【作者】 闫金成；

【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】 神经纤维瘤病(NF1)是常见的常染色体显性遗传性疾病,国外报道,其发病率高达1:3000。这种疾病是由于NF1基因的突变而发生的。NF1是一个肿瘤抑制基因,它编码神经纤维瘤蛋白(Neurofibromin)的表达。NF1基因全长350kb,含有60个外显子,可转录成11-13kb的mRNA,其蛋白产物为神经纤维瘤蛋白。由外显子21-27编码的约360个氨基酸为RasGTP酶激活蛋白(Ras GAP),此基因片段被称为RasGTP激活蛋白相关功能区(Ras-GRD)。Ras信号途径是一种常见的细胞分子信号传导途径,Ras是一条多肽链组成的胞浆蛋白,处于传导途径的上游,在细胞因子的作用下Ras由无活性的RasGDP转化为有活性的RasGTP,激活下游的信号传导通路,引起细胞的增殖、分化,并影响细胞的功能。Ras蛋白具有内在的GTP酶活性,能使GTP降解为GDP而失活,但其酶活性较低,而GTP酶激活蛋白(GAP)能促进GTP酶活性,使Ras蛋白水解的速度提高1万倍。神经纤维瘤蛋白有RasGTP酶激活蛋白(Ras GAP)活性,负向调控肿瘤基因Ras,将活性的RasGTP水解为无活性的RasGDP。NF1病人基因突变导致GTP结合形式的Ras聚集,造成了Ras下游的多种信号传递途径活跃,从而使得细胞增生活跃,功能亢进,引发肿瘤形成。神经纤维瘤病(NF1)是一个涉及神经、血液、骨骼等多器官的疾病,常于儿童期和青春期发病。临床统计表明,约50%的NF1患者出现骨骼疾患。最近美国和欧洲有4个研究室连续报道了NF1患者出现明显的骨量减少和骨质疏松。在NF1患者中,临床上常见的骨病是脊柱侧弯,发生率为10-70%。脊柱侧弯是脊柱的不正常弯曲,包括脊柱后凸畸形,侧凸畸形和脊柱的扭转,引起肺活动受限,疼痛等严重并发症。此外,假关节形成也是一种常见的临床表现。患儿出生后1岁左右出现胫骨弓状畸形,进行性加重,大约2岁时发生骨折,而且骨折不易骨性愈合,形成假关节,造成功能和形态异常,一些患者需要因此而截肢,这给患者带来了巨大痛苦和经济负担。然而迄今为止,NF1骨疾患的发生机制还不清楚,更无有效的预防和治疗措施。骨组织一直处于自我更新中。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。这种平衡一旦被破坏,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质。破骨细胞则来源于造血干细胞。在一定条件下,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,极化,形成有活性的破骨细胞,发挥骨吸收作用。研究表明,M-CSF和RANKL调控诱导破骨细胞的增殖和分化,对破骨细胞的生理和病理功能起着关键的作用。Yang的实验小组在小鼠体外试验中表明,单一Nf1基因缺失小鼠(Nf1+/-)出现了成骨细胞的分化降低,这种成骨细胞的分化降低与Ras的功能活跃直接相关。Karsenty小组的研究表明,单一Nf1基因缺失所致成骨细胞功能改变不足以造成体内骨的异常。最近,Yang的实验小组又报道了破骨细胞和成骨细胞同时受损的小鼠出现明显的骨量减少,这与NF1患者的骨密度显著降低的临床表现相符。NF1患者和Nf1+/-小鼠的破骨细胞具有明显的Ras活性增加,由此导致了破骨细胞功能的变化。通过以上的研究我们不难看出NF1( Nf1)基因缺失后破骨细胞功能异常在NF1( Nf1)骨异常中起着重要的作用。另有研究表明,肌动蛋白直接影响着破骨细胞的移动、骨表面附着和骨吸收。肌动蛋白是细胞骨架结构的基本组成部分,而细胞骨架结构、功能以及肌动蛋白的动态变化是由Rho基因家族调控的。Rho基因家族由Rac,Cdc42和Rho三大部分组成。普遍认为Rho基因家族在哺乳动物细胞中调节运动、附着以及变形等功能。另外,Rho基因活性化后能够激活许多控制细胞周期、基因表达、细胞存活及细胞变性的信号传导途径。Rac则被认为是该家族中的核心成分,有Rac1、Rac2和Rac3三个亚型。其中,Rac1在各种组织和细胞中普遍表达,Rac2只在造血系统表达,而Rac3则表达于神经系统。Yang等发现Rac的活性成分Rac-GTPase对于肥大细胞的移动、吸附、及脱颗粒等起着关键性的调节作用,肥大细胞和破骨细胞都来源于单核造血干细胞。由此,我们设想NF1病人破骨细胞功能的增强也与Rac活性增高有关。本实验研究了NF1病人和Nf1+/-小鼠模型中破骨细胞的功能变化以及Rac1在神经纤维瘤病破骨细胞中的作用,为临床上治疗NF1骨疾患提供可靠依据。基于上述表述,本实验研究分为三个部分。第一部分Nf1+/-小鼠破骨细胞测定目的:测定Nf1+/-小鼠破骨细胞。方法:选取同源对应基因型WT小鼠和Nf1+/-小鼠3对,取其骨髓单核细胞(BMMCs),分别行破骨细胞克隆形成试验、破骨细胞形成试验及破骨细胞功能测定试验,测定WT小鼠和Nf1+/-小鼠破骨细胞克隆形成和破骨细胞的形成、增殖、移动、骨吸收和Actin环形成并对结果行统计学分析。简言之,应用干细胞克隆测定方法测定WT小鼠和Nf1+/-小鼠破骨细胞克隆形成。在小鼠重组M-CSF和RANKL的作用下诱导小鼠骨髓单核细胞分化为多核破骨细胞,行TRAP染色测定破骨细胞的形成,通过胸腺嘧啶核苷与破骨细胞前驱细胞共同培养试验测定破骨细胞前驱细胞的增殖功能。应用24孔Transwell系统评价破骨细胞趋化因子诱导的破骨细胞前驱细胞的移动功能。骨吸收试验测定小鼠破骨细胞骨吸收功能。通过FITC结合的鬼笔环肽染色,应用荧光显微镜和Confocal扫描观察Actin环的形成。应用以上试验方法重复3次以上试验。对试验结果进行t检验,P<0.05有显著性差异。结果:Nf1+/-小鼠破骨细胞前驱细胞克隆数量是同一基因的正常WT小鼠的2倍,有显著性差异。在破骨细胞形成试验中,Nf1+/-小鼠破骨细胞形成显著高于WT小鼠。Nf1+/-小鼠破骨细胞增殖显著高于WT小鼠。Nf1+/-小鼠破骨细胞移动能力显著高于WT小鼠。Nf1+/-小鼠的骨吸收显著高于WT小鼠。Nf1+/-小鼠破骨细胞Actin环形成显著高于WT小鼠。结论: Nf1+/-小鼠破骨细胞形成增多,功能增强。第二部分Nf1+/-小鼠破骨细胞形成增加和功能增强的分子机理研究目的:NF1是一种肿瘤抑制基因,它编码神经纤维瘤蛋白(Neurofibromin)的表达。神经纤维瘤蛋白(Neurofibromin)可以激活活性化的三磷酸鸟苷(GTP酶)使之变成非活性的二磷酸鸟苷(GDP酶),负向调控肿瘤基因Ras的活性。Ras在一系列信号传导途径的上游,调控着骨髓来源细胞的功能。由于NF1基因的突变使细胞中的RasGTP聚集,造成了Ras下游的多种信号传递途径活跃,从而使得细胞增生活跃,功能亢进,Rac1是Ras下游的重要组成,调节细胞骨架的形成,活性Akt和活性Erk对破骨细胞的存活和功能起着重要的作用。本研究旨在通过Rac1敲除研究Rac1在Nf1+/-小鼠破骨细胞形成和功能中的作用以及Nf1+/-小鼠破骨细胞形成增多、功能增强的信号传导途径,研究NF1破骨细胞形成增加和功能增强的分子机理研究,为治疗NF1骨损害提供理论依据。方法:Rac1条件基因敲除小鼠(Rac1flox/flox)包含有LoxP位点外显子I,通过Cre介导敲除其等位基因获得Rac1-/-小鼠。Rac1flox/flox与MxCre转基因C57BL/6J小鼠杂交,得到Nf1+/- ;Rac1flox/floxMxcre+小鼠。3月龄时行多聚I:C(PI:PC)(Sigma,St Louis,MO) 300μg(应用灭菌PBS稀释成1ml溶液)连续3天的腹膜内注射,得到Nf1+/- ;Rac1-/-小鼠。最后一次注射后48小时内处死小鼠取骨髓细胞。实验前经聚合酶链反应PCR测定明确基因型,选取同源对应基因型小鼠3对即WT小鼠、Nf1+/-小鼠、Rac1-/-小鼠和Nf1+/- ;Rac1-/-小鼠各三只,首先,应用PAK pulldown测定法测定Rac1-GTPase和Rac2-GTPase活性。然后分别进行破骨细胞克隆形成试验和破骨细胞形成和功能试验,测定WT小鼠、Nf1+/-小鼠、Rac1-/-小鼠和Nf1+/- ;Rac1-/-小鼠破骨细胞克隆形成和破骨细胞的形成、增殖、移动、骨吸收和Actin环形成,并对结果行统计学分析。简言之,应用干细胞克隆测定方法测定WT小鼠、Nf1+/-小鼠、Rac1-/-小鼠和Nf1+/- ;Rac1-/-小鼠破骨细胞克隆形成。在小鼠重组M-CSF和RANKL的作用下诱导小鼠骨髓单核细胞分化为破骨细胞,行TRAP染色测定破骨细胞的形成,通过胸腺嘧啶核苷与破骨细胞前驱细胞共同培养试验测定破骨细胞前驱细胞的增殖功能。应用24孔Transwell系统评价破骨细胞趋化因子诱导的破骨细胞前驱细胞的移动功能。骨吸收试验测定小鼠破骨细胞骨吸收能力。通过FITC结合的鬼笔环肽染色通过荧光显微镜和Confocal观察Actin环的形成。通过应用特异性抗体行Western blot来测定活性Erk和活性Akt的水平。简言之,破骨细胞前驱细胞饥饿9小时后应用M-CSF(30ng/ml)刺激不同的时间,每一个独立的条带的颜色深浅通过NIH图像分析软件测定。应用PD98059处理培养破骨细胞,进一步测定破骨细胞功能亢进的信号传导途径。在破骨细胞培养中加入Ras-Erk信号途径中Mek的特异性抑制剂PD98059,测定破骨细胞的形成。每个试验均应用同样的试验方法进行3次以上试验,将试验结果进行t检验和方差分析,P<0.05有显著性差异。结果:Nf1+/-小鼠破骨细胞前驱细胞的Rac1-GTPase活性显著增强,将Nf1+/-小鼠Rac1基因敲除后纠正了破骨细胞前驱细胞克隆形成和破骨细胞的形成,使其回到正常水平。将Nf1+/-小鼠Rac1基因敲除后纠正了Nf1+/-小鼠破骨细胞增殖、移动、骨吸收和Actin环形成。我们比较了Nf1+/-和WT小鼠细胞中活性Akt和活性Erk。和WT相比,在Nf1+/-破骨细胞中M-CSF刺激的活性Akt和活性Erk水平显著升高。应用Ras-Erk信号传导途径中Erk上游Mek的特异性抑制剂PD98059,Nf1+/-和WT破骨细胞的形成均显著减少,这些数据说明,抑制Ras-Erk信号传导途径能够使亢进的Nf1+/-破骨细胞功能恢复正常。结论: Rac1对于Nf1+/-小鼠破骨细胞数量增多、功能亢进起着重要的作用。Rac1基因敲除后纠正了Nf1+/-破骨细胞增多和增强的功能。Nf1+/-小鼠破骨细胞增多和功能增强是通过Rac1/Erk和Rac1/Akt信号传导途径传导的。敲除Rac1能够恢复亢进Nf1+/-破骨细胞的形成和功能至正常水平。第三部分NF1病人破骨细胞数量和功能测定及其机理研究目的:研究NF1病人破骨细胞功能情况,探讨NF1病人骨损害原因方法:根据WHO的NF1诊断标准,收集32个NF1骨病患者外周血(每个患者10ml)。同时收集32个相同地区、性别及年龄的健康人作为正常对照组,取等量血液标本。从血液中分离单核细胞,在人重组M-CSF和RANKL的作用下诱导形成破骨细胞,行TRAP染色测定破骨细胞的形成。应用24孔Transwell系统评价破骨细胞趋化因子诱导的破骨细胞前驱细胞的移动功能。测定破骨细胞的附着功能。骨吸收试验测定小鼠破骨细胞骨吸收能力。通过FITC结合的鬼笔环肽染色通过荧光显微镜和Confocal观察Actin环的形成。我们应用Western blot测定NF1病人破骨细胞M-CSF刺激后活性Akt、活性Erk水平以及活性Rac1水平。应用t检验进行统计学处理,P<0.05有显著性差异。结果:在破骨细胞形成试验中,NF1破骨细胞形成显著高于正常对照组。NF1破骨细胞移动和附着能力显著高于正常对照组。NF1的骨吸收显著高于正常对照组。NF1破骨细胞Actin环形成显著高于正常对照组。与正常健康对照相比,NF1病人破骨细胞活性Akt和活性Erk水平显著增高,有显著性差异。NF1患者活性Rac1显著增高。结论: NF1病人破骨细胞前驱细胞形成增多、功能增强(包括移动功能、附着功能和骨吸收功能增强),活性Akt和活性Erk水平增强。NF1患者破骨细胞中Rac1-GTPase增加。更多还原

【Abstract】 Neurofibromatosis type 1 (NF1) is a common, autosomal-dominant disorder caused by mutations in the NF1 tumor suppressor gene. One in 3500 individuals worldwide is born with NF1. The NF1 gene is extremely large and complex. It spans 350 kilobases of the genome and there are 60 exons. There are three genes within the intron of 27b of the NF1 gene that are transcribed in the opposite orientation of the NF1 gene for which no function has been ascribed. The only region of NF1 for which a function has been ascribed is the GAP Related domains of NF1 which accelerate the hydrolysis of Ras thousands of fold.The NF1 gene encodes for the protein neurofibromin, and is expressed in a broad range of cell and tissue types. Neurofibromin negatively regulates the activity of an intracellular signaling molecule p21ras (Ras) by functioning as a GTPase activating protein (Ras-GAP). The Ras-GAP function of neurofibromin has been linked to a variety of the clinical symptoms associated with NF1. Ras is on the upstream of any singal pathway. Haploinsufficiency or a complete deficiency in NF1 results in a dose-dependent elevation in Ras activity, which in turn can activate a variety of signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway, and the phosphatidylinositol-3-phosphate kinase (PI-3K) pathway. These pathways have the capacity to affect cellular proliferation and differentiation in a cell type specific manner.NF1 is involved nervous, vascular, and skeletal system compoments and it always occur in children or adolescent. Fifty percent of NF1 patients have osseous manifestations including a high incidence of osteoporosis. Recently, in USA and Euroupe, 4 labs reported NF1 patient show osteoporosis and osteopenia. Some NF1 patients suffer scoliosis, its prevalence is 10-70%. Spine abnormal manifestation involves kyphoscoliosis scoliosis and cervical twist. Additionally, pseudarthrosis is another common manifestation in NF1 patient. When the child is 1 year old, his tibia presents bow, and about 2 year old Fracture occured, the Fracture is difficult to heal and pseudarthrosis occurs. Pseudarthrosis make function and appearance abnormal,at last some patients have to take amputation. But until now, the mechanism underlying NF1 bone defects is not clear and no effective treatment.Bone renew continuously. Old bone resorps and new bone forms. This process is the preventive maintenance of mechanical strength by continuously replacing fatigued bone by new‘‘fresh’’bone. The bone is the main reservoir of calcium ions, and the remodeling is critical for Ca ?uxes into and from the extracellular ?uid to maintain an appropriate level of blood calcium. Bone homeostasis is maintained by balancing skeletal matrix formation and resorption. Osteoclasts are specialized cells derived from the myeloid monocyte/macrophage lineage that successively adhere to bone matrix and resorb bone, while osteoblasts generate new skeletal matrix. Imbalances in the morphogenesis and remodeling of bone are known to lead to pathological perturbations of skeletal structure and function. Osteoclasts are multinucleared cells that derive from hematopoietic progenitors in the bone marrow which also give rise to monocytes in peripheral blood, and to the various types of tissue macrophages. Osteoclasts are formed by the fusion of precursor cells. The exptriment show that M-CSF and RANKL mediate ostocalst proliferation and differentiation, which play a critical role in physiological and pathological function of osteoclast.Yang’s reresults show that, in vitro, there is a decresed differentiation in osteoblast of Nf1+/- mice, and this change is directly associated with Ras activity. Karsenty found that only change of osetobalst function can not cause bone diseases. Recently, Yang reported when both abnormal in ostobalst and ostocals caused the osteoporosis, which is similar to the clinical result. NFpatient and Nf1+/-mice have Ras hyperactitive, which induced osteoclast function haperactitive. Based on these results, we presume osteoclast as a important role in NF1 skeletal lesion. The osteoclast function includes migration, adhesion and resorption, and these functions are associated with the skeletal lesion. Another study showed that acin formation influences osteoclast migration, adhesion and resorption. Acin compose cytoskeletal. Cytoskeletal formation, function are controled by Rho gene.Rho family includes Rac, Cdc42, and Rho. In general, Rho gene controls motion, adhesion, and deformation in mammal. Furthermore, Rho gene controls the signal pathway of cell cycle, expression, survival, and aptosis. Rac is regard as the core in Rho family. it have three isofome: Rac1, Rac2 and Rac3. Rac1 express in general tissues, Rac2 in haemopoiesis and Rac3 in nervous system. Yang found that Rac-GTPase control not only affects cell appearance and function, but also plays a important role in adjusting mast cell migration, adhesion and degranulation. Mast cell and osteoclast are both derived from monohaemopoietic stem cell. So, we think that ostocalst gain-in-function is associated with Rac. This study focus on the osteoclast function of NF1patient and Nf1+/- mice, the functionof Rac1, and exploring a effective treatment of NF1.This study is composed of three parts:Part one Nf1+/- mice osteoclast assayObject: to investigate Nf1+/- mice osteoclast formation and function in Nf1+/- mice.Method: the marrow mononuclear cells (BMMNCs) were taken from 3 paires gene WT and Nf1+/- mice for osteoclast colony formation, osteoclast formation, proliferation, migration, bone resorption and Actin ring formation. In the presence of M-CSF and RANKL, BMMCs was induced to osteoclast. Each experiment conducted on more than three independent occasions. Student’s t-test was used to evaluate statistical difference. P-values less than 0.05 were considered significant.Result: Nf1+/–mice showed doubled osteoclast and osteoclast progenitor number and have a significant increase in migration, proliferation, actin ring formation , bone resorption of the Nf1+/– osteoclast compared with wild type. Conclusion: Nf1+/–mice has a increased number and function of osteoclast.Part two The mechanism of the osteoclast gains-in-function in Nf1+/- mice Object: to investigate mechanism in increased Nf1+/- mice osteoclast formation and functionMethod: Rac1 conditional (Rac1?ox/?ox) and null alleles were generated by Dr Kwiatkowski as previously reported. The conditional Rac1 allele contains loxP sites ?anking exon 1; upon cre-mediated excision this allele generates a null allele. Rac1 conditional knockout mice were crossed with MxCre-transgenic C57BL/6 mice. Animals received three intraperitoneal injections at 3 months of age with 300μg poly I:-poly C (poly I:C) diluted in sterile phosphate-buffered saline (PBS). Mice were sacrificed for bone marrow cell harvest 48 h after the last injection. Take comparable 3 paires gene WT, Nf1+/-, Rac1-/-and Nf1+/-;Rac1-/-mice, take the bone marrow mononuclear cells (BMMNCs). To examine the Rac1-GTPase and Rac2 GTPase activity, Rac activation was determined using a PAK pulldown assay. Then to assay osteoclast colony formation, osteoclast formation, proliferation, migration, bone resorption and Actin ring formation. In the present of M-CSF and RANKL, BMMCs induce to osteoclasts, we utilized clonogenic assays to measure colony-forming-unit-macrophage (CFU-M) and osteoclast progenitors following tartrate resistant acid phosphatase (TRAP) staining (TRAP+ CFU-M) from freshly isolated bone marrow cells, Through Thymidine incorporation assay proliferation. Utilize Transwell assay migration and through dentine resoption assay to test bone resoption. Utilize FITC-phyloitine and confocol scan observe Actin ring formation. Erk phosphorylation and Akt phosphorylation were determined by western blot using phosphospecific antibody. Osteoclasts were deprived of serum and growth factors for 9h followed by stimulation with 30 ng/ml M-CSF for various amounts of time. Densitometry of individual bands was conducted using NIH Image software. To validate our biochemical results, PD98059, a pharmacological inhibitor of Mek within the Ras-Erk signaling pathway was utilized in the osteoclast culture and the effects on f-actin organization were examined. Each experiment conducted on more than three independent occasions. Student’s t-test and ANOVA were used to evaluate statistical difference. P-values less than 0.05 were considered significant.Result: our data showed that the Rho-GTPase Rac1 is a crucial Ras-mediated effector in Nf1 haploinsufficient (Nf1+/-) osteoclasts. Nf1+/- mice were intercrossed with conditional Rac1?ox/?oxMxcre+ (Rac1-/-) mice to generate Nf1+/-; Rac1-/- mice. Increased Rac1 activation was seen in Nf1+/- osteoclasts as compared to that in control cells, Genetic disruption of Rac1 restored the pathological increase in osteoclast progenitor cells in Nf1+/- mice and was sufficient to correct the increased Nf1+/- osteoclast function and osteoclast belt formation, an Actin structure observed in mature osteoclasts critical for bone resorption and lytic activity. The increased Akt and Erk phosphorylation correlates with the gain in proliferation, migration, and bone resorption observed in Nf1+/- osteoclasts. Hyperactivation of Akt and Erk phosphorylation in Nf1+/- osteoclast progenitors is normalized by deletion of Rac1. Upon culture with PD98059 (50 mM), the size and the number of osteoclasts observed in the Nf1+/- cultures were reduced and identical to that observed in the WT cultures.Conclusion: these data demonstrate that Rac1 critically contributes to increased osteoclast function induced by haploinsufficiency of Nf1. Genetic disruption of Rac1 restored the pathological increase in osteoclast progenitor cells in Nf1+/- mice and was sufficient to correct the increased Nf1+/- osteoclast function and osteoclast belt formation. Rac1-Erk/ Rac1-Akt pathway is the increased osteoclast function signal pathway. Hyperactivation of Erk and Akt in Nf1+/- osteoclast progenitors is normalized by deletion of Rac1 and inhibition of the Ras-Erk pathway corrects the osteoclast formation observed in Nf1+/- osteoclasts. This implicated Rac1 as a rational therapeutic target for osteoporosis. Part three NF1 patient osteoclast assayObject: to investigate NF1 patient osteoclast formation and function in NF1 patient and its mechanismMethod: According to WHO NF1 diognosis criteria, harvest 32 pairs of compared normal and NF1 person, take peripheral blood 10ml each person. Get mononuclear cells from peripheral blood (PBMNCs) after centrifugalization. Osteoclast formation and function assay, includes osteoclast formation, migration, adhension, resorption and actin ring formation. In the presence of M-CSF and RANKL, PBMNCs was induced to osteoclast. We utilized osteoclast progenitors following tartrate resistant acid phosphatase (TRAP) staining from freshly isolated PBMNCs. 24-Transwell assay was used for migration and dentine resoption assay to test bone resoption. We used FITC-phyloitine and confocol scan observe Actin ring formation. Osteoclasts were deprived of serum and growth factors for 9 hours and stimulated with 30ng/ml M-CSF. Akt and Erk phosphorylation were determined by Western blot using phospho-specific antibody for Akt and Erk. Densitometry of individual bands was conducted using NIH Image software. To examine the Rac1-GTPase activity, a PAK pulldown assay was performed.Each experiment conducted on more than three independent occasions. Student’s t-test was used to evaluate statistical difference. P-values less than 0.05 were considered significant.Result: The individuals with NF1 had increased osteoclast formation following M-CSF and RANKL stimulation as compared to control individuals. In addition, osteoclasts of individuals with NF1 had increased migration, adhension and bone resorption. The gain in bone absorption is correlated with the elevated Actin ring formation and Rac1-GTP activity. Furthermore, NF1 osteoclasts had elevated phosphorylation of Erk and Akt, consistent with elevated cellular functions.Conclusion: NF1 patient showed osteoclast formation, migration, adhension, bone resorption, actin ring formation have significant inceased compared with health person. The increased Akt and Erk phosphorylation correlates with the gain in formation, migration, actin ring formation and bone resorption observed in NF1 osteoclasts. In addition, increased Rac1 activation was also seen in NF1 osteoclasts as compared to that in health person, consistent with hypermotility and increased actin ring formation in NF1 osteoclasts. Rac1-Erk/ Rac1-Akt pathway is the increased osteoclast function signal pathway.更多还原