Gadd45a基因对小鼠造血干细胞功能的影响

发布时间：2018-06-09 02:56

本文选题：Gadd45α + 单克隆　；参考：《北京协和医学院》2012年硕士论文

【摘要】：Gadd45为生长抑制和DNA损伤基因(Growth arrest and DNA damage inducible gene Gadd45)是由Fornace等[’]于1988年发现。Gadd45家族包括三个成员：Gadd45α (Gadd45α/Gadd45)、Gadd45b(Gadd45β/MyD118)、Gadd45g(Gadd45γ/CR6),它们是DNA损伤修复的重要相关基因,在细胞凋亡中发挥重要的作用[2-3]。Gadd45基因家族序列非常相似大约有57%基因同源性,通常在环境损伤因子作用后,随之DNA修复途径的启动而诱导产生。 Gadd45a蛋白是一个小球状酸性蛋白(分子量18000KD)主要表达在细胞核内,与各种细胞周期相关的核蛋白质相互作用,如丝裂素活化蛋白激酶的激酶(mitogen activated protein kinase kinase,MAPKK/MTK1),细胞分裂调控蛋白2(cell dicision control2,Cdc2),增殖细胞核抗原(PCNA),p21以及核心组蛋白等发生相互作用。Gadd45a基因的第三个内含子区段,存在一个高度保守的p53结合位点,野生型p53可以与此作用位点结合,促进Gadd45a基因的转录表达,各种影响p53因素均可导致Gadd45a基因启动子活性的下降,如cmyc锌指蛋白ZBRK1.Gadd45α是Gadd45基因家族中唯一的p53靶基因,它在细胞的生命过程中起到重要的作用,其中包括遗传物质稳定性的维持、细胞周期的调控、核苷酸剪切修复以及凋亡调控等。造血细胞包括造血干细胞和造血祖细胞,对放射线照射非常敏感。5-10Gy的放射线照射剂量很可能导致小鼠造血系统破坏从而导致死亡。已知Gadd45α作为压力刺激敏感反应基因,Gadd45家族中的唯一p53下游靶基因,而p53作为放射线照射反应的重要信号通路,调节细胞的增殖、DNA修复以及凋亡,从而我们通过Gadd45a基因敲除小鼠的体外骨髓造血干细胞(Haematopoietic stem cells,HSCs)单克隆培养、体内竞争性移植、细胞免疫荧光等实验研究Gadd45a在HSC自我更新能力和损伤修复能力中的作用。实验结果显示在正常的生理水平下Gadd45a基因缺失小鼠骨髓造血干／祖细胞表型分析并无差异。全骨髓竞争性连续移植及干细胞竞争性移植实验显示,Gadd45a基因缺失小鼠骨髓长期造血干细胞造血重建能力升高,而祖细胞水平功能下降。在放射线损伤情况下,Gadd45a基因缺失小鼠骨髓造血干／祖细胞表型分析仍无差异。体外单克隆培养显示,Gadd45a基因缺失小鼠骨髓长期／短期／多潜能祖细胞均抵抗低剂量放射线损伤；全骨髓移植后受体小鼠给予8.5Gy放射线照射损伤显示,Gadd45a基因缺失HSC对放射线敏感性无影响；竞争性骨髓移植受体小鼠给予连续两次4.5Gy放射线处理实验显示,在应激损伤条件下,Gadd45a基因缺失有利于小鼠HSC自我更新,并由凋亡减少而导致的造血干细胞数目增多；低剂量放射线损伤,体外检测Gadd45a基因缺失小鼠骨髓造血干细胞凋亡无差异,增殖升高,同时低剂量放射线损伤证实Gadd45a基因缺失小鼠骨髓造血干细胞DNA损伤修复较快。无论是在正常情况下还是应激反应中Gadd45a基因缺失均导致HSC功能上升,提示Gadd45a基因在维持HSC稳态方面起重要作用。
[Abstract]:Gadd45 is a gene for growth inhibition and DNA damage (Growth arrest and DNA damage inducible gene Gadd45) was found in Fornace, etc. in 1988, the.Gadd45 family included three members. The important role of the [2-3].Gadd45 gene family is very similar to about 57% gene homology, which is usually induced by the initiation of the DNA repair pathway after the action of the environmental damage factor.
Gadd45a protein is a small Spherular acidic protein (molecular weight 18000KD) expressed mainly in the nucleus, interacting with nuclear proteins associated with various cell cycles, such as mitogen activated protein kinase kinase, MAPKK/MTK1, cytokinesis regulated protein 2 (cell dicision control2, Cdc2), and proliferation. Nuclear antigen (PCNA), p21 and core histone have third intron segments that interact with.Gadd45a genes. There is a highly conserved p53 binding site. Wild type p53 can combine with this site to promote the transcriptional expression of the Gadd45a gene. All kinds of p53 factors can lead to the activity of the Gadd45a gene promoter. Drop, such as cmyc zinc finger protein ZBRK1.Gadd45 alpha, is the only p53 target gene in the Gadd45 gene family, which plays an important role in the cell life process, including the maintenance of the stability of the genetic material, the regulation of cell cycle, the repair of nucleotide shear and the regulation of apoptosis.
Hematopoietic cells include hematopoietic stem cells and hematopoietic progenitor cells. Radiation exposure dose of.5-10Gy, which is very sensitive to radiation exposure, is likely to cause damage to the hematopoietic system in mice and lead to death. Gadd45 alpha is known as the pressure sensitive response gene, the only p53 downstream target gene in the Gadd45 family, and p53 as a radiation exposure response The important signal pathway regulates cell proliferation, DNA repair and apoptosis, thus we study Gadd45a in the self-renewal capacity and damage repair ability of HSC through the monoclonal culture of bone marrow hematopoietic stem cells (Haematopoietic stem cells, HSCs) in Gadd45a knockout mice, in vivo competitive transplantation and cell immunofluorescence. The role.
The experimental results showed that there was no difference in the phenotype analysis of bone marrow hematopoietic stem / progenitor cells in Gadd45a gene deletion mice at normal physiological level. Competitive continuous transplantation and competitive stem cell transplantation experiments showed that the hematopoietic rebuilding ability of bone marrow stem cells in Gadd45a gene deletion mice increased and the level of progenitor cells decreased.
The phenotypic analysis of bone marrow hematopoietic stem / progenitor cells in Gadd45a gene deletion mice remained unchanged under radiation damage. In vitro monoclonal culture showed that the long / short term / multipotential progenitor cells of Gadd45a gene deletion mice were all resistant to low dose radiation injury, and the recipient mice were given 8.5Gy radiation injury after all bone marrow transplantation. Gadd45a gene deletion HSC had no effect on Radiosensitivity; competitive bone marrow transplant recipient mice given two consecutive 4.5Gy radiation treatments showed that the deletion of Gadd45a gene was beneficial to the self-renewal of HSC in mice and the increase of the number of hematopoietic stem cells induced by the decrease of apoptosis; low dose radiation. There was no difference in the apoptosis of bone marrow hematopoietic stem cells in Gadd45a gene deletion mice, and the proliferation increased. At the same time, low dose radiation damage confirmed that the DNA damage of the bone marrow hematopoietic stem cells of Gadd45a gene deletion mice was faster. The Gadd45a gene deletion in normal or stress responses led to the increase of HSC function, suggesting Gad. The d45a gene plays an important role in maintaining the homeostasis of HSC.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

【参考文献】