突变型FoxO1基因的体外和体内表达研究

发布时间：2018-02-26 05:31

本文关键词： 突变型FoxO1 瘦素转基因动物 CMV启动子甲基化　出处：《河南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：代谢障碍综合征包括肥胖症、糖尿病、高血压和心脑疾病等,严重危害着人类的健康。研究发现,FoxO1(forkhead box O1)参与的瘦素抵抗在代谢障碍综合征的发生机制中具有重要的地位,FoxO1与其靶基因的相互作用就成为能量代谢领域的研究焦点之一。FoxO1可以作用于神经肽基因前黑素细胞皮质素原(Pro-opiomelanocortin,POMC)的启动子上,抑制POMC基因的转录,从而阻断瘦素的信号通路,影响动物的能量代谢。为了研究DNA结合结构域(DNA Binding Domain,DBD)在FoxO1功能中所起的作用,实验室构建了敲除DBD的突变体,构建成质粒pCMV5-Myc-FoxO1Δ4-5(pFΔ4-5)。在此基础上,本论文开展了此突变体的体内体外表达及其功能验证试验。首先,我们将pFΔ4-5进行扩增、酶切分析和序列测定;然后将相关质粒转染至293Rb细胞(含瘦素受体的293细胞),western blot的检测结果显示细胞中有蛋白表达,表明该表达载体构建成功;进一步的荧光素酶活性检测结果表明,FoxO1能够明显地抑制POMC启动子的活性,而缺失了DBD的FΔ4-5不但丧失了抑制作用,反而明显地增强了POMC启动子的活性,表明DBD是FoxO1发挥抑制所必需的;最后,将突变体基因进行显微注射获得了转基因小鼠。将所得的6个F0代阳性转基因小鼠与野生型小鼠进行交配繁育,基因型鉴定结果表明6个F0转基因小鼠的子代中均存在阳性,表明FΔ4-5已整合至转基因小鼠基因组中。然后,选取阳性转基因小鼠,对其各组织中转基因的表达情况进行检测,western blot的结果显示目的蛋白在转基因小鼠的下丘脑、肝、白色脂肪、棕色脂肪、脾、心、肺、肾等组织中均未表达,说明FΔ4-5虽然整合至转基因小鼠的基因组中,但却发生了基因沉默现象。为了明确目的基因是否是因为发生了启动子甲基化而导致了基因表达抑制,我们进一步应用亚硫酸氢盐测序法检测了转基因小鼠肝脏基因组DNA中CMV启动子的甲基化情况,结果显示CMV启动子上的7个Cp G位点均发生了较高程度的甲基化,证实了我们的推测。随后将不同剂量的地西他滨(5-aza-2deoxycytidine,5-aza)对小鼠进行腹腔注射,进行CMV启动子的去甲基化研究。CMV启动子甲基化分析和目的蛋白的western blot结果显示,在注射5-aza后,虽然有去甲基化现象的发生,但目的蛋白仍未表达。结论:1.体外实验结果证明DBD是FoxO1分子中抑制POMC启动子活性的关键结构域;2.CMV启动子控制突变型FoxO1基因在293Rb细胞内表达;3.突变型FoxO1基因在转基因动物中沉默可能是由CMV启动子甲基化导致。
[Abstract]:Metabolic disorders include obesity, diabetes, high blood pressure, heart and brain diseases, It has been found that leptin resistance involved in leptin resistance plays an important role in the pathogenesis of metabolic disorder syndrome. The interaction between FoxO1 and its target gene has become the research focus in energy metabolism field. FoxO1 can act on the Pro-opiomelanocortinin (POMC) promoter of the neuropeptide gene Proopiomelanocortin. In order to study the role of DNA binding domain Binding domain DNA in FoxO1 function, a mutant of DBD knockout was constructed in order to inhibit the transcription of POMC gene, thus blocking leptin signaling pathway and affecting energy metabolism in animals. The plasmid pCMV5-Myc-FoxO1 螖 4-5 pF 螖 4-5 was constructed. On the basis of this, the expression of the mutant in vitro and in vivo and its function verification were carried out. Firstly, we amplified PF 螖 4-5, analyzed its enzyme digestion and sequenced it. Then the related plasmids were transfected into 293Rb cells (Western blot analysis of 293Rb cells containing leptin receptor) showed that there was protein expression in the cells, which indicated that the expression vector was successfully constructed. The results of further luciferase activity test showed that FoxO1 could significantly inhibit the activity of POMC promoter, while the absence of F 螖 4-5 of DBD not only lost its inhibitory effect, but also significantly enhanced the activity of POMC promoter. The results showed that DBD was necessary for the inhibition of FoxO1. Finally, the transgenic mice were obtained by microinjection of the mutant gene. 6 F 0 generation positive transgenic mice were mated and bred with wild-type mice. The results of genotypic identification showed that F 螖 4-5 was integrated into the genome of 6 F 0 transgenic mice, and F 螖 4-5 was integrated into the genome of the transgenic mice. Then, the positive transgenic mice were selected. The results of western blot analysis showed that the target protein was not expressed in hypothalamus, liver, white fat, brown fat, spleen, heart, lung and kidney of transgenic mice. The results showed that F 螖 4-5 was integrated into the genome of transgenic mice, but gene silencing occurred. In order to determine whether the target gene was inhibited by promoter methylation, The methylation of CMV promoter in the genomic DNA of transgenic mice was detected by bisulfite sequencing. The results showed that the 7 CpG sites on the CMV promoter were methylated to a higher degree. Our hypothesis was confirmed. Then the mice were injected intraperitoneally with different doses of dietabine 5-aza-2deoxycytidine 5-aza. The demethylation of the CMV promoter was studied. The methylation analysis of the promoter and the western blot of the target protein showed that after 5-aza injection, Although demethylation has occurred, Conclusion in vitro, DBD is the key domain of FoxO1 to inhibit the activity of POMC promoter. 2.CMV promoter controlled mutant FoxO1 gene is expressed in 293Rb cells. The mutant FoxO1 gene is expressed in the transcriptional gene. Silencing in animals may be caused by methylation of the CMV promoter.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.2

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