【作者】 刘水冰；

【导师】 梅其炳； 田琼；

【作者基本信息】 第四军医大学 ， 药理学， 2008， 博士

【摘要】 目的:成年哺乳动物骨髓存在两类干细胞:造血干细胞和间充质干细胞(造血微环境的重要组分)。造血细胞的增殖和分化依赖于造血微环境,造血微环境中的造血细胞因子和细胞之间的相互作用,促进了造血细胞的增殖和分化。γ射线不仅可以导致急性骨髓抑制,而且还会引起长期的造血损伤后遗症。长期造血损伤后遗症的产生主要是因为γ射线损害了造血干细胞的自我更新。我们先前的研究已经表明:重组人骨形成蛋白-2(rhBMP-2)能修复γ射线引起的小鼠急性造血损伤,其作用与其促进造血干细胞的增殖有关。为了进一步证实rhBMP-2修复γ射线引起的小鼠急性造血损伤的机制,我们设计本实验以期证明rhBMP-2调控的间充质干细胞增殖和分化在治疗造血损伤时起重要的作用。方法:(1)用XTT的方法检测rhBMP-2对体外培养的间充质干细胞的增殖作用。(2)用real-timePCR的方法检测rhBMP-2对间充质干细胞分泌的造血细胞因子mRNA表达的作用。(3)用ELISA试剂盒检测rhBMP-2对间充质干细胞分泌造血细胞因子的蛋白含量的作用。(4)将绿色荧光蛋白转基因小鼠骨髓中分离纯化的间充质干细胞用Hoechst33342进行标记,然后移植给BALB/c小鼠,移植后6周,分别用免疫荧光染色和流式细胞分析检测间充质干细胞在体内的分化情况。结果:(1)rhBMP-2在浓度为10ng/ml-1×105ng/ml之间能显著促进MSCs的增殖,呈剂量依赖性。然而,这种增殖作用随着rhBMP-2浓度进一步增加到1×106 ng /ml而开始减弱。(2)rhBMP-2能够显著地促进造血细胞因子IL-7、IL-11、G-CSF、M-CSF、SCF的mRNA表达。(3)用rhBMP-2处理体外培养的MSCs后,培养上清中造血细胞因子IL-6、IL-7、IL-11以及M-CSF的蛋白含量明显高于对照组。(4)当小鼠受到γ射线照射后,MSCs能够归巢到辐射损伤的造血组织并分化为造血细胞,rhBMP-2能显著地增强MSCs的归巢与分化,从而促进造血重建进程。结论:正常的造血依赖于造血细胞和造血微环境的相互作用。造血微环境可以支持并调控造血细胞的定居、增殖、分化、发育及成熟。MSCs在造血重建过程中起着重要作用。rhBMP-2能够促进MSCs的增殖并分化为造血细胞,其作用不仅能促进造血微环境修复,而且还能加速受照射小鼠造血细胞的生成。这就是rhBMP-2修复造血损伤的重要机制。更多还原

【Abstract】 Aim: Adult mammalian bone marrow contains two types of stem cells:haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) that arethe key composition in hematopoietic microenvironment. The proliferation anddifferentiation of hematopoiesis depend on hematopoietic microenvironment.Interaction of hematopoietic cytokines and cells in hematopoieticmicroenvironment generates various signals, which supports the proliferationanddifferentiationofhematopoieticcells.Itiswellknownthatexposureto?-raynot only causes acute bone marrow suppression but also leads to long-termresidual hematopoieticinjury.Thelatteris duetodamageofhematopoietic stemcells (HSCs) self-renewal. Our previous studies have shown that recombinationhuman bone morphogenetic protein-2 (rhBMP-2) can rescue acutehematopoietic damage of mice exposed to ?-ray, which is concerned withpromotion of rhBMP-2 in the proliferation of HSCs. In order to further confirmthe mechanism of rhBMP-2’s rescuing acute hematopoietic injury in mice exposed toγ-ray, we carried out this experiment to demonstrate that theproliferation and differentiation of MSCs regulated by rhBMP-2 plays a crucialroleinrescuinghematopoieticinjuryinmiceexposedto?-ray.Methods: (1) The XTT method was used to detect the effect of rhBMP-2on the proliferation of MSCs cultured in vitro. (2) The real-time quantitativePCR was used to detect the effect of rhBMP-2 on the hematopoietic cytokinesmRNAexpressionsofMSCsculturedinvitro.(3)TheELISAwasusedtodetectthe effect of rhBMP-2 on the hematopoietic cytokines protein level of MSCscultured in vitro. (4) MSCs from Green Fluorescent Protein (GFP) transgenicmice were marked with Hoechst33342 before transplanted into BALB/c mice.Immunofluorescent staining and flow cytometry were employed to detect thedifferentiationofMSCsinvivoaftertransplantationfor6weeks.Results: (1) rhBMP-2 significantly promoted MSCs proliferation in doseranging from 10 ng/ml - 1×105 ng/ml in a dose-dependent manner. However,this proliferation effect turned down with further increased dose of rhBMP-2 to1×106 ng /ml. (2) rhBMP-2 significantly promoted hematopoietic cytokinesmRNA expression such as IL-7, IL-11, G-CSF, M-CSF, and SCF. (3) Theprotein levels of hematopoietic cytokines such as IL-6, IL-7, IL-11, and M-CSFin the supernatant of cultured MSCs with rhBMP-2 were higher than those ofculturedMSCs without rhBMP-2.(4)MSCs couldhometotheradiation-injuredhematopoietic tissues and differentiate to hematopoietic cells when the micewere exposed toγ-ray. The effect of MSCs was enhanced byrhBMP-2, whichacceleratedthehematopoieticrestoration.Conclusion: Normal hematopoiesis depends on interaction ofhematopoietic cells and hematopoietic microenvironment. Hematopoieticmicroenvironment can support and regulate establishment, proliferation, differentiation, development and maturity of hematopoietic cells. In conclusion,MSCs play an important role in hematopoietic restoration. The effect ofrhBMP-2 promotes the proliferation and differentiation of mesenchymal stemcells, which not only improved the haematopoietic microenvironment, but alsoand accelerated the production of haematopoietic cells in mice exposed to ?-ray,which may be important mechanism of rhBMP-2 rescuing the hematopoieticinjuryofmice.更多还原