【作者】 杨岩；

【导师】 王冠军；

【作者基本信息】 吉林大学 ， 内科学， 2007， 博士

【摘要】 低剂量辐射(LDR)具有兴奋性效应和适应性反应,兴奋性效应以免疫增强作用最受关注。LDR对造血系统的作用研究极少,仅有几篇文献报道,显示对造血系统有兴奋性效应,而LDR对骨髓间充质干细胞(BM-MSC)的影响国内外均未见报道。BM-MSC是一种具有多向分化潜能的干细胞,能在体外培养扩增,且能长期维持其遗传学性状稳定,在体外能使脐血造血祖细胞扩增,在体内与造血干细胞共移植,能加速造血重建。本文首先建立大规模BM-MSC体外培养及扩增体系,并对其生物学特性进行检测,确定为纯化的BM-MSC;再以X线为照射源,研究LDR后BM-MSC生长曲线,对数生长期群体倍增时间(PDT)的变化,来观察LDR是否对BM-MSC有兴奋性效应;确定有兴奋性效应,为探讨其机制,应用流式细胞术检测细胞周期及调亡的变化,应用ELISA方法检测细胞因子表达量的变化;最后应用差异蛋白质组学的方法分析75mGy X射线照射BM-MSC 48h后蛋白质差异表达的变化。结果表明:体外培养扩增的BM-MSC均一性好,无造血细胞表型表达;LDR后BM-MSC有兴奋性效应,表现为细胞生长加速,PDT缩短;LDR后BM-MSC,G0/G1百分率逐渐减少,S期、G2/M期百分率逐渐增多,S期百分率明显增多,细胞迅速进入增殖周期;LDR-MSC早期调亡的百分率有增多趋势,但72h后调亡即减少,这是MSC生长加快的机制;LDR后BM-MSC在一定时间内SCF、IL-6、M-CSF表达量增加,M-CSF含量增加以75mGy照射后最明显,IL-6含量增加,以50mGy照射后最明显;即LDR对BM-MSC从细胞生长曲线,细胞周期及凋亡,细胞因子的分泌量,不同层次上表现出兴奋性效应;2-DE显示有27个表达差异蛋白点,其中12个上调点,12个下调蛋白点,3个消失点,对这27个差异点进行MADI-TOT-MS质谱鉴定,结果发现12个点有统计学意义的13种蛋白质,分析认为:氨基肽酶、线粒体延长因子、P43、G蛋白、核糖核蛋白A2/B1亚型A2表达上调可能与LDR后BM-MSC产生的兴奋性效应有关,而且P43表达上调与IL-6表达增多可能有关,其它蛋白质表达改变与LDR后BM-MSC产生兴奋性效应关系不清楚。更多还原

【Abstract】 There are many evidences shown that low dose radiation has hormesis effect and adaptive response. Hormesis effect may manifest as stimulating growth of animals and plants, prolonging the lifespan of organisms, immune enhancement and anti-cancer effect. Among them, the immunostimulation is mostly considered. Adaptive response means that the organism can endure larger dose of radiation if it has been exposed to LDR. Serial reports have focused on the effects on hematopoietic system of LDR. And there are few studies shown that LDR has significant hormesis effect to the proliferation of hematopoietic progenitor cells and the secretion of cytokines of cord blood. However, nobody has reported the effect on bone marrow mesenchymal stem cells (BM-MSC). Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the main two kinds of stem cells in bone marrow. MSCs have multi-differentiation potency and can be amplified in vitro while maintaining the genetics traits. MSCs can differentiate to neuron cells, osteoblasts, adipocytes, cartilage cells and muscle cells and so on. MSCs can generate several hematopoietic growth factors, adhesion molecules and promote the amplification of hematopoietic cells of cord blood in vitro, it also can shorten the time of hematopoiesis recovery if co-transplanted with HSCs. Here we researched the changes of BM-MSC pretreated with LDR in growth properties, cell cycle and apoptosis and expression of cytokines. Further explore the mechanisms of hormesis effect of BM-MSC induced by LDR, we analyzed the alteration of proteome treated by LDR by the proteomics method. And the results supply the theoretical basis for MSC being used to cord blood transplantation and co-transplantation with HSCs after treated with LDR.Part one The study on the cultivation and biological traits of human mesenchymal stem cells (hMSC) in vitro. hMSC were harvested by bone marrow aspiration on the posterior superior iliac spine of health adult donors. The mononuclear cells were separated by percol separating medium (r=1.073g/ml) and then adherent cultured in DMEM-LG containing 10% FBS. The traits of shape were observed by microscope, the growth curves were drawn by cell count method, the cell cycle and surface markers were determined by FACS. All the experiments were repeated 3 times and the matched t test and mono-factor variance analysis was used for statistical treatment. We found that the hMSC were homogeneous, spindle-shaped and fibroblast-like cells. The cells can be propagated and extensively amplified. The amount of amplified hMSC from different donors reached to (3.46~3.60)×107/L when the cells were transfered to P3 and there was no statistical significance among the amounts of cells (P=0.896). the amplification times of primary cells from different donors varies from 9d to 15d with statistical significant deviation(P=0.000). The proliferation rates of the MSC from different donors speed up after serial subcultivation, however, maintaining significant deviation at every generation(P=0.000).The FACS results show that the hMSC express CD29, CD44, CD90, CD105, without CD34, CD45. the cell cycle analysis show that there is 90.03% G0/G1 stage cells and 9.97% S+G2+M stage cells.Part two Study on the effect on the biological traits of human mesenchymal stem cells from bone marrow pretreated with LDR. The cells were the P4, P5 and P6 MSCs from the part one. They were exposed to X rays at the dose of 50mGy,75mGy、100mGy (dose rate 12.5mGy/min). The growth curve, cell cycle and apopotosis ,expression variation of cytokines of MSC treated by LDR were investigated. The results were treated with t test using SPSS10.0 statistics software. The growth rates of MSC treated by LDR obviously increase from d3. Population doubling times (PDT) at logarithmic growth phase are shorten .The cell cycle and apoptosis were examined with FACS. The results show that the G0/G1 stage cells decrease after exposure to LDR, compared with the same time control groups.The following groups have significant difference (P<0.05): 50mGy dose group at 48h, 75mGy at 48h and 72h.,100mGy at 48h . The percent of G0/G1 stage cells of 75mGy at 72h is the lowest (30.86%). The percent of G2-M stage cells increase (the peak was at 48h) after exposed to 50mGy X-rays. The G2-M stage cells increase at 24h while decrease at 48h and 72h in 75mGy groups. However, the G2-M stage cells decrease after exposed to 100mGy X-rays. The S stage cells percentage gradually increase at 24h, 48h and 72h. The most one is 75mGy group at 72h, which reaches to 68.88%. The 75mGy group at 48h,72h and the 100mGy group at 24h, 48h, 72h have significant difference (P<0.05) compared with the same time point control group respectively. The S stage cells are found decreased only in 50mGy and 75mGy at 24h. The apoptosis percentages have increased tendency at 24h and 48h in all dose groups, especially in 100mGy at 24h (25.99%), while have decreased tendency at 72h and the most decreased group is the 50mGy (6.8%). These results indicate that the LDR has hormesis effect on BM-MSC: increasing growth rate, shortening PDT, increasing percentage of S stage cells, transient enhancement of apoptosis in the early stage and soon being decreased, enhancement of hematopoitic growth factors. The content changes of SCF, IL-6, M-CSF secreted by BM-MSC after treated by LDR were determined by ELISA method. The contents of SCF increase at 24h, 48h after X-rays irradiated with 50mGy, 75mGy, 100mGy. The contents of SCF decrease at 72h,. As for IL-6, the contents in different dose groups at 24h and 48h have up-regulation. These groups, 50mGy at 24h, 48h, 75mGy at 24h, 48h, 100mGy at 24h have statistical difference (P<0.05) compared with their control groups respectively. The content of IL-6 has greatest enhancement at dose of 50mGy. Similarly, the IL-6 content decrease at 72h and the 100mGy dose groups have statistical difference compared with their control groups. The contents of M-SCF in all the groups at 24h, 48h and 72h except for the 50mGy dose at 72h have also been found increased. The greatest increased content occur in the 75mGy dose group at 72h.Part three Study on the mechanisms of hormesis effect of human bone marrow mesenchymal stem cells pretreated with low dose irradiation by proteomics method. For further explore the mechanisms of hormesis effect of BM-MSC induced by LDR, we dynamically multi-analyzed the alteration of proteome treated by LDR with the proteomics method. The MSCs were the P5 BM-MSC in the part one, and were irradiated with 75mGy X-rays. We adopted the 48h after irradiation as the observation point for experimental group. The the non-irradiation group was the control group. We extracted the whole proteins of the two groups respectively, set up the 2-DE conditions with excellent repetitiveness, Then the proteins formed both groups experience electrophoresis at the same conditions. The result shows there are 27 differential expression protein points, 12 of them are up-regulated, 12 of them are down-regulated and the other 3 disappear. We have determined all of the altered proteins with MALDI-TOF-MS, searched with the Mascot software, and found 12 protein points with 13 proteins had the statistical significance (score>64 means statistical significance). we guess aminopeptidase ,the mitochondrial elongation factor Tu ,P43,G protein and ribonucleoproteinA2/B1 isoform A2 may contribute to the hormesis effects of human BM-MSCs pretreated with low dose irradiation ,However, whether it is also related to the other proteins is not known.更多还原