【作者】 李会杰；

【导师】 张英泽； 杨逢春；

【作者基本信息】 河北医科大学 ， 外科学， 2009， 博士

【摘要】 Ⅰ型神经纤维瘤病是一种常见的起源于神经嵴发育异常的常染色体基因遗传病,其发病率在1:3000。该病的发生是由于神经纤维瘤病基因(NF1)突变引起的。神经纤维瘤病基因已经被确定为一种抑癌基因,它编码的蛋白是Ras-GTP酶激动蛋白(Ras -GAP),被命名为神经纤维瘤病蛋白。神经纤维瘤病蛋白在功能上和结构上都和p120rasGAP同源。GAP蛋白能通过激活Ras活化的GTP结合形式转变成为无活性的GDP结合形式而终止Ras介导信号传导通路。因此,NF1的肿瘤抑制功能被认为主要是依赖它的下调原癌基因ras而实现的。Ras通过MEK/ERK和PI3K传导通路调节细胞的增殖,分化和生存能力。Ⅰ型神经纤维瘤病病人的特征性表现分为肿瘤症状和非肿瘤症状,非肿瘤症状包括高血压,头颅巨大,心脑血管畸形,皮肤皱褶处的间擦型雀斑,智力发育异常,精神症状和骨骼方面的异常。其骨骼病变表现常见的有脊柱侧弯,椎体发育异常,身材短小,蝶骨翼结构异常,胫骨弯曲或假关节形成,局部生长过度,胸廓发育异常,膝内翻,膝外翻,骨囊肿,骨硬化,肋骨融合,髌骨阙如,并指畸形,和一些其他骨的先天畸形,这些骨改变提示神经纤维瘤病基因的突变会引起骨代谢和骨发育的异常。最近有研究表明NF1病人存在明显的骨质疏松和骨量减少。骨组织一直处于自我更新过程中。为了适应骨力学强度及骨代谢平衡的双重需要,骨组织不断被吸收,新骨不断形成并改建。这个过程在骨修复和骨再生时就变得更为活跃。而骨吸收和骨形成是由成骨细胞和破骨细胞之间的平衡来调控的。这两种细胞的平衡一旦被破坏,就会引起骨形成异常,造成骨疾病。成骨细胞来源于间充质干细胞,它的作用是形成新的骨基质。破骨细胞则来源于造血干细胞,其直接前驱细胞又被称为巨噬细胞。在特定的环境下,骨髓中的造血干细胞首先分化成巨噬细胞克隆形成单位(CFU-M)再分化成巨噬细胞,巨噬细胞从骨髓中游离出来,进一步分化成单核破骨细胞。这些单核破骨细胞进而融合成为多核破骨细胞,附着在骨表面,发挥骨吸收作用。Yang等研究表明,Nf1+/-小鼠骨骼中破骨细胞数量明显较野生型小鼠破骨细胞增多,并通过体外培养破骨细胞证明ras通路和PI3K通路活性增强是造成Nf1小鼠破骨细胞功能增强的原因。由于Nf1基因完全敲除的小鼠会在胚胎13天左右因心脏发育异常而死亡,所以不适合用来研究神经纤维瘤病的骨表现。而对Nf1基因杂合型小鼠的研究为研究神经纤维瘤病提供了合适的模型,例如, Nf1+/-小鼠会在出生后第二年自发幼年型粒单核细胞白血病,类似人类神经纤维瘤病病人自然病程中发生恶性肿瘤。Nf1基因半缺失可以造成肥大细胞的增殖,生存能力增强,克隆形成增多,肥大细胞增多症患者有骨质疏松,肥大细胞的增多可能与神经纤维瘤病的骨量减少有关。Nf1基因杂合型上调ras活性,影响细胞功能,已在造血细胞,雪旺细胞,肥大细胞,黑素细胞中证实。骨桥蛋白(Osteopontin)是一种含有Arg-Gly-Asp (RGD)的带负电的非胶原性骨基质糖蛋白,OPN由多种组织细胞合成与分泌,广泛的分布于多种组织和细胞中,包括骨、肾、肌肉、膀胱及自然杀伤细胞。骨细胞、破骨细胞、血管内皮细胞、平滑肌细胞、多种肿瘤细胞及不同成熟阶段的成骨细胞均能合成和分泌OPN分子,OPN具有细胞黏附蛋白和细胞因子的双重作用,它通过与细胞表面的整合素(integrin)如αvβ3、αvβ1、αvβ5、α4β1、α9β1和CD44结合,通过RGD序列结合于细胞表面,从而介导细胞-细胞、细胞-基质的相互作用,传导细胞外信号。CD44作为OPN的受体,在雪旺细胞与神经突黏附、雪旺细胞凋亡及完成信号传导方面有重要作用,这也提示了OPN在Nf1的发病机制中发挥一定作用。在破骨细胞骨吸收过程中,OPN与破骨细胞膜上结合,激发αvβ3钙调素相关的钙泵从而促进破骨细胞极化。破骨细胞膜上αvβ3结合可以引起酪氨酸激酶的活化,从而使破骨细胞形成皱褶缘,促进破骨细胞的骨吸收作用。Taeko Ishii通过对关节炎模型小鼠破骨细胞的研究,发现在关节炎模型小鼠的破骨细胞培养液中OPN含量明显增多,给与OPN中和抗体可明显抑制关节炎小鼠破骨细胞的功能,并证明OPN可增加基质细胞的RANKL的分泌,从而刺激破骨细胞功能增强。本实验研究了1型神经纤维瘤病小鼠模型的破骨细胞功能变化以及骨桥蛋白在其中的作用,并为治疗提供思路。第一部分1型神经纤维瘤病基因型小鼠的破骨细胞功能研究目的:研究?型神经纤维瘤病基因型小鼠的破骨细胞功能变化,探讨?型神经纤维瘤病引起骨质疏松的发病机制。方法:选取神经纤维瘤病基因杂合型(Nf1+/-)和野生型(Nf1+/+)小鼠为研究对象,取胫骨干骺端行抗酒石酸酸性磷酸酶(TRAP)染色,比较两组小鼠骨内破骨细胞含量,并进行统计学分析。体外实验取两种基因型小鼠的骨髓单核细胞,观察在巨噬细胞集落刺激因子(M-CSF, macroPhage colony-stimulating factor)和细胞核因子κB受体活化因子配基(RANKL, receptor activator of NF-κB ligand)诱导下的破骨细胞分化能力,测定两组破骨细胞形成、分化、贴附、迁移和骨吸收功能,并进行统计学比较。结果: Nf1+/-小鼠骨内成熟破骨细胞数量比Nf1+/+增多,细胞体积明显增大, ( TRAP阳性区面积占总面积的百分比分别为Nf1+/+, 0.88%±0.0014; Nf1+/-, 2.33%±0.0013,P<0.01)有显著性差异;体外培养的Nf1+/-破骨细胞形成增多,(每1×105骨髓单核细胞形成的破骨细胞数分别为Nf1+/+,41.75±13.14 ; Nf1+/-, 61.17±18.17, P<0.01)与Nf1+/+有显著性差异;体外诱导培养的Nf1+/-破骨细胞的粘附、迁移、骨吸收功能强(粘附细胞数量Nf1+/+,53±11.08; Nf1+/-,108±11.67, P<0.01;迁移细胞数量Nf1+/+,88.33±12.40; Nf1+/-,239.83±67.77, P<0.01;骨吸收面积百分比Nf1+/+,18.37%±0.0367; Nf1+/-, 40.44,%±0.1052, P<0.01)二者有显著性差异。结论:破骨细胞形成增多,功能增强是?型神经纤维瘤病引起骨质疏松的发病机制之一。第二部分外源性骨桥蛋白对1型神经纤维瘤病小鼠破骨细胞功能影响目的:研究外源性骨桥蛋白对?型神经纤维瘤病基因型小鼠的破骨细胞功能的影响,探讨?型神经纤维瘤病破骨细胞功能增强,引起骨改变的发病机制。方法:选取神经纤维瘤病基因杂合型(Nf1+/-)和野生型(Nf1+/+)小鼠为研究对象,取小鼠的骨髓基质细胞,在一定M-CSF和RANKL的刺激下向破骨细胞分化,并给与外源性骨桥蛋白,研究测定两组破骨细胞形成、分化、黏附、迁移和骨吸收功能,并进行统计学比较。结果:经三次以上独立的实验结果统计,在骨桥蛋白的作用下Nf1+/-基因型小鼠破骨细胞的形成、分化、黏附、迁移、和骨吸收功能强于Nf1+/+基因型小鼠,破骨细胞形成(Trap+细胞计数Nf1+/+不加OPN 39.25±11.9加入OPN 47±2.34;Nf1+/-不加OPN 51.5±5.90加入OPN 70.75±0.81, p<0.05 )黏附功能(黏附细胞数Nf1+/+不加OPN 52.20±11.79,加入OPN,69.80±7.65;Nf1+/-不加OPN 80.40±13.61,加入OPN 162.50±10.52, p<0.05),迁移功能(迁移细胞计数Nf1+/+不加OPN 87.20±5.17,加入OPN 238.17±28.20 ; Nf1+/-不加OPN 223.17±29.02 ,加入OPN 582.13±51.51, p<0.05),骨吸收能力(骨吸收面积百分比Nf1+/+不加OPN 4.18%±0.0011加入OPN 17.08%±0.018;Nf1+/-不加OPN 9.11%±0.008,加入OPN 72.14%±0.083, p<0.05),二者有显著性差异。结论:骨桥蛋白诱导的破骨细胞功能增强是?型神经纤维瘤病骨改变的发病机制之一,骨桥蛋白是治疗?型神经纤维瘤病骨改变的候选靶向分子。第三部分Nf1+/-小鼠破骨细胞自分泌骨桥蛋白在其破骨细胞功能增强中的作用目的:研究体外培养的Nf1+/-小鼠破骨细胞的分泌合成骨桥蛋白的能力,应用骨桥蛋白中和抗体抑制破骨细胞分泌的骨桥蛋白,并与野生型破骨细胞比较,分析骨桥蛋白在Nf1+/-小鼠破骨细胞功能增强中的作用。方法:采取体外培养的Nf1+/-小鼠破骨细胞不同时期的上清液,做ELISA分析检测OPN浓度,并与Nf1+/+破骨细胞比较;在加入OPN中和抗体的条件下,测定体外培养的Nf1+/-小鼠破骨细胞的形成及迁移、粘附、骨吸收能力,并与Nf1+/+破骨细胞比较。结果:在体外培养的Nf1+/-小鼠破骨细胞上清液中,OPN含量比野生型小鼠破骨细胞上清液中含量明显增高,与培养3天的破骨细胞上清液相比,培养6天的破骨细胞上清液中OPN含量的差别更为明显,(Nf1+/+ 3天,54.60±6.72ng/ml;6天,80.04±11.11ng/ml;Nf1+/-3天,71.34±9.18ng/ml, 6天,120.73±3.4ng/ml p<0.05)二者有显著性差异。在体外培养破骨细胞时加入有活性的OPN中和抗体,可以抑制Nf1+/-破骨细胞的形成, (Trap+细胞数Nf1+/+无OPN抗体42.0±6.08加入OPN抗体34.67±6.23;Nf1+/-无OPN抗体72.33±5.75加入OPN抗体41.67±11.86, p<0.01)二者有显著性差异。贴附功能(贴附细胞数Nf1+/+无OPN抗体39.25±11.9加入OPN抗体27±2.34;Nf1+/-无OPN抗体51.5±5.90加入OPN抗体10.75±0.81, p<0.01)二者有显著性差异。迁移功能(迁移细胞数Nf1+/+无OPN抗体150.20±35.28加入OPN抗体114.8±24.05 ; Nf1+/-无OPN抗体241.60±37.10加入OPN抗体111.80±21.13, p<0.01)二者有显著性差异。骨吸收功能(骨吸收面积百分比Nf1+/+无OPN抗体4.18%±0.0011加入OPN抗体2.08%±0.0041;Nf1+/-无OPN抗体19.11%±0.046加入OPN抗体1.81%±0.002, p<0.01)二者有显著性差异。结论:自分泌骨桥蛋白在Nf1+/-小鼠破骨细胞功能增强过程中发挥重要作用,OPN是治疗神经纤维瘤病骨质疏松的靶向治疗候选靶分子之一。第四部分Nf1+/-小鼠成骨细胞旁分泌骨桥蛋白在破骨细胞功能增强中的作用目的:研究体外培养的Nf1+/-小鼠成骨细胞的分泌合成骨桥蛋白的能力,应用骨桥蛋白中和抗体抑制成骨细胞分泌的骨桥蛋白,并与破骨细胞共同培养,分析成骨细胞旁分泌骨桥蛋白在Nf1+/-小鼠破骨细胞功能增强中的作用。方法:采取体外培养的Nf1+/-小鼠成骨细胞的上清液,做ELISA分析检测OPN浓度,并与Nf1+/+破骨细胞比较;在加入OPN中和抗体的条件下,测定加入Nf1+/-成骨细胞条件培养液体外培养的小鼠破骨细胞的形成及迁移、粘附、骨吸收能力,并与Nf1+/+破骨细胞比较。结果:在体外培养的Nf1+/-小鼠成骨细胞条件培养液中,OPN含量比野生型小鼠成骨细胞细胞条件培养液中含量明显增高。(Nf1+/+141.09±11.31ng/ml;Nf1+/-241.08±18.63ng/ml, p<0.01)二者有显著性差异。在体外培养破骨细胞时加入成骨细胞条件培养液,可促进破骨细胞形成,功能增强,以Nf1+/-小鼠成骨细胞条件培养液作用更明显。同时加入有活性的OPN中和抗体,可以抑制Nf1+/-成骨细胞条件培养液促进破骨细胞的形成的作用,二者有显著性差异。结论:成骨细胞旁分泌骨桥蛋白在Nf1+/-小鼠破骨细胞功能增强过程中发挥重要作用,OPN是治疗神经纤维瘤病骨质疏松的靶向治疗候选靶分子之一。更多还原

【Abstract】 Neurofibromatosis type 1 (NF1) is a common, autosomal-dominant disorder caused by mutations in the NF1 tumor suppressor gene. Individuals with NF1 have a high incidence of both malignant as well as non-malignant complications. Many laboratories are pursuing concentrated efforts to understand the pathogenesis of the peripheral and central nervous system tumors that affect a wide range of NF1 patients. Despite NF1 being considered a neurocristopathy, NF1 functions as a GTPase in mesothelial derived tissues including blood cells, fibroblasts and osteoprogenitor cells that are relevant to skeletal development. However, less effort to date has been focused on understanding the molecular mechanisms underlying the pathogenesis of many of the non-malignant manifestations of NF1, particularly the skeletal manifestations that cumulatively affect up to 50% of all NF1 patients. Recent clinical studies have found that individuals with NF1 are at significant risk for both generalized and focal skeletal abnormalities. Several groups have reported that NF1 have reduced including a controlled trial using WHO criteria for osteoporosis and osteopenia, have provided evidence that NF1 patients have a significantly higher incidence of osteoporosis and osteopenia.Osteoclasts play a central role in the regulation of bone mass. Osteoclasts are terminally differentiated multinucleated giant cells with a principal bone resorptive activity. Bone resorption appears to proceed by the intricate coordination of the processes of attachment to bone, polarized secretion of acid and proteases, and active motility of osteoclasts along the bone surface. As osteoclasts crawl over bone surfaces they require rapid attachment and release from the extracellular matrix. The adhesion structures, podosomes, are uniquely designed for this purpose. After osteoclasts attach to the bone surface, podosomes develop highly polarized cytoplasmic organization in their ruffled borders. Recently, we reported a gain-of–function in the osteoclast bone resorption derived from Nf1+/- mice.Osteopontin (OPN) has been shown to be multifunctional, with activities in cell migration, cell survival, inhibition of calcification, regulation of immune cell function, and tumor cell metastasis. Increased levels of OPN protein have been linked to many disease states including atherosclerosis, transformation, tumor metastasis and renal diseases. Osteopontin (OPN) has also been reported involved in bone resorption, angiogenesis, wound healing, and tissue injuries following binding with type I collagen , fibronectin , and osteocalcin. OPN is a secreted, non-collagenous, sialic acid-rich phosphog- lycoprotein and acts both as chemokine and cytokine . Recently, OPN has been shown interacts with several integrins and CD44 variants which causes cell adhesion, migration, extracellular matrix invasion, and cell proliferation. In bone, it is predominantly expressed in osteoblasts and in terminally differentiated chondrocytes at the chondroosseous border. OPN-deficient (OPN-/-) mice are resistant to ovariectomy-induced bone resorption. Yu et al. have shown that haploinsufficiency of Nf1 enhances osteopontin expression in bone marrow-inducible osteoprogenitors and cortical bone. Kolanczyk et al. reported a significant upregulation of osteopontin in chondrocytes derived from a conditional Nf1 knockout mice, Nf1Prx1 mice. This upregulation of OPN expression may regulated by Ras. These studies imply an association between OPN and gain in functions in Nf1 haploinsufficient osteoclasts. In the present study, we proposed to investigate the role of OPN in regulating multiple cellular functions of Nf1+/- osteoclasts and related signaling axis. Our study indicates that Nf1 haploinsufficient osteoclasts are hypersensitive to OPN, which leads to increased osteoclast migration, adhesion, and bone resorption. Further, hyper phosphorylation of Ras/MAPK pathway is responsible for the gain in the osteoclast functions associated with OPN. Target of OPN may be a new treatment for the osseous manifestations in NF1 patients. PART 1Osteoclast hyperfunction in neurofibromatosis type 1 murine modelObjective: Investigate cellular functions of osteoclasts derived from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice,and analyze the mechanism of the osteoprosis in Neurofibromatosis type 1.Method: Stain histological sections of tibias from Nf1+/- and Nf1+/+ mice with H&E or tartrate-resistant acid phosphatase (TRAP) activity, and measure the osteoclasts contained in metaphysis; Culture the low density bone marrow cells from Nf1+/- and Nf1+/+ mice with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), examine the frequency of osteoclast progenitor, and evaluate multiple functions, including migration, adhesion, and bone absorption.Result: Nf1+/- mice contain numerous enlarged multinucleated osteoclasts in vivo than Nf1-/- mice.(TRAP+ area/total area Nf1+/+, 0.88%±0.0014; Nf1+/-, 2.33%±0.0013,P<0.01 ) , The number of Nf1+/- osteoclast formated from 1×105 low dencity bone marrow cells are higher than that of Nf1+/+ with significant difference. (Nf1+/+,41.75±13.14 ; Nf1+/-, 61.17±18.17, P<0.01). The mutiple functions of osteoclasts derived from Nf1 heterozygote are higher than those of wild type with significant difference(adhesion Nf1+/+,53±11.08; Nf1+/-,108±11.67, P<0.01; migration Nf1+/+,88.33±12.40; Nf1+/-,239.83±67.77, P<0.01; pit formation Nf1+/+, 18.37%±0.0367; Nf1+/-,40.44%±0.1052, P<0.01).Conclusion: The hyperfuctions of osteoclast in Nf1+/- mice may be responsible for the pathogenesis of NF1 osteoprosis.PART 2 OSTEOPONTIN PREFERENTIALLY PROMOTES MULTIPLE FUNCTIONS OF MURINE Nf1+/- OSTEOCLASTObjective: Test the osteoclast function of neurofibromatosis type 1 mice with exogenous osteopontin, analysis the mechanism of the bone change in neurofibromatosis type 1.Method: We culture the low density bone marrow cells from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice ( 4~6weeks old) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand( RANKL), with or without exogenous osteopontin,The function of osteoclast and osteoclast progenitor in formation, migration, adhesion, and bone absorption are tested. Osteoclast formation(Trap+cells Nf1+/+ without OPN 39.25±11.9with OPN 47±2.34;Nf1+/- without OPN 51.5±5.90 with OPN 70.75±0.81),adhesion(adhesiion cellsNf1+/+ without OPN 52.20±11.79, with OPN,69.80±7.65 ; Nf1+/- without OPN 80.40±13.61, with OPN 162.50±10.52), migration(migration cells Nf1+/+ without OPN 87.20±5.17, with OPN 238.17±28.20;Nf1+/- without OPN 223.17±29.02 ,with OPN 582.13±51.51),pit formation(area of bone absorptionNf1+/+ without OPN 4.18%±0.0011 with OPN 17.08%±0.018;Nf1+/- without OPN 9.11%±0.008, with OPN 72.14%±0.083)the difference is significant.Result: Given exogenous osteopontin, the osteoclast functions of Nf1 heterozygote are higher than wild type. The difference is significant.Conclusion: The hyperfunction of osteoclast in Nf1 heterozygote are related with osteopontin, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1.PART 3 AUTOCRINE OSTEOPONTIN PROMOTES MULTIPLE FUNCTIONS OF MURINE Nf1+/- OSTEOCLASTObjective: Test the osteoclast function of neurofibromatosis type 1 mice with exogenous osteopontin antibody, analysis the mechanism of the bone change in neurofibromatosis type 1. Method: Measure the OPN concentration in osteoclast culture superenant with ELISA. culture the low density bone marrow cells from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice ( 4~6weeks old ) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand( RANKL), with or without exogenous Anti-osteopontin,The function of osteoclast and osteoclast progenitor in formation, migration, adhesion, and bone absorption are tested.Result: The concentration of OPN in osteoclast culture media (Nf1+/+ 3days,54.60±6.72ng/ml ; 6day , 80.04±11.11ng/ml ; Nf1+/-3days ,71.34±9.18ng/ml, 6days,120.73±3.4ng/ml p<0.05) the difference is significant. Osteoclast formation (Trap+cells Nf1+/+ without anti-OPN antibody42.0±6.08with anti-OPN antibody ; Nf1+/- without anti-OPN antibody72.33±5.75with anti-OPN antibody 41.67±11.86), adhesion(adhesiion cellsNf1+/+ without anti-OPN antibody 39.25±11.9 with anti-OPN antibody, 27±2.34 ; Nf1+/- without anti-OPN antibody 51.5±5.90, with anti-OPN antibody 10.75±0.81,), migration(migration cellsNf1+/+ without anti-OPN antibody150.20±35.28, with anti-OPN antibody 114.8±24.05;Nf1+/- without anti-OPN antibody241.60±37.10 ,with anti-OPN antibody111.80±21.13),pit formation ( area of bone absorptionNf1+/+ without anti-OPN antibody4.18%±0.0011 with anti-OPN antibody2.08%±0.0041 ; Nf1+/- without anti-OPN antibody19.11%±0.046, with anti-OPN antibody1.81%±0.002).the difference are significant.Conclusion: The hyperfunction of osteoclast in Nf1 heterozygote are related with autocrine osteopontin, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1. PART 4 Paracrine secretion of osteopontin by Nf1+/- osteoblasts promote osteoclast activation in a neurofibromatosis type I murine modelObjective: Test the osteoclast function of neurofibromatosis type 1 mice with osteoblast conditioned media, analysis the relationship of osteoclast hyperfunction with the paracrine of osteopontin by haploinsufficient loss of Nf1 osteoblast.Method: Measure the OPN concentration in osteoblast conditioned media with ELISA. culture the low density bone marrow cells from Nf1 heterozygote (Nf1+/-) and wild type (Nf1+/+) mice (age at 4~6weeks ) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand(RANKL), with or without OBCM and exogenous Anti-osteopontin antibody,The function of osteoclast and osteoclast progenitor in formation, migration, adhesion, and bone absorption are tested.Result: The concentration of OPN in osteoblast conditioned media ((Nf1+/+141.09±11.31ng/ml;Nf1+/-241.08±18.63ng/ml,p<0.01) the difference is significant. Conditioned media from Nf1+/- osteoblasts promotes the Nf1+/- osteoclast formation. Neutralizing antibody for OPN inhibited bioactivity of Nf1+/- OBCM. the difference are significant.Conclusion: The hyperfunction of osteoclast in Nf1 heterozygote are related with paratocrine osteopontin by osteoblast, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1.更多还原