本文關(guān)鍵詞： 突變型FoxO1 瘦素 轉(zhuǎn)基因動(dòng)物 CMV啟動(dòng)子 甲基化　出處：《河南農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：代謝障礙綜合征包括肥胖癥、糖尿病、高血壓和心腦疾病等,嚴(yán)重危害著人類的健康。研究發(fā)現(xiàn),FoxO1(forkhead box O1)參與的瘦素抵抗在代謝障礙綜合征的發(fā)生機(jī)制中具有重要的地位,FoxO1與其靶基因的相互作用就成為能量代謝領(lǐng)域的研究焦點(diǎn)之一。FoxO1可以作用于神經(jīng)肽基因前黑素細(xì)胞皮質(zhì)素原(Pro-opiomelanocortin,POMC)的啟動(dòng)子上,抑制POMC基因的轉(zhuǎn)錄,從而阻斷瘦素的信號(hào)通路,影響動(dòng)物的能量代謝。為了研究DNA結(jié)合結(jié)構(gòu)域(DNA Binding Domain,DBD)在FoxO1功能中所起的作用,實(shí)驗(yàn)室構(gòu)建了敲除DBD的突變體,構(gòu)建成質(zhì)粒pCMV5-Myc-FoxO1Δ4-5(pFΔ4-5)。在此基礎(chǔ)上,本論文開展了此突變體的體內(nèi)體外表達(dá)及其功能驗(yàn)證試驗(yàn)。首先,我們將pFΔ4-5進(jìn)行擴(kuò)增、酶切分析和序列測(cè)定;然后將相關(guān)質(zhì)粒轉(zhuǎn)染至293Rb細(xì)胞(含瘦素受體的293細(xì)胞),western blot的檢測(cè)結(jié)果顯示細(xì)胞中有蛋白表達(dá),表明該表達(dá)載體構(gòu)建成功;進(jìn)一步的熒光素酶活性檢測(cè)結(jié)果表明,FoxO1能夠明顯地抑制POMC啟動(dòng)子的活性,而缺失了DBD的FΔ4-5不但喪失了抑制作用,反而明顯地增強(qiáng)了POMC啟動(dòng)子的活性,表明DBD是FoxO1發(fā)揮抑制所必需的;最后,將突變體基因進(jìn)行顯微注射獲得了轉(zhuǎn)基因小鼠。將所得的6個(gè)F0代陽性轉(zhuǎn)基因小鼠與野生型小鼠進(jìn)行交配繁育,基因型鑒定結(jié)果表明6個(gè)F0轉(zhuǎn)基因小鼠的子代中均存在陽性,表明FΔ4-5已整合至轉(zhuǎn)基因小鼠基因組中。然后,選取陽性轉(zhuǎn)基因小鼠,對(duì)其各組織中轉(zhuǎn)基因的表達(dá)情況進(jìn)行檢測(cè),western blot的結(jié)果顯示目的蛋白在轉(zhuǎn)基因小鼠的下丘腦、肝、白色脂肪、棕色脂肪、脾、心、肺、腎等組織中均未表達(dá),說明FΔ4-5雖然整合至轉(zhuǎn)基因小鼠的基因組中,但卻發(fā)生了基因沉默現(xiàn)象。為了明確目的基因是否是因?yàn)榘l(fā)生了啟動(dòng)子甲基化而導(dǎo)致了基因表達(dá)抑制,我們進(jìn)一步應(yīng)用亞硫酸氫鹽測(cè)序法檢測(cè)了轉(zhuǎn)基因小鼠肝臟基因組DNA中CMV啟動(dòng)子的甲基化情況,結(jié)果顯示CMV啟動(dòng)子上的7個(gè)Cp G位點(diǎn)均發(fā)生了較高程度的甲基化,證實(shí)了我們的推測(cè)。隨后將不同劑量的地西他濱(5-aza-2deoxycytidine,5-aza)對(duì)小鼠進(jìn)行腹腔注射,進(jìn)行CMV啟動(dòng)子的去甲基化研究。CMV啟動(dòng)子甲基化分析和目的蛋白的western blot結(jié)果顯示,在注射5-aza后,雖然有去甲基化現(xiàn)象的發(fā)生,但目的蛋白仍未表達(dá)。結(jié)論:1.體外實(shí)驗(yàn)結(jié)果證明DBD是FoxO1分子中抑制POMC啟動(dòng)子活性的關(guān)鍵結(jié)構(gòu)域;2.CMV啟動(dòng)子控制突變型FoxO1基因在293Rb細(xì)胞內(nèi)表達(dá);3.突變型FoxO1基因在轉(zhuǎn)基因動(dòng)物中沉默可能是由CMV啟動(dòng)子甲基化導(dǎo)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.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.2

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