骨重塑在造血干细胞动员中的机制及应用研究

【作者】 李斯丹；

【导师】 邱录贵； 翟琼莉； 邹德慧；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【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。更多还原

【Abstract】 The majority of HSPC reside in the bone marrow surrounded by specialized bone-shielded environment. The specialized microenvironment or niche not only provides a favorable habitat for HSPC maintenance and development but also governs stem cell function. Here we investigated the potential role of bone remodeling osteoblasts and osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Herein, our results shown that1. Short term G-CSF treatment leads to decreased number and activity of endosteal and trabecular osteoblasts in mice as well as in humam beings. Mouse osteocalcin mRNA expression was sharply reduced 27±6 fold (P＜0.01) after 3 days treatment of G-CSF, and returned to normal levels on the fifth day of G-CSF cessation. The number of CD45-Ter119-OPN+osteoblast was significantly reduced (control nonmobilized,4085±135 cells/femur mobilized, d3,1118±80 cells/femur; mobilized, d5,1032±55 cells/femur;P=0.02). A significant decrease in serum osteocalcin protein was detected in G-CSF-treated mice (control nonmobilized,59.44±3.16 ng/ml; mobilized, d3,39.21±4.49 ng/ml; mobilized, d5,42.36±2.23 ng/ml;P＜0.01)2. Diminished osteoblast leads to SDF-1, SCF, and OPN falling, mouse osteoblast SDF-1 mRNA expression decline 3.44±0.3 fold (P＜0.05) on the third day of G-CSF mobilization.3. G-CSF treatment induced osteoblasts retraction is partly due to osteoblast apoptosis but not inhibits osteoblast differentiation inhibition through Wnt pathway.4. G-CSF acts through an indirect pathway to suppress osteoblasts, osteocalcin levels were 1.53±0.02 ng/mL (with G-CSF) vs 1.47±0.08 ng/mL (without G-CSF), P=0.89.5. G-CSF administration stimulates osteoclast activity, serum TRAP-5b levels showed lightly drop down on day 3 both in human (5.04±0.43 U/L on day 0 vs 3.45±0.37 U/Lon day 3, P=0.03) and mouse (5.43±1.2 U/L on day 0 vs 4.04±0.86 U/L on day 3, P=0.47) while increased gradually after that day (5.04±0.43 U/L on day 0 vs 6.87±0.57 on day 5,P=0.04) in human and (5.43±1.2 U/L on day 0 vs 13.06±1.65 on day 5, P=0.02) in mouse.In conclusion:Mobilization-induced changes in the osteoblast compartment were apparent before changes in HSCs. The loss of mature osteoblast may be the underlying reason of stem cell mobilization. Above findings indicated that bone remodeling plays an important role during HS cell mobilization. We further tested the hypothesis that targeting the niche might improve stem cell-based therapies using six mouse models to mimic the multiple rounds of chemotherapy followed by autologous HSC transplantation in a clinical setting. Herein, we show that1. Multiple rounds treatment of cytotoxic drugs influence osteoblasts and HSPCs, osteocalcin mRNA expression was reduced 9.32±0.3 fold (P＜0.01) in CTLs group and 16.82±0.8 fold (P＜0.01) in Gs group compared with untreated mouse, circulation osteocalcin decreased in CTLs group (33.81±1.99 ng/ml) and Gs group (27.18±1.09 ng/ml) compared with untreated mice (59.44±3.16 ng/ml) P＜0.01. The number of bone marrow HSPCs reduced significantly in comparison to untreated mice (29.17±1.22U, untreated vs 21.16±1.35U, CTLs vs 13.00±1.71U, Gs, P=0.01)2. Pharmacologic use of PTH or RANKL increases the number of HS cells mobilized into the peripheral blood for stem cell harvests and protects stem cells from repeated exposure to cytotoxic chemotherapy.These data provide evidence that targeting the HSC niche may enhance stem cell-based therapies.In conclusion:Cytotoxic chemotherapy markedly depletes HS cells in bone marrow. Targeting the niche can protect and expand the resident HS cell pool in the bone marrow during chemotherapy rounds, especially if co-administered with G-CSF.更多还原