本文選題：低劑量輻射 + 骨髓間充質(zhì)干細(xì)胞　； 參考：《吉林大學(xué)》2011年博士論文

【摘要】：低劑量輻射作用于生物體可以引起與大劑量輻射不同的特殊的的生物學(xué)效應(yīng)。其中興奮性效應(yīng)與適應(yīng)性反應(yīng)是最為常見。興奮性效應(yīng)表現(xiàn)為接受低劑量輻射以后細(xì)胞增殖加快,機(jī)體免疫力提高等。適應(yīng)性反應(yīng)可以使細(xì)胞或機(jī)體對隨后接觸的高劑量輻射產(chǎn)生一定的適應(yīng),減輕受到的損傷。既往的研究表明這些生物學(xué)效應(yīng)在很多正常的細(xì)胞出現(xiàn),但在造血系統(tǒng)尤其是骨髓間充質(zhì)干細(xì)胞中的研究還未深入。本研究旨在揭示低劑量輻射對骨髓間充質(zhì)干細(xì)胞的生物學(xué)效應(yīng),并從信號轉(zhuǎn)導(dǎo)通路的角度對其機(jī)制進(jìn)行探討。 本實(shí)驗(yàn)首先從大鼠骨髓分離并擴(kuò)增了骨髓間充質(zhì)干細(xì)胞并利用其表面標(biāo)志特征和多向分化潛能等生物學(xué)特性進(jìn)行了鑒定。然后證實(shí)了其在低劑量輻射的誘導(dǎo)下出現(xiàn)了興奮性效應(yīng)和適應(yīng)性反應(yīng)。并應(yīng)用多種實(shí)驗(yàn)技術(shù)手段在蛋白水平研究了興奮性效應(yīng)和適應(yīng)性反應(yīng)的機(jī)制。證實(shí)興奮性效應(yīng)與MAPK/ERK、PI3K/AKT信號轉(zhuǎn)導(dǎo)通路的激活引起細(xì)胞周期調(diào)控有著密切的關(guān)系。適應(yīng)性反應(yīng)與ATM磷酸化后引起的DNA損傷修復(fù)加強(qiáng)有關(guān)。 骨髓造血干細(xì)胞的分離純化和擴(kuò)增。選用全骨髓培養(yǎng)法,在原代培養(yǎng)開始24-36小時(shí)首次換液從骨髓中將間充質(zhì)干細(xì)胞分離培養(yǎng),細(xì)胞貼壁生長良好,呈成纖維細(xì)胞狀,魚群樣或旋渦狀排列。傳代至第三代后進(jìn)行流式細(xì)胞術(shù)進(jìn)行表面標(biāo)志檢測,結(jié)果為骨髓間充質(zhì)干細(xì)胞表面標(biāo)志CD44,CD90陽性,CD34,CD45陰性。純度達(dá)95%以上。經(jīng)成脂、成骨誘導(dǎo)可分化為脂肪細(xì)胞,成骨細(xì)胞,分別用油紅染料及茜素紅染料染色證實(shí)。 低劑量輻射誘導(dǎo)骨髓間充質(zhì)干細(xì)胞興奮性效應(yīng)的發(fā)生與機(jī)制。低劑量輻射后24小時(shí)進(jìn)行Brdu ELISA法檢測及細(xì)胞計(jì)數(shù)顯示細(xì)胞增殖加快,75 mGy劑量引起的增殖最為明顯,證明骨髓間充質(zhì)干細(xì)胞在低劑量輻射誘導(dǎo)下出現(xiàn)興奮性效應(yīng)。流式細(xì)胞術(shù)檢測細(xì)胞周期可見G1期細(xì)胞比例減少,進(jìn)入S期細(xì)胞比率明顯增多。Western blot檢測75mGy X射線照射后P-c-Raf,P-MEK,P-ERK均表達(dá)增強(qiáng),且在輻射后6小時(shí)最為明顯。應(yīng)用MEK阻滯劑U0126可阻滯細(xì)胞增殖,證明MAPK/ERK信號轉(zhuǎn)導(dǎo)通路的激活參與低劑量輻射誘導(dǎo)興奮性反應(yīng)的形成。P-AKT及P-PDK1也表達(dá)增強(qiáng),特異性阻滯劑LY294002可抑制細(xì)胞增殖,證明PI3K/AKT信號轉(zhuǎn)導(dǎo)通路也參與興奮性效應(yīng)的產(chǎn)生。應(yīng)用PI3K阻滯劑LY294002后p-ERK的表達(dá)也降低,證明MAPK/ERK信號轉(zhuǎn)導(dǎo)通路與PI3K/AKT信號轉(zhuǎn)導(dǎo)通路在低劑量輻射誘導(dǎo)的興奮性反應(yīng)中存在協(xié)同效應(yīng)。 低劑量輻射誘導(dǎo)適應(yīng)性反應(yīng)及機(jī)制的研究�？寺⌒纬蓪�(shí)驗(yàn)顯示4 Gy X線輻射前6小時(shí)給予75 mGy的照射使得克隆存活率較直接給予4 Gy X線輻射明顯提高,產(chǎn)生適應(yīng)性反應(yīng)。單細(xì)胞凝膠電泳實(shí)驗(yàn)發(fā)現(xiàn)給予4 Gy X線輻射前6小時(shí)接受75 mGy的照射能減輕DNA雙鏈斷裂損傷。免疫熒光檢測磷酸化H2AX與p-ATM焦點(diǎn)完全重合,且在預(yù)先接受低劑量輻射時(shí)較直接進(jìn)行高劑量輻射數(shù)量少,修復(fù)時(shí)間也明顯縮短。應(yīng)用ATM特異性阻滯劑KU55933后適應(yīng)性反應(yīng)消失。Western blot實(shí)驗(yàn)檢測H2AX與p-ATM結(jié)果也與上述結(jié)果符合。 據(jù)以上結(jié)果分析,在DNA雙鏈斷裂損傷中起重要作用的ATM,H2AX的磷酸化等參與適應(yīng)性反應(yīng)的形成。綜上所述,低劑量輻射可以誘導(dǎo)骨髓間充質(zhì)干細(xì)胞產(chǎn)生興奮性效應(yīng)和適應(yīng)性反應(yīng),且發(fā)生機(jī)制分別與調(diào)節(jié)細(xì)胞增殖相關(guān)的MAPK/ERK、PI3K/AKT信號轉(zhuǎn)導(dǎo)通路及DNA損傷修復(fù)相關(guān)蛋白ATM, H2AX磷酸化有關(guān)。
[Abstract]:The effects of low - dose radiation on bone marrow mesenchymal stem cells in hematopoietic system , especially bone marrow mesenchymal stem cells , have not been studied . The present study is aimed at revealing the biological effects of low - dose radiation on bone marrow mesenchymal stem cells , and to probe into the mechanism of the signal transduction pathways .


In this experiment , the bone marrow mesenchymal stem cells were isolated from the bone marrow of rats and the biological characteristics of bone marrow mesenchymal stem cells were identified by using their surface marker features and multi - directional differentiation potential .


The bone marrow hematopoietic stem cells were isolated , purified and amplified . The whole marrow culture method was used to separate the mesenchymal stem cells from the bone marrow for the first time 24 to 36 hours after the initial culture . The results showed that the surface markers CD44 , CD90 were positive , CD34 and CD45 were negative in the bone marrow mesenchymal stem cells .


The activation of MAPK / ERK signal transduction pathway was enhanced . The expression of MAPK / ERK signal transduction pathway was also decreased after irradiation of 75 mGy X - ray .


Low dose radiation induced adaptive response and mechanism . The results showed that irradiation of 75 mGy 6 hours before irradiation with 4 Gy X - ray irradiation increased the survival rate of 4 Gy X - ray radiation significantly , which resulted in the adaptive response . Immunofluorescence detection phosphorylating H2AX was completely coincident with the p - ATM focal point , and the adaptive response disappeared after receiving low - dose radiation . The results of Western blot showed that H2AX and p - ATM were consistent with the above results .


In conclusion , low - dose radiation can induce excitatory and adaptive responses to bone marrow mesenchymal stem cells , and the mechanisms are related to MAPK / ERK signaling pathway and DNA damage repair related protein ATM , H2AX phosphorylation related to the regulation of cell proliferation , respectively .
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R329;R81

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相關(guān)期刊論文 前2條

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本文編號：1943671