不同類型成體干細胞對DNA損傷應答反應研究
發(fā)布時間：2018-09-08 10:23
【摘要】：當今社會老齡化狀況日益嚴重,衰老迫使我們要對衰老及衰老相關疾病的分子機制有更為深刻的理解。衰老相關機制之一是端粒縮短,G3Terc-/-是研究端粒縮短誘發(fā)衰老的較好的實驗動物模型,G3Terc-/-由于失去端粒酶的保護作用而出現染色體末端端粒長度嚴重縮短,進而激活干細胞DNA損傷應答通路,不但可以引起細胞內出現不同的損傷應答檢定點變化,還能夠引起細胞外環(huán)境損傷而導致干細胞功能受損。 錯配修復相關基因核酸外切酶(Exonucleasel,Exo1)是端�？s短激活DNA損傷信號通路中的檢定點之一,敲除Exo1基因可以阻斷G3Terc-/-小鼠小腸隱窩干細胞DNA損傷信號通路激活,進而改善小腸隱窩干細胞損傷情況。然而Exo1基因敲除對G3Terc-/小鼠其他組織干細胞作用如何尚未見報道。 我們的研究表明,Exo1基因敲除不能改善G3Terc-/-小鼠造血干細胞損傷。骨髓移植實驗提示,與野生型對照組比較,G3Terc-/-供體來源HSC移植重建能力明顯下降,Exo1基因敲除不能改善這種重建能力降低的情況。Exo1基因敲除也不能改善G3Terc-/-小鼠神經干細胞損傷。 小鼠神經干細胞初次及第二次自我更新能力明顯下降,Exol基因敲除不能改善G3Terc-/-小鼠神經干細胞自我更新能力降低的情況。同樣,Exo1基因敲除不能改善G3Terc-/小鼠骨髓間充質干細胞損傷,G3Terc-/-小鼠骨髓間充質干細胞增殖能力降低,G3Terc-/-Exo1-/-雙基因敲除小鼠骨髓間充質干細胞增殖能力并未改善,其DNA損傷信號通路未能被阻斷。因此第一次提出這樣的證據：不同類型成體干細胞對端�？s短激活DNA損傷信號通路相關的檢定點存在不同的應答反應；這對研究其他基因對不同組織器官干細胞功能影響差異提供新的實驗依據。也對不同組織器官干細胞衰老及再生研究提供新的研究策略啟示,具有潛在的實際應用價值。 端�？s短激活DNA損傷應答通路可以引起細胞外環(huán)境(系統(tǒng)大環(huán)境)損傷,影響HSC分化能力,導致細胞向淋系細胞分化減少和向髓系細胞分化增加,和B淋巴細胞及T淋巴細胞生成障礙。之前的研究表明系統(tǒng)大環(huán)境影響造血干細胞的分化,而系統(tǒng)大環(huán)境(尤其是血清成分)對造血干細胞自我更新及重建能力影響尚未見報道。 我們的研究提示,與同年齡野生型小鼠血清對比,正常年輕野生型小鼠HSC在體外與G3Terc-/-小鼠血清共培養(yǎng)后,造血干細胞增殖明顯增加,更多的造血干細胞離開靜止期進入細胞周期。與G3Terc-/-小鼠血清共培養(yǎng)的骨髓干細胞移植進入經致死性放射線照射過的小鼠,造血干細胞重建能力顯著下降。調控細胞周期G1期的基因p21和調控細胞周期G0期的基因TGF-β在正常年輕野生型小鼠與G3Terc-/-小鼠血清共培養(yǎng)后表達均顯著下調,但是分別去掉這兩個基因的干擾后,與G3Terc-/-小鼠血清共培養(yǎng)的HSC增殖仍然增多,說明端�？s短激活DNA損傷信號通路引起造血干細胞功能損傷是多因素調控結果。因此,這一部分工作第一次提出這樣的觀點：系統(tǒng)大環(huán)境中血清成分改變直接影響造血干細胞自我更新能力。這也為臨床開展干細胞治療提供新的研究策略及研究啟示。 綜上,本項研究第一次提出不同類型成體干細胞面對細胞內相同的DNA損傷檢定點表現不同,同時也第一次提出細胞外環(huán)境改變影響造血干細胞自我更新及重建功能。從而為干細胞損傷的調節(jié)機制研究提供新的研究思路與研究策略。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363
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