GATA2与Smad4相互作用对TGFβ信号通路负调控的初步研究

发布时间：2018-03-18 01:29

  本文选题：GATA2　切入点：TGF-β信号通路　出处：《安徽医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：GATA2属于锌指类转录因子GATA家族成员之一。该蛋白广泛表达于造血干细胞(HSCs),参与调控HSCs增殖、分化与自我更新等生理功能。TGFβ信号通路是参与细胞生长、分化、增殖及肿瘤生成等多种生理病理学过程的重要信号通路,同时参与了HSCs的自我更新过程。Smad4是TGFβ通路中重要的节点分子,能够与其他Smads家族成员结合,介导细胞外信号传递至细胞核,并且调节下游靶基因的表达。实验室前期利用大规模酵母双杂交技术发现GATA2与Smad4具有高度可信相互作用,进一步研究验证了GATA2与Smad4相互作用确实存在,并且共定位于细胞核,但GATA2与Smad4相互作用的意义以及机制并不明确。因此本研究利用外源性免疫共沉淀实验验证了GATA2与Samd4存在相互作用。利用荧光素酶报告基因实验,证明了GATA2能够抑制Smad4及其家族成员中R-Smads的转录活性;K562细胞中过表达GATA2,能够抑制内源性Smad4的转录活性;同时GATA2能够抑制TGFβ通路下游靶基因PAI-1的表达。上述实验表明,GATA2通过抑制Smad4的转录活性,从而抑制TGFβ通路。利用实验室前期根据GATA2结构域所构建的不同缺失体进行荧光素酶报告基因检测,证明GATA2对Smad4的抑制作用是通过其结构域中的N端及其锌指结构。本研究验证了TGFβ因子刺激能够抑制Huh7细胞的增殖。通过构建GATA2慢病毒表达载体,包装慢病毒并建立GATA2过表达的Huh7稳定细胞系,证明GATA2能够抑制Huh7细胞对TGFβ刺激的敏感性。为进一步在HSCs细胞中检测GATA2功能及作用机制,构建了人GATA2的慢病毒RNAi干涉载体,并对慢病毒进行了包装,获得了GATA2 RNAi慢病毒颗粒,建立了K562,TF-1的siGATA2稳定细胞系,为后续的研究打下良好的基础。GATA2通过与Smad4发生相互相互作用,发挥转录抑制活性,从而负调控TGFβ信号通路,并且抑制下游靶基因的表达。GATA2对TGFβ信号通路的负调控作用可能在造血干细胞的自我更新与分化过程中具有重要生理意义。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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