骨重塑在造血干细胞动员中的机制及应用研究

发布时间：2018-01-13 17:28

  本文关键词：骨重塑在造血干细胞动员中的机制及应用研究　出处：《中国协和医科大学》2010年博士论文　论文类型：学位论文

  更多相关文章： 成骨细胞 破骨细胞 造血干细胞动员 干祖细胞动员 甲状旁腺激素 NF-KB配体的受体

【摘要】： 目的骨髓中大多数造血干细胞位于骨组织构成的骨龛中。成骨细胞是骨内膜表面的内衬细胞,生理条件下HSC及移植后归巢至骨髓的HSC与之密切接触,这种解剖定位提示成骨细胞可能调节HSC的功能。这一观点首先在成骨细胞和HSC的体外共培养中得到证实,随即一系列的研究几乎同时确定：成骨细胞是HSC龛中一关键组成成分,目前人们将之命名为‘成骨细胞龛’或‘骨内膜龛’。通过调节‘骨内膜龛’的大小,可以控制HSC的数量,维持HSC的稳定状态。本研究以干细胞动员中的成骨细胞及破骨细胞为研究对象,观察干细胞动员过程中成破骨细胞数量和功能的变化及其与动员的关系,进一步研究造血干细胞动员的机制。 方法利用流式细胞术检测动员0、3、5天小鼠外周血干细胞数量,评价动员效果；通过实时定量PCR的方法比较动员0、3、5天小鼠成骨细胞特异性基因OCN.OPN.SDF-1.SCF的水平；并通过免疫组化及流式细胞术比较动员0、3、5天人类及小鼠成骨细胞数量功能的差异；利用细胞化学TRACP染色的方法比较动员0、3、5天小鼠破骨细胞数量和功能的差异；利用连续切片标记Caspase3的方法检测人类及小鼠成骨细胞的凋亡情况;利用ELISA的方法检测人类及小鼠循环中OCN. TRAP-5b蛋白水平反应成破骨细胞活性；共培养小鼠成骨细胞和小鼠骨髓有核细胞,检测成骨细胞活性。 结果 1.短期应用G-CSF可导致人类和小鼠动员模型中干骺端成骨细胞数量减少活性下降：动员前小鼠成骨细胞OCNmRNA水平为动员第三天的27±6倍(P＜0.01)；且动员后第3天骨内膜成骨细胞数量明显减少,形态由立方形变为梭形；CD45-Ter119-OPN成骨细胞数量在动员后3天及5天明显减少(动员前,4085±135cells／femur;动员后3天,1118±80 cells／femur:动员后5天,1032±55cell s/femur；P=0.02);动员3天和5天成骨细胞活性明显下降,OCN水平分别为59.44±3.16 ng/ml(动员前),39.21±4.49 ng/ml(动员后第3天)42.36±2.23 ng/ml(动员后第5天),P＜0.01。进一步检测正常供者和自体移植患者标本同样显示动员后成骨细胞数量减少并伴有活性下降。然而在小鼠G-CSF动员第3天外周血LSK细胞比例并未明显增加,动员前、动员3天及动员5天分别为0.40±0.07%、0.55±0.05%和2.9±0.32%,因此成骨细胞的变化发生于动员之前。 2.成骨细胞数量减少导致SDF-1、SCF、OPN等蛋白表达的减少,动员前小鼠成骨细胞SDF-1 mRNA的表达水平为动员后3天的3.44±0.3倍,进而引起动员的发生。 3.G-CSF诱导的成骨细胞数量减少功能下降部分是由于动员过程中成骨细胞发生了凋亡,但成骨细胞的分化并未受到抑制,供者血清DKK1水平在动员前和动员后5天并无明显差异,13621.13±1081.99 pg/ml和10079.83±2055.82pg/ml,P=0.22。 4.共培养成骨细胞和骨髓有核细胞发现,加或不加G-CSF两组成骨细胞OCNmRNA表达情况无明显差别,P=0.69。G-CSF通过间接途径抑制成骨细胞。 5.动员后3天供者(动员前5.04±0.43 U／L,动员后3天3.45±0.37 U／L,P=0.03)及小鼠(动员前5.43±1.2 U／L,动员后3天4.04±0.86 U／L,P=0.47)血清TRAP-5b水平稍有下降,随后血清TRAP-5b水平明显上升(供者动员前为5.04±0.43 U／L,动员后5天为6.87±0.57U／L,P=0.04；小鼠动员前5.43±1.2U／L,小鼠动员后5天13.06±1.65,P=0.02)。动员过程中破骨细胞的活性逐渐增强。 结论动员过程中成骨细胞数量活性的下降导致了动员的发生,并伴随有破骨细胞的活化。 目的造血干细胞移植不仅是恶性血液病、严重免疫系统疾病及部分实体肿瘤主要甚至唯一的治愈手段,而且随着对干细胞及其细胞和组织工程研究的深入,正逐渐应用到心脑血管疾病、神经系统疾病等领域。 我们在第一部分的研究中证明了骨重塑在造血干细胞动员过程中发挥了重要的作用,在第二部分的研究中我们用6种小鼠模型模拟临床上的自体干细胞移植过程,研究应用甲状旁腺激素(PTH)或NF-KB配体的受体(RANKL)靶向于成骨细胞或破骨细胞是否能够增加干细胞数量,保护干细胞造血重建的功能。 方法利用细胞毒药物CTX处理小鼠,建立贫动员模型,进行造血干祖细胞培养,比较正常动员和贫动员小鼠模型中干细胞功能,并利用流式计数、RQ-PCR、ELISA的方法检测贫动员小鼠成骨细胞数量和功能。模拟临床自体移植过程,用PTH、RANKL干预贫动员小鼠(具体分组见材料与方法图1),利用竞争性移植模型(CRA)检测不同用药组小鼠干细胞移植16周后外周血中CD45.2阳性细胞比例。 结果 1.多次细胞毒药物化疗严重影响了成骨细胞和造血干细胞功能：自体干细胞移植患者化疗后血清OCN水平明显下降(化疗前：22.19±1.08 ng/mL和化疗后：16.08±2.12 ng/mL,P=0.01),小鼠模型中同样证明了上述观点,成骨细胞数量在多次应用细胞毒药物小鼠中明显减少,功能下降。且多次应用细胞毒严重影响了造血干祖细胞功能。CTLs/Gs组小鼠集落形成实验结果分别为21.16±1.35U,和13.00±1.71U,正常未用药小鼠为29.17±1.22U,三者相比P=0.01。 2.骨髓CRA结果显示给予G-CSF支持的小鼠干细胞功能明显下降(G组比CTL组,P=0.01)。然而,应用PTH后可显著改善该组小鼠干细胞造血重建的功能(PTH组比G组,P＜0.01;PTH组比CTL组,P＜0.05)。外周血CRA结果同样证明应用G-CSF支持治疗的G组小鼠仅有极少数周血干细胞能进行造血重建(G组比CTL组,P＜0.05)。而应用PTH的P+G组小鼠外周血干细胞造血重建的能力明显增加(P+G组比G组,P＜0.01;P+G组比CTL组,P＜0.05)。与CTL组或G组相比,P+R及P+R+G组周血干细胞同样显示出了更强的林髓系造血重建能力。RANKL同G-CSF一样可有效地动员骨髓中的造血干细胞(P+G组比P+R组,P＞0.05)因此,应用PTH和RANKL可增加小鼠模型中动员至外周血中的造血干细胞数量并且保护多次应用细胞毒药物后造血干细胞功能。 结论靶向于骨龛的药物可有效地改善多次应用细胞毒药物,尤其是联用G-CSF日寸干细胞治疗的效果。
[Abstract]:The majority of hematopoietic stem cells in the bone marrow to the niche structure of the bone tissue. Osteoblasts are the cells lining the endosteal surface, under physiological conditions and HSC after transplantation of bone marrow HSC homing to close contact with them, the anatomical locations suggest the osteoblast may regulate the function of HSC. The first point in the bone cells and HSC cultured in vitro were confirmed, then a series of studies identified almost at the same time: osteoblast is a key component in the HSC niche, at present people named the 'osteoblast niche' or '. By adjusting the endosteal niches'' niche endosteal size, we can control the number. "HSC, maintain the stable state of HSC. In this study, stem cell mobilization in osteoblasts and osteoclasts as the research object, observe the mobilization of stem cells in the process of change as the number and function of osteoclasts and mobilization, into the One step is to study the mechanism of hematopoietic stem cell mobilization.
Methods using flow cytometry to detect the mobilization of peripheral blood stem cells in mice of 0,3,5 days, the mobilization effect evaluation; through real-time quantitative PCR method to compare the mobilization of 0,3,5 days of mouse osteoblast specific gene OCN.OPN.SDF-1.SCF levels; and by immunohistochemistry and flow cytometry compared mobilization 0,3,5 days of mice and humans into different bone cells the number of function; difference method using cytochemical TRACP staining compared mobilization 0,3,5 days mouse osteoclast number and function of bone cells; detection of mice and humans into apoptosis of bone cells using the method of serial sections labeled with Caspase3; the levels of OCN. TRAP-5b protein in human and mouse cycle reaction using ELISA method into osteoclast activity; bone cells and mouse bone marrow mononuclear cells into mice were cultured to detect the osteoblast activity.
Result
1. short term application of G-CSF can lead to the mobilization of human and mouse end of reducing the number of osteoblast activity decreased diaphysis model: mobilization of mouse osteoblast OCNmRNA level for the mobilization of third days of 27 + 6 times (P < 0.01); and the third day after the mobilization of endosteal osteoblasts decrease by cubic shape deformation spindle; CD45-Ter119-OPN the number of osteoblasts in the mobilization after 3 days and 5 days was significantly reduced (before mobilization, 4085 + 135cells / femur; mobilization after 3 days, 1118 + 80 cells / femur: mobilization after 5 days, 1032 + 55cell s/femur; P=0.02); 3 days and 5 days of bone cell activity decreased mobilization OCN, ng/ml levels were 59.44 + 3.16, 39.21 + (mobilization before) 4.49 ng/ml (third days after the mobilization of 42.36 + 2.23 (ng/ml) fifth days after mobilization), P < 0.01. for further detection of normal specimens from patients with autologous transplantation and also showed that after mobilization as the number of cells with reduced bone Activity decreased. However, the proportion of LSK cells in peripheral blood did not increase significantly after mobilization of G-CSF for third days. Mobilization for 3 days and mobilization for 5 days were 0.40 0.40, 0.07%, 0.55 + 0.05% and 2.9 0.32%, respectively, so the change of osteoblasts occurred before mobilization.
2., the number of osteoblasts decreased, resulting in a decrease in protein expression of SDF-1, SCF and OPN. The expression level of SDF-1 mRNA in rat osteoblasts before mobilization was 3.44 + 0.3 times on the 3 day after mobilization, which led to mobilization.
3.G-CSF induced by reducing the number of osteoblast function decline is due in part to the mobilization process of osteoblast apoptosis, but the osteoblast differentiation was not inhibited, the level of serum DKK1 in donors before mobilization and mobilization after 5 days there was no significant difference, 13621.13 + 1081.99 and 10079.83 + pg/ml, 2055.82pg/ml, P=0.22.
4. co culture of osteoblasts and bone marrow nucleated cells revealed that there was no significant difference in OCNmRNA expression between G-CSF cells and bone marrow cells, and P=0.69.G-CSF inhibited osteoblasts indirectly through P=0.69.G-CSF.
For 3 days and 5. after mobilization (mobilization 5.04 + 0.43 U / L, 3 days after the mobilization of 3.45 + 0.37 U / L, P=0.03) and mice (5.43 mobilization + 1.2 U / L, 3 days after the mobilization of 4.04 + 0.86 U / L, P=0.47) level of serum TRAP-5b decreased slightly, then serum the level of TRAP-5b increased significantly (donors before mobilization of 5.04 + 0.43 U / L, 5 days after the mobilization of 6.87 + 0.57U / L, P=0.04; mice mobilization 5.43 + 1.2U / L mice after 5 days, the mobilization of 13.06 + 1.65, P=0.02). In the process of mobilization of osteoclast activity gradually increased.
Conclusion the decrease of the number of osteoblasts in the mobilization process leads to the mobilization and the activation of osteoclast.
The purpose of hematopoietic stem cell transplantation is not only the malignant blood disease, serious diseases and some solid tumor immune system mainly or only cure, and with the stem cells and cell and tissue engineering research, is gradually applied to the field of cardiovascular and cerebrovascular diseases, diseases of the nervous system.
We show that bone remodeling in hematopoietic stem cell mobilization plays an important role in the process of the first part of the study, in the second part of the study we used 6 mouse model to simulate clinical autologous stem cell transplantation, application of parathyroid hormone (PTH) or NF-KB ligand receptor (RANKL) targeted to whether osteoblasts or osteoclasts can increase the number of stem cells, stem cells protect hematopoietic reconstruction function.
Cytotoxic drugs in mice treated with CTX, using the method of establishment, lean mobilization model, hematopoietic stem progenitor cells, stem cell mobilization and mobilization function of normal lean mouse model, and using flow cytometry counting, RQ-PCR, ELISA to detect poor mobilization of mouse osteoblast number and function. PTH used to simulate the process of clinical transplantation RANKL, lean mice (specific mobilization intervention group see materials and methods in Figure 1), using competitive transplantation model (CRA) detection of different treatment group mice transplantation of stem cell proportion in peripheral blood of CD45.2 positive cells after 16 weeks.
Result
1. times of cytotoxic chemotherapy affect osteoblasts and hematopoietic stem cell function: autologous stem cell transplantation after chemotherapy in patients with serum OCN levels were significantly decreased (22.19 + 1.08 ng/mL before chemotherapy and after chemotherapy: 16.08 + 2.12 ng/mL, P=0.01), mouse model also proves the point, the number of osteoblasts obviously reduce the repeated application of cytotoxic drugs in mice and multiple function decline. Ofcytotoxic seriously affected the function of hematopoietic stem and progenitor cells in.CTLs/Gs mice colony formation experiment results were 21.16 + 1.35U and 13 + 1.71U, normal untreated mice was 29.17 + 1.22U, compared to three P=0.01.
2.楠ㄩ珦CRA缁撴灉鏄剧ず缁欎簣G-CSF鏀�鎸佺殑灏忛紶骞茬粏鑳炲姛鑳芥槑鏄句笅闄�(G缁勬瘮CTL缁，

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