different parts(epididymis,subcutaneous,retroperitoneal,brown fat)and liver of mice were measured.The size,quantity and damage of fat were observed by H&E and oil red O staining;RT-PCR and Western blot were used to detect the expression of mRNA and protein related to lipogenesis and lipolysis by the overexpression of TIGAR.Conventional culture and differentiation of 3T3-L1 preadipocytes in vitro.The small RNA interference chain was transferred into the cells by liposome method,observation of the effect of TIGAR knockdown on the number and size of lipid droplets by oil red O staining;RT-PCR and Western blot were used to detect the expression of mRNA and protein related to lipogenesis and lipolysis by the knockdown of TIGAR.Results:In vivo,compared with wild-type mice,the body weight of TIGAR transgenic mice was significantly increased(P<0.05),the glucose tolerance and insulin sensitivity were decreased(P<0.01);TIGAR transgenic mice had higher oxygen consumption and carbon dioxide production(P<0.05),but there was no significant difference in energy consumption and breathing rate(P>0.05);There were significant increases in the weight of fat tissue and liver,lipid drops and liver damage in TIGAR transgenic mice(P<0.01);TIGAR transgenic mice increased the levels of blood glucose and blood lipid(P<0.01);The mRNA levels and protein expression of lipogenesis related factors increased(FAS,ACC,PPAR-y)and the mRNA level and protein expression level of fat decomposition related factors decreased(HSL,ACOX)in TIGAR transgenic mice(P<0.05).In vitro,TIGAR knockdown could decrease lipid droplets,mRNA levels and protein expression of lipogenesis related factors,but the mRNA levels and protein expression of lipolysis related factors increased(P<0.05).Conclusions:Compared with wild-type mice,TIGAR transgenic mice had increased body weight,food and water intake,increased content of adipose tissue and liver with different degrees of damage,increased blood lipid and glucose,decreased glucose tolerance and sensitivity of insulin,accelerated oxygen expenditure and carbon dioxide outout.Overexpression of TIGAR was conducive to fat synthesis and inhibition of fat decomposition.In this study,we focused on the direct regulation of TIGAR on fat synthetase and catabolic enzyme to find out the new function of TIGAR,which provided a new concept for regulation of lipid metabolism and prevention of obesity.更多还原