發(fā)布時(shí)間：2018-05-28 06:00

  本文選題：粉塵誘導(dǎo)基因 + 蛋白質(zhì)組學(xué)��； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：粉塵誘導(dǎo)基因mdig(又稱mina53, MINA或N052)是在煤礦工人巨噬細(xì)胞中發(fā)現(xiàn)的環(huán)境誘導(dǎo)基因。mdig的表達(dá)升高與多種腫瘤的發(fā)病與預(yù)后相聯(lián)系。然而mdig如何作用于細(xì)胞代謝和腫瘤發(fā)展仍有待闡明。有研究指出mdig蛋白通過其JmjC結(jié)構(gòu)域,參與組蛋白H3在甘氨酸H3K9me3的去甲基化作用。然而不同于其他經(jīng)典的去甲基化酶,mdig缺乏與染色質(zhì)接合的蛋白結(jié)構(gòu)域。于是ndig對染色質(zhì)或組蛋白的作用更可能是通過與其他染色質(zhì)接合蛋白的相互作用而完成的。第一部分蛋白質(zhì)組學(xué)分析粉塵誘導(dǎo)基因蛋白與細(xì)胞內(nèi)功能蛋白的相互作用目的：本研究旨在通過蛋白質(zhì)組學(xué)分析,發(fā)掘細(xì)胞中與粉塵誘導(dǎo)基因ndig相互作用的功能蛋白,以探查mdig在細(xì)胞代謝和腫瘤發(fā)生過程中所起的作用。方法：在人肺癌細(xì)胞A549和人支氣管上皮細(xì)胞BEAS-2B中,運(yùn)用免疫共沉淀技術(shù)分別沉淀mdig和對照組IgG的抗體蛋白復(fù)合物。該蛋白復(fù)合物在SDS-PAGE膠中電泳分離,用考馬斯亮藍(lán)染色觀察mdig蛋白的條帶模式。在四組獨(dú)立實(shí)驗(yàn)中,應(yīng)用兩種不同的多段高效液相色譜-電噴霧-串聯(lián)質(zhì)譜(Orbitrap Fusion和OrbitrapXL)分析與mdig相互結(jié)合的蛋白。篩選出的蛋白,用免疫共沉淀結(jié)合免疫印跡的方法加以驗(yàn)證。最后用IPA軟件統(tǒng)計(jì)分析A549細(xì)胞和BEAS-2B細(xì)胞中與mdig相互作用蛋白的分子通路。結(jié)果：在考馬斯亮藍(lán)染色的蛋白質(zhì)分離膠中,三至五條特異性條帶穩(wěn)定的出現(xiàn)在mdig抗體沉淀的蛋白樣本中,而在對照組IgG中未被發(fā)現(xiàn)。蛋白質(zhì)譜分析結(jié)果顯示,在mdig抗體沉淀的蛋白復(fù)合物中可檢測到一些DNA修復(fù)相關(guān)蛋白和染色質(zhì)連接蛋白,包括XRCC5, XRCC6, RBBP4, CBX8, PRMT5,和TDRD等。免疫印跡實(shí)驗(yàn)進(jìn)一步確認(rèn)了mdig與這些蛋白的結(jié)合。在BEAS-2B細(xì)胞中,mdig蛋白的相互作用與A549細(xì)胞相似,但仍有一些蛋白,如CBX3和CBX5等,僅在BEAS-2B細(xì)胞中被觀察到與mdig有相互作用。結(jié)論：mdig通過與伴侶蛋白的相互作用完成其在表觀遺傳學(xué)、DNA修復(fù)、DNA復(fù)制和細(xì)胞生長調(diào)節(jié)的功能。第二部分粉塵誘導(dǎo)基因蛋白通過DNA非同源末端接合通路參與DNA損傷修復(fù)目的：DNA雙鏈斷裂(DSBs)的修復(fù),是在哺乳動物細(xì)胞中維持基因組完整性和預(yù)防腫瘤形成的關(guān)鍵。蛋白質(zhì)組學(xué)研究發(fā)現(xiàn)粉塵誘導(dǎo)基因Mdig與DSBs的非同源末端連接修復(fù)(NHEJ)的關(guān)鍵蛋白XRCC6、XRCC5和DNA-PK等有相互結(jié)合作用。本研究旨在探索mdig在NHEJ修復(fù)通路中所起的作用,并驗(yàn)證mdig通過與功能蛋白XRCC6的相互作用,完成其對DNA損傷修復(fù)的影響。方法：建立穩(wěn)定的mdig過表達(dá)細(xì)胞系、mdig空載體傳染細(xì)胞系、mdig shRNA轉(zhuǎn)染的基因沉默細(xì)胞系和對照shRNA轉(zhuǎn)染細(xì)胞系。分別用30 gM腐草霉素(phleomycin)處理各細(xì)胞0,0.25,0.5,1,2,4小時(shí)后,應(yīng)用免疫印跡實(shí)驗(yàn)測定各細(xì)胞系中NHEJ通路蛋白的變化情況,并應(yīng)用免疫熒光法觀察DNA斷裂標(biāo)記物γH2AX和pDNA-PKcs的表達(dá)。彗星實(shí)驗(yàn)檢測各細(xì)胞系對phleomycin處理的DNA損傷敏感程度。結(jié)果：Mdig可與DNA損傷的非同源末端接合修復(fù)(NHEJ)蛋白XRCC6相互作用,并影響NHEJ通路的關(guān)鍵酶pDNA-PK和pATM在phleomycin處理后的表達(dá)。與此同時(shí),Mdig過表達(dá)細(xì)胞系對phleomycin所誘導(dǎo)的DNA雙鏈斷裂更敏感。相反的,用shRNA沉默ndig,可減緩DNA的雙鏈斷裂。結(jié)論：mdig通過與XRCC6的相互作用影響細(xì)胞DNA損傷的NHEJ修復(fù)通路,并導(dǎo)致phleomycin引起的DNA雙鏈斷裂更加敏感。
[Abstract]:Dust induced gene mdig (also known as mina53, MINA or N052) is associated with the increase of the expression of environmental induced gene.Mdig found in coal miners' macrophages and the incidence and prognosis of a variety of tumors. However, the effect of mdig on cell metabolism and tumor development remains to be elucidated. Studies have indicated that mdig proteins participate in the group through its JmjC domain. The demethylation of protein H3 in the glycine H3K9me3. However, unlike other classical demethylation enzymes, mdig lacks the protein domain that conjugates with chromatin. Thus the effect of ndig on chromatin or histone is more likely to be accomplished by interaction with other chromatin conjugproteins. The interaction between the dust induced gene protein and the intracellular functional proteins: This study aims to explore the functional proteins interacting with the dust induced gene ndig by proteomic analysis to explore the role of mdig in cell metabolism and tumor development. Square method: on human lung cancer cells A549 and human bronchus The antibody protein complex of mdig and IgG in the control group was precipitated by immunoprecipitation in the skin cell BEAS-2B. The protein complex was separated in SDS-PAGE gel, and the strip pattern of mdig protein was observed by coloring of komomesi blue. In the four groups of independent experiments, two different multi segment high performance liquid chromatography electrospray ionization tandem mass was used. Protein (Orbitrap Fusion and OrbitrapXL) analysis of proteins that are combined with mdig. The proteins screened were screened by immunoblotting and immunoblotting. Finally, the IPA software was used to analyze the molecular pathway of mdig interacting protein in A549 and BEAS-2B cells. Fruit: protein separation gel stained with Coomassie blue Three to five specific bands were stable in the protein samples of the mdig antibody precipitation and were not found in the control group IgG. The protein mass spectrometry analysis showed that some DNA repair related proteins and chromatin linked egg white could be detected in the protein complex of the mdig antibody precipitation, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD. Western blot experiments further confirmed the binding of mdig to these proteins. In BEAS-2B cells, the interaction of mdig proteins is similar to that of A549 cells, but some proteins, such as CBX3 and CBX5, are observed only in BEAS-2B cells and interact with mdig. Conclusion: mdig through interaction with chaperone proteins is apparent. The functions of genetics, DNA repair, DNA replication and cell growth regulation. Second part of the dust induced gene protein is involved in the repair of DNA damage through the DNA non homologous terminal junction pathway: the repair of DNA double strand breaks (DSBs) is the key to maintain genomic integrity and prevent tumor formation in mammalian cells. Proteomics research This study aims to explore the role played by mdig in the NHEJ repair pathway and to verify the effect of mdig through the interaction with functional protein XRCC6, and the effect of mdig on the repair of DNA damage. Method: establishment of the effect of mdig in NHEJ repair pathway, and the effect of mdig on the repair of DNA damage. Methods: establishment of Mdig. Stable mdig overexpressed cell lines, mdig empty carrier infectious cell lines, mdig shRNA transfected gene silencing cell lines and control shRNA transfected cell lines. The changes of NHEJ pathway proteins in each cell line were measured by immunoblotting experiments with 30 gM humomycin (phleomycin), respectively. The expression of DNA fracture markers - gamma H2AX and pDNA-PKcs was observed by the immunofluorescence method. Comet assay detected the DNA damage sensitivity of each cell line to phleomycin treatment. Results: Mdig could interact with the non homologous terminal joint repair (NHEJ) protein XRCC6 interaction with DNA damage, and affect the key enzyme pDNA-PK of the NHEJ pathway and the table after the pATM. At the same time, Mdig overexpressed cell lines were more sensitive to the DNA double strand breaks induced by phleomycin. Conversely, the use of shRNA to silence ndig could slow the double strand breaks of DNA. Conclusion: mdig affects the NHEJ repair pathway of DNA damage in cells through the interaction with XRCC6, and leads to greater sensitivity of DNA double strand breaks caused by phleomycin.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R135.2

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