低剂量辐射诱导骨髓间充质干细胞生物学效应及机制研究

发布时间：2018-05-27 20:39

本文选题：低剂量辐射 + 骨髓间充质干细胞　；参考：《吉林大学》2011年博士论文

【摘要】：低剂量辐射作用于生物体可以引起与大剂量辐射不同的特殊的的生物学效应。其中兴奋性效应与适应性反应是最为常见。兴奋性效应表现为接受低剂量辐射以后细胞增殖加快,机体免疫力提高等。适应性反应可以使细胞或机体对随后接触的高剂量辐射产生一定的适应,减轻受到的损伤。既往的研究表明这些生物学效应在很多正常的细胞出现,但在造血系统尤其是骨髓间充质干细胞中的研究还未深入。本研究旨在揭示低剂量辐射对骨髓间充质干细胞的生物学效应,并从信号转导通路的角度对其机制进行探讨。本实验首先从大鼠骨髓分离并扩增了骨髓间充质干细胞并利用其表面标志特征和多向分化潜能等生物学特性进行了鉴定。然后证实了其在低剂量辐射的诱导下出现了兴奋性效应和适应性反应。并应用多种实验技术手段在蛋白水平研究了兴奋性效应和适应性反应的机制。证实兴奋性效应与MAPK/ERK、PI3K/AKT信号转导通路的激活引起细胞周期调控有着密切的关系。适应性反应与ATM磷酸化后引起的DNA损伤修复加强有关。骨髓造血干细胞的分离纯化和扩增。选用全骨髓培养法,在原代培养开始24-36小时首次换液从骨髓中将间充质干细胞分离培养,细胞贴壁生长良好,呈成纤维细胞状,鱼群样或旋涡状排列。传代至第三代后进行流式细胞术进行表面标志检测,结果为骨髓间充质干细胞表面标志CD44,CD90阳性,CD34,CD45阴性。纯度达95%以上。经成脂、成骨诱导可分化为脂肪细胞,成骨细胞,分别用油红染料及茜素红染料染色证实。低剂量辐射诱导骨髓间充质干细胞兴奋性效应的发生与机制。低剂量辐射后24小时进行Brdu ELISA法检测及细胞计数显示细胞增殖加快,75 mGy剂量引起的增殖最为明显,证明骨髓间充质干细胞在低剂量辐射诱导下出现兴奋性效应。流式细胞术检测细胞周期可见G1期细胞比例减少,进入S期细胞比率明显增多。Western blot检测75mGy X射线照射后P-c-Raf,P-MEK,P-ERK均表达增强,且在辐射后6小时最为明显。应用MEK阻滞剂U0126可阻滞细胞增殖,证明MAPK/ERK信号转导通路的激活参与低剂量辐射诱导兴奋性反应的形成。P-AKT及P-PDK1也表达增强,特异性阻滞剂LY294002可抑制细胞增殖,证明PI3K/AKT信号转导通路也参与兴奋性效应的产生。应用PI3K阻滞剂LY294002后p-ERK的表达也降低,证明MAPK/ERK信号转导通路与PI3K/AKT信号转导通路在低剂量辐射诱导的兴奋性反应中存在协同效应。低剂量辐射诱导适应性反应及机制的研究。克隆形成实验显示4 Gy X线辐射前6小时给予75 mGy的照射使得克隆存活率较直接给予4 Gy X线辐射明显提高,产生适应性反应。单细胞凝胶电泳实验发现给予4 Gy X线辐射前6小时接受75 mGy的照射能减轻DNA双链断裂损伤。免疫荧光检测磷酸化H2AX与p-ATM焦点完全重合,且在预先接受低剂量辐射时较直接进行高剂量辐射数量少,修复时间也明显缩短。应用ATM特异性阻滞剂KU55933后适应性反应消失。Western blot实验检测H2AX与p-ATM结果也与上述结果符合。据以上结果分析,在DNA双链断裂损伤中起重要作用的ATM,H2AX的磷酸化等参与适应性反应的形成。综上所述,低剂量辐射可以诱导骨髓间充质干细胞产生兴奋性效应和适应性反应,且发生机制分别与调节细胞增殖相关的MAPK/ERK、PI3K/AKT信号转导通路及DNA损伤修复相关蛋白ATM, H2AX磷酸化有关。
[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 .
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329;R81

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