本文選題：GATA2　切入點：TGF-β信號通路　出處：《安徽醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：GATA2屬于鋅指類轉(zhuǎn)錄因子GATA家族成員之一。該蛋白廣泛表達于造血干細胞(HSCs),參與調(diào)控HSCs增殖、分化與自我更新等生理功能。TGFβ信號通路是參與細胞生長、分化、增殖及腫瘤生成等多種生理病理學過程的重要信號通路,同時參與了HSCs的自我更新過程。Smad4是TGFβ通路中重要的節(jié)點分子,能夠與其他Smads家族成員結(jié)合,介導細胞外信號傳遞至細胞核,并且調(diào)節(jié)下游靶基因的表達。實驗室前期利用大規(guī)模酵母雙雜交技術(shù)發(fā)現(xiàn)GATA2與Smad4具有高度可信相互作用,進一步研究驗證了GATA2與Smad4相互作用確實存在,并且共定位于細胞核,但GATA2與Smad4相互作用的意義以及機制并不明確。因此本研究利用外源性免疫共沉淀實驗驗證了GATA2與Samd4存在相互作用。利用熒光素酶報告基因?qū)嶒?證明了GATA2能夠抑制Smad4及其家族成員中R-Smads的轉(zhuǎn)錄活性;K562細胞中過表達GATA2,能夠抑制內(nèi)源性Smad4的轉(zhuǎn)錄活性;同時GATA2能夠抑制TGFβ通路下游靶基因PAI-1的表達。上述實驗表明,GATA2通過抑制Smad4的轉(zhuǎn)錄活性,從而抑制TGFβ通路。利用實驗室前期根據(jù)GATA2結(jié)構(gòu)域所構(gòu)建的不同缺失體進行熒光素酶報告基因檢測,證明GATA2對Smad4的抑制作用是通過其結(jié)構(gòu)域中的N端及其鋅指結(jié)構(gòu)。本研究驗證了TGFβ因子刺激能夠抑制Huh7細胞的增殖。通過構(gòu)建GATA2慢病毒表達載體,包裝慢病毒并建立GATA2過表達的Huh7穩(wěn)定細胞系,證明GATA2能夠抑制Huh7細胞對TGFβ刺激的敏感性。為進一步在HSCs細胞中檢測GATA2功能及作用機制,構(gòu)建了人GATA2的慢病毒RNAi干涉載體,并對慢病毒進行了包裝,獲得了GATA2 RNAi慢病毒顆粒,建立了K562,TF-1的siGATA2穩(wěn)定細胞系,為后續(xù)的研究打下良好的基礎。GATA2通過與Smad4發(fā)生相互相互作用,發(fā)揮轉(zhuǎn)錄抑制活性,從而負調(diào)控TGFβ信號通路,并且抑制下游靶基因的表達。GATA2對TGFβ信號通路的負調(diào)控作用可能在造血干細胞的自我更新與分化過程中具有重要生理意義。
[Abstract]:GATA2 is a member of the zinc finger transcription factor GATA family. This protein is widely expressed in hematopoietic stem cells. TGF- 尾 signaling pathway is involved in cell growth and differentiation, which is involved in regulating the proliferation, differentiation and self-renewal of HSCs. Proliferation and tumorigenesis are important signaling pathways in many physiological and pathological processes, and Smad4 is an important nodal molecule in the TGF 尾 pathway, which is involved in the self-renewal of HSCs, and can bind to other members of the Smads family. To mediate the transmission of extracellular signal to the nucleus, and to regulate the expression of downstream target genes. In the early stage of laboratory, we found that GATA2 and Smad4 have highly credible interaction with Smad4 by using large-scale yeast two-hybrid technique. Further studies have confirmed that the interaction between GATA2 and Smad4 exists and is co-located in the nucleus. However, the significance and mechanism of the interaction between GATA2 and Smad4 were not clear. Therefore, the interaction between GATA2 and Samd4 was verified by exogenous immunoprecipitation experiments. The luciferase reporter gene experiment was used. It is proved that GATA2 can inhibit the transcription activity of R-Smads in Smad4 and its family members, and inhibit the transcription activity of endogenous Smad4 in K562 cells. At the same time, GATA2 could inhibit the expression of PAI-1, the downstream target gene of TGF 尾 pathway. In order to inhibit the TGF 尾 pathway, luciferase reporter genes were detected using different deletions constructed according to the GATA2 domain in early laboratory. It was proved that the inhibitory effect of GATA2 on Smad4 was mediated by N terminal and zinc finger structure in its domain. This study demonstrated that TGF 尾 stimulated Huh7 cell proliferation. GATA2 lentivirus expression vector was constructed. Packaging lentivirus and establishing Huh7 stable cell line with overexpression of GATA2, it was proved that GATA2 could inhibit the sensitivity of Huh7 cells to TGF 尾 stimulation. In order to further detect the function and mechanism of GATA2 in HSCs cells, a human GATA2 lentivirus RNAi interference vector was constructed. The lentivirus was packaged, GATA2 RNAi lentivirus particles were obtained, and the stable cell line of siGATA2 of K562TF-1was established, which laid a good foundation for further study. GATA2 acted as a transcription suppressor by interacting with Smad4. Therefore, negative regulation of TGF 尾 signaling pathway and inhibition of downstream target gene expression. GATA2 may play an important physiological role in the process of self-renewal and differentiation of hematopoietic stem cells.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

【參考文獻】

相關(guān)期刊論文 前1條

1 TOMOMI Takahashi;TORU Nakano;;CREB-binding proteins (CBP) as a transcriptional coactivator of GATA-2[J];Science in China(Series C:Life Sciences);2008年03期

，

本文編號：1627407