TGH在猪脂肪细胞中的表达及其脂解作用的初步研究

发布时间：2018-04-20 13:26

本文选题：甘油三酯水解酶(TGH) + 敏感脂肪酶(HSL)　；参考：《西北农林科技大学》2007年硕士论文

【摘要】： 脂肪细胞内甘油三酯的水解对机体能量代谢有着重要影响。脂解的平衡不仅可以有效地为机体提供能量,而且可以防止因体脂过度沉积引发的肥胖以及胰岛素抵抗等一系列代谢性疾病,因此对脂解的调控研究十分关键。脂肪分解酶是脂解反应中的关键因素。甘油三酯水解酶(TGH)是继激素敏感脂酶(HSL)之后发现的又一脂肪分解关键酶。以往对于TGH的功能研究主要集中于啮齿类动物,有关哺乳动物中TGH的脂解作用研究尚未见报道;TGH是和HSL一样是在激素刺激作用下发挥作用,还是主要介导基础状态下的脂解,目前尚不明确。因此,本实验以与人类生理生化方面最为相似的猪为模式动物,利用细胞培养、甘油三酯含量检测、甘油释放量测定,半定量RT-PCR、Western-Blot以及载体构建等细胞生物学和分子生物学技术研究了TGH在猪原代脂肪细胞分化中的表达规律以及细胞饥饿和重新恢复营养状态下的脂解及TGH的表达变化;并且通过在异丙肾上腺素刺激作用下与HSL的对比,初步探讨了其在脂肪分解中的作用方式;此外,构建了TGH的RNA干扰载体,为实验室以后进一步研究TGH的功能作出铺垫。所取得的结果如下: 1检测了猪原代脂肪细胞分化过程中TGH的表达。TGH mRNA在前脂肪细胞不表达,在诱导分化第4天表达量最高,之后表达量相对稳定,到第10天表达量略有下降。TGH的蛋白表达和其mRNA表达不尽相同,TGH蛋白在第0天几乎不表达,第二天表达量较低,第六天表达量最高,随后表达有所降低,然后基本趋于稳定。 2检测了细胞饥饿及重新恢复营养状况下脂肪分解的变化及TGH的表达改变,分析该过程中TGH的作用。细胞缺乏营养状态下,即用无糖无血清培养基培养时甘油释放量升高,即脂解作用增强,同时TGHmRNA和蛋白表达与对照相比升高;当恢复营养(完全培养基培养)后其表达又有所下降,但脂解下降不显著。 3研究了异丙肾上腺素(Isoproterenol)刺激状态下脂肪分解及TGH的表达变化,并分析该状态下TGH的作用。低浓度的异丙肾上腺素(0.1μmol/L,1μmol/L)没有检测到脂肪细胞释放到培养基中的甘油含量的改变,随异丙肾上腺素浓度的升高,甘油释放量显著上升,同时检测到细胞中甘油三酯含量下降,即脂解作用增强。此过程中HSL mRNA及蛋白表达均随异丙肾上腺素浓度的升高以及作用时间的不同而呈剂量和时间依赖性改变,但TGHmRNA和蛋白的表达在此过程中没有显著变化。 4构建了pSilencer-TGH载体并筛选了潮霉素的最佳致死浓度。对原质粒进行酶切、回收,连接、转化、质粒提取,最后测序鉴定表明TGH RNA干扰载体已经构建成功。200μg/mL是潮霉素抗性筛选的最佳浓度。
[Abstract]:The hydrolysis of triglycerides in adipocytes plays an important role in energy metabolism. The balance of lipids can not only provide energy for the body, but also prevent obesity and insulin resistance caused by excessive body fat deposition. Therefore, the regulation of lipid hydrolysis is very important. Lipase is a key factor in the lipopolysis reaction. Triglyceride hydrolase (TGH) is a key lipolytic enzyme after hormone sensitive lipase (HSL). In the past, the functional studies of TGH were mainly focused on rodents. It has not been reported that whether TGH plays the same role as HSL in hormone stimulation or mainly mediates the lipid hydrolysis in basic state. It is not clear yet. Therefore, in this experiment, pig, which is the most similar to human physiology and biochemistry, was used as the model animal. The cell culture, triglyceride content and glycerol release were used in this experiment. Semi-quantitative RT-PCR Western-Blot and vector construction were used to study the expression of TGH in primary adipocyte differentiation, and the changes of lipid and TGH expression in the condition of starvation and restoration of nutrition. By comparing with HSL stimulated by isoproterenol, the mechanism of its action in adipose decomposition was preliminarily discussed. In addition, the RNA interference vector of TGH was constructed to pave the way for further study on the function of TGH in laboratory. The results were as follows: 1 the expression of TGH was detected during the differentiation of porcine primary adipocytes. TGH mRNA was not expressed in preadipocytes, but the expression level was the highest in preadipocytes on the 4th day after differentiation, and then the expression level was relatively stable. On the 10th day, the expression of TGH was slightly decreased. The expression of TGH and its mRNA were not the same. On the second day, the expression of TGH was lower, the expression of TGH was the highest on the sixth day, then the expression decreased, and then it tended to be stable. (2) the changes of lipid decomposition and TGH expression were detected under the condition of cell starvation and recovery of nutrition, and the role of TGH in the process was analyzed. Under the condition of lack of nutrition, the amount of glycerol released increased in the culture medium without glucose and serum, and the expression of TGHmRNA and protein was higher than that of the control. When the nutrition was restored (complete culture medium), its expression decreased again, but the lipolysis did not decrease significantly. 3 the changes of adipose decomposition and TGH expression in Isoproterenol stimulated by isoprenaline were studied, and the role of TGH in this state was analyzed. Low concentration of isoproterenol (0.1 渭 mol / L ~ (-1) 渭 mol / L) did not detect the change of glycerol content released by adipocytes into the medium. With the increase of isoproterenol concentration, glycerol release increased significantly, and the content of triglyceride decreased. In other words, the lipopolysis is enhanced. The expression of HSL mRNA and protein changed in a dose-and time-dependent manner with the increase of isoproterenol concentration and the time of action, but the expression of TGHmRNA and protein did not change in this process. PSilencer-TGH vector was constructed and the optimum lethal concentration of hygromycin was screened. The plasmids were digested, recovered, ligated, transformed, and the plasmid was extracted. The result of sequencing indicated that TGH RNA interference vector had been successfully constructed. 200 渭 g/mL was the best concentration for hygromycin resistance screening.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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