不同类型成体干细胞对DNA损伤应答反应研究
发布时间：2018-09-08 10:23
【摘要】：当今社会老龄化状况日益严重,衰老迫使我们要对衰老及衰老相关疾病的分子机制有更为深刻的理解。衰老相关机制之一是端粒缩短,G3Terc-/-是研究端粒缩短诱发衰老的较好的实验动物模型,G3Terc-/-由于失去端粒酶的保护作用而出现染色体末端端粒长度严重缩短,进而激活干细胞DNA损伤应答通路,不但可以引起细胞内出现不同的损伤应答检定点变化,还能够引起细胞外环境损伤而导致干细胞功能受损。 错配修复相关基因核酸外切酶(Exonucleasel,Exo1)是端粒缩短激活DNA损伤信号通路中的检定点之一,敲除Exo1基因可以阻断G3Terc-/-小鼠小肠隐窝干细胞DNA损伤信号通路激活,进而改善小肠隐窝干细胞损伤情况。然而Exo1基因敲除对G3Terc-/小鼠其他组织干细胞作用如何尚未见报道。 我们的研究表明,Exo1基因敲除不能改善G3Terc-/-小鼠造血干细胞损伤。骨髓移植实验提示,与野生型对照组比较,G3Terc-/-供体来源HSC移植重建能力明显下降,Exo1基因敲除不能改善这种重建能力降低的情况。Exo1基因敲除也不能改善G3Terc-/-小鼠神经干细胞损伤。 小鼠神经干细胞初次及第二次自我更新能力明显下降,Exol基因敲除不能改善G3Terc-/-小鼠神经干细胞自我更新能力降低的情况。同样,Exo1基因敲除不能改善G3Terc-/小鼠骨髓间充质干细胞损伤,G3Terc-/-小鼠骨髓间充质干细胞增殖能力降低,G3Terc-/-Exo1-/-双基因敲除小鼠骨髓间充质干细胞增殖能力并未改善,其DNA损伤信号通路未能被阻断。因此第一次提出这样的证据：不同类型成体干细胞对端粒缩短激活DNA损伤信号通路相关的检定点存在不同的应答反应；这对研究其他基因对不同组织器官干细胞功能影响差异提供新的实验依据。也对不同组织器官干细胞衰老及再生研究提供新的研究策略启示,具有潜在的实际应用价值。 端粒缩短激活DNA损伤应答通路可以引起细胞外环境(系统大环境)损伤,影响HSC分化能力,导致细胞向淋系细胞分化减少和向髓系细胞分化增加,和B淋巴细胞及T淋巴细胞生成障碍。之前的研究表明系统大环境影响造血干细胞的分化,而系统大环境(尤其是血清成分)对造血干细胞自我更新及重建能力影响尚未见报道。 我们的研究提示,与同年龄野生型小鼠血清对比,正常年轻野生型小鼠HSC在体外与G3Terc-/-小鼠血清共培养后,造血干细胞增殖明显增加,更多的造血干细胞离开静止期进入细胞周期。与G3Terc-/-小鼠血清共培养的骨髓干细胞移植进入经致死性放射线照射过的小鼠,造血干细胞重建能力显著下降。调控细胞周期G1期的基因p21和调控细胞周期G0期的基因TGF-β在正常年轻野生型小鼠与G3Terc-/-小鼠血清共培养后表达均显著下调,但是分别去掉这两个基因的干扰后,与G3Terc-/-小鼠血清共培养的HSC增殖仍然增多,说明端粒缩短激活DNA损伤信号通路引起造血干细胞功能损伤是多因素调控结果。因此,这一部分工作第一次提出这样的观点：系统大环境中血清成分改变直接影响造血干细胞自我更新能力。这也为临床开展干细胞治疗提供新的研究策略及研究启示。 综上,本项研究第一次提出不同类型成体干细胞面对细胞内相同的DNA损伤检定点表现不同,同时也第一次提出细胞外环境改变影响造血干细胞自我更新及重建功能。从而为干细胞损伤的调节机制研究提供新的研究思路与研究策略。
[Abstract]:Nowadays, aging is becoming more and more serious. Aging forces us to have a deeper understanding of the molecular mechanism of aging and aging-related diseases.One of the mechanisms of aging is telomere shortening. The severely shortened telomere length at the end of chromosome can activate the DNA damage response pathway of stem cells. It can not only induce different damage response checkpoints in cells, but also cause damage to the extracellular environment and damage the function of stem cells.
Exonuclease (Exo1), a mismatch repair-related gene, is one of the checkpoints in the signaling pathway of telomere shortening activating DNA damage. Knocking out Exo1 can block the activation of DNA damage signaling pathway in G3Terc-/-mouse intestinal crypt stem cells and improve the damage of small intestinal crypt stem cells. However, Exo1 knockout can improve the damage of G3Terc-/mouse intestinal crypt stem cells. The role of stem cells in other tissues has not been reported.
Our study showed that Exo1 knockout did not ameliorate G3Terc - / - mouse hematopoietic stem cell injury. Bone marrow transplantation experiments showed that G3Terc - / - donor-derived HSC transplantation and reconstruction ability were significantly decreased compared with wild-type control group, and Exo1 knockout did not ameliorate the reduction of this ability. Exo1 knockout did not ameliorate G3Terc - / - mouse hematopoietic stem cell injury either. Injury of neural stem cells in mice.
Exol knockout did not improve the self-renewal ability of G3Terc - / - mice. Similarly, Exo 1 knockout did not ameliorate the damage of G3Terc - / mice bone marrow mesenchymal stem cells and the proliferation of G3Terc - / - mice bone marrow mesenchymal stem cells decreased. G3Terc-/-Exo1-/-binuclear knockout mice showed no improvement in the proliferation of bone marrow mesenchymal stem cells and their DNA damage signaling pathways could not be blocked. Other genes provide new experimental evidence for the difference of stem cell function in different tissues and organs, and provide new research strategies for the study of stem cell senescence and regeneration in different tissues and organs.
Shortened telomere activation of DNA damage response pathways can cause extracellular (systemic) damage, affect the ability of HSC to differentiate, reduce cell differentiation into lymphocytes and myeloid cells, and impede the production of B lymphocytes and T lymphocytes. The effects of systemic environment (especially serum components) on self-renewal and reconstitution of hematopoietic stem cells have not been reported.
Our study suggests that HSC from normal young wild type mice co-cultured with G3Terc-/- mouse serum in vitro increases the proliferation of hematopoietic stem cells and more hematopoietic stem cells leave the stationary phase and enter the cell cycle. Bone marrow stem cell transplantation co-cultured with G3Terc-/-mouse serum enters the menstrual cycle. The ability of hematopoietic stem cells to reconstitute was significantly decreased in mice irradiated with lethal radiation. The expression of p21 gene regulating G1 phase and TGF-beta gene regulating G0 phase of cell cycle were significantly down-regulated after co-culturing with G3Terc-/- mice serum, but after the interference of these two genes was removed, the expression of p21 gene regulating G1 phase and TGF-beta gene regulating G0 phase of cell cycle was significantly down-regulated. The proliferation of HSC co-cultured with rat serum is still increasing, indicating that telomere shortening activating DNA damage signaling pathway leads to hematopoietic stem cell dysfunction is the result of multiple factors. The bed provides new research strategies and research implications for stem cell therapy.
In conclusion, this study is the first to suggest that different types of adult stem cells exhibit different DNA damage checkpoints in the same cells, and that changes in the extracellular environment affect the self-renewal and reconstitution of hematopoietic stem cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
[Abstract]:Nowadays, aging is becoming more and more serious. Aging forces us to have a deeper understanding of the molecular mechanism of aging and aging-related diseases.One of the mechanisms of aging is telomere shortening. The severely shortened telomere length at the end of chromosome can activate the DNA damage response pathway of stem cells. It can not only induce different damage response checkpoints in cells, but also cause damage to the extracellular environment and damage the function of stem cells.
Exonuclease (Exo1), a mismatch repair-related gene, is one of the checkpoints in the signaling pathway of telomere shortening activating DNA damage. Knocking out Exo1 can block the activation of DNA damage signaling pathway in G3Terc-/-mouse intestinal crypt stem cells and improve the damage of small intestinal crypt stem cells. However, Exo1 knockout can improve the damage of G3Terc-/mouse intestinal crypt stem cells. The role of stem cells in other tissues has not been reported.
Our study showed that Exo1 knockout did not ameliorate G3Terc - / - mouse hematopoietic stem cell injury. Bone marrow transplantation experiments showed that G3Terc - / - donor-derived HSC transplantation and reconstruction ability were significantly decreased compared with wild-type control group, and Exo1 knockout did not ameliorate the reduction of this ability. Exo1 knockout did not ameliorate G3Terc - / - mouse hematopoietic stem cell injury either. Injury of neural stem cells in mice.
Exol knockout did not improve the self-renewal ability of G3Terc - / - mice. Similarly, Exo 1 knockout did not ameliorate the damage of G3Terc - / mice bone marrow mesenchymal stem cells and the proliferation of G3Terc - / - mice bone marrow mesenchymal stem cells decreased. G3Terc-/-Exo1-/-binuclear knockout mice showed no improvement in the proliferation of bone marrow mesenchymal stem cells and their DNA damage signaling pathways could not be blocked. Other genes provide new experimental evidence for the difference of stem cell function in different tissues and organs, and provide new research strategies for the study of stem cell senescence and regeneration in different tissues and organs.
Shortened telomere activation of DNA damage response pathways can cause extracellular (systemic) damage, affect the ability of HSC to differentiate, reduce cell differentiation into lymphocytes and myeloid cells, and impede the production of B lymphocytes and T lymphocytes. The effects of systemic environment (especially serum components) on self-renewal and reconstitution of hematopoietic stem cells have not been reported.
Our study suggests that HSC from normal young wild type mice co-cultured with G3Terc-/- mouse serum in vitro increases the proliferation of hematopoietic stem cells and more hematopoietic stem cells leave the stationary phase and enter the cell cycle. Bone marrow stem cell transplantation co-cultured with G3Terc-/-mouse serum enters the menstrual cycle. The ability of hematopoietic stem cells to reconstitute was significantly decreased in mice irradiated with lethal radiation. The expression of p21 gene regulating G1 phase and TGF-beta gene regulating G0 phase of cell cycle were significantly down-regulated after co-culturing with G3Terc-/- mice serum, but after the interference of these two genes was removed, the expression of p21 gene regulating G1 phase and TGF-beta gene regulating G0 phase of cell cycle was significantly down-regulated. The proliferation of HSC co-cultured with rat serum is still increasing, indicating that telomere shortening activating DNA damage signaling pathway leads to hematopoietic stem cell dysfunction is the result of multiple factors. The bed provides new research strategies and research implications for stem cell therapy.
In conclusion, this study is the first to suggest that different types of adult stem cells exhibit different DNA damage checkpoints in the same cells, and that changes in the extracellular environment affect the self-renewal and reconstitution of hematopoietic stem cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
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