ADAM10在长骨发育障碍和骨肉瘤进展中的作用
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摘要
不同类型细胞在骨骺区动态相互作用，通过软骨内骨化的方式促进长骨生长，骨骺区还是骨肉瘤好发部位。其中最常见的细胞类型为骨骺区一侧的肥大软骨细胞以及另一侧的侵入破骨细胞、成骨细胞、及血管内皮细胞。目前对于软骨细胞、成骨细胞、及破骨细胞之间的相互调节在软骨内骨化中的作用了解较多，但对血管内皮细胞在其中的作用了解甚少。我们此前发现内皮细胞敲除ADAM10会导致小鼠长骨生长缺陷，但此生长缺陷的潜在原因并不清楚，此外对于细胞表面金属蛋白酶ADAM10是否在骨肉瘤中起作用仍不明确。ADAM10是Notch信号的主要调节因子，内皮细胞缺乏ADAM10(ADAM10△EC)小鼠视网膜血管结构显示出特征性的血管分支增加，也是Notch信号缺陷所特有的现象。大多数ADAM10△EC小鼠可以存活数月，该动物模型因此提供了研究内皮细胞缺乏ADAM10如何影响长骨生长的独特机会。另一方面，近期研究表明Notch信号通路促进骨肉瘤的形成和肿瘤侵袭，提示作为Notch受体主要脱落酶的ADAM10在骨肉瘤的进展中可能通过影响肿瘤血管生成起作用。本课题旨在比较ADAM10△EC小鼠和对照小鼠不同发育阶段的长骨生长情况，重点研究骨骺区血管内皮细胞、软骨细胞、及破骨细胞可能的形态及分布异常，此外还研究了ADAM10与骨肉瘤进展及血管生成的相关性。
第一部分：ADAM10在小鼠长骨发育障碍中的作用
材料与方法
1.内皮细胞ADAM10敲除(ADAM10△EC)模型小鼠的建立及鉴定。通过将ADAM10lox/lox小鼠与表达内皮细胞特异性Tie2-Cre转基因小鼠杂交，建立ADAM10内皮细胞敲除小鼠(ADAM10△EC，基因型为ADAM10lox/loxTie2-Cre+/-)及正常对照小鼠(Control,基因型为ADAM10lox/loxTie2-Cre-/-)；提取小鼠尾部DNA，PCR法通过检测loxP及Tie2产物鉴定小鼠型别(ADAM10△EC小鼠或对照小鼠)。
2. ADAM10△EC小鼠长骨发育缺陷的动态改变。采用Faxitron X射线仪监测ADAM10△EC/对照小鼠不同发育时间点多种长骨发育缺陷的动态变化，并计算松质骨密度改变；茜素红-阿辛蓝大体骨-软骨染色确定敲除ADAM10对长骨的骨及软骨的大体影响；小鼠膝关节骨组织切片HE染色、番红精-固绿软骨染色，确定股骨及胫骨生长板区软骨代谢及软骨细胞改变，并计算内皮细胞敲除ADAM10后对生长板内软骨细胞增生的影响。
3.内皮细胞敲除ADAM10对小鼠长骨骨骺区血管发育的影响。利用小鼠内皮细胞特异性endomucin抗体行膝关节骨组织切片免疫荧光染色，对比检测内皮细胞敲除ADAM10后对股骨骨骺区血管形态的改变；通过甲基丙烯酸酯血管铸型构建股骨骨骺区血管三维模型，研究内皮细胞敲除ADAM10对小鼠长骨骨骺区血管发育的三维改变。
4. ADAM10在小鼠内皮细胞缺失后对骨骺区破骨细胞的影响及与血管异常的联系。小鼠膝关节骨组织切片TRAP染色，确定内皮细胞缺失ADAM10后骨骺区破骨细胞数目的改变；endomucin-TRAP双重染色，研究ADAM10内皮细胞敲除后血管发育异常与破骨细胞改变的相关性。
5.敲除小鼠内皮细胞ADAM10对破骨细胞分化的影响。分离ADAM10△EC/对照小鼠股骨及胫骨骨髓细胞，体外用巨噬细胞-击落刺激因子(M-CSF)分化培养为巨噬细胞，加入/不加入RANKL继续分化为破骨细胞，TRAP染色确定破骨细胞数目及形态改变。
6.小鼠内皮细胞敲除ADAM10对骨骺区RANKL/OPG信号通路的影响。分离ADAM10△EC/对照小鼠上下肢骨骺区软骨并提取RNA，实时定量逆转录PCR(real timequantitative RT-PCR)mRNA水平检测RANKL/OPG比值改变，以确定RANKL/OPG信号通路对ADAM10内皮细胞敲除引起的破骨细胞改变的影响。
结果
1. ADAM10内皮细胞敲除导致小鼠多个长骨发育障碍。Faxitron X线摄片长度测量分析显示，小鼠内皮细胞缺失ADAM10导致股骨及胫骨最早于生后7天明显短缩(P7)，其中股骨短缩随时间进展明显加重，肱骨、尺骨、桡骨于生后14天开始明显短缩，而上肢掌骨在所有监测时间点(P7-P183)均无明显短缩；骨密度分析显示ADAM10△EC小鼠股骨及胫骨生长板下骨松质密度均显著增高，分别始于P28及P21。
2. ADAM10敲除导致长骨生长板结构异常。小鼠膝关节骨组织切片HE染色显示，与对照小鼠相比ADAM10△EC小鼠股骨生长板生后7天及14天未见明显异常，生长板后方在生后21天则开始出现不连续，至生后28天生长板后方进展为局部中断，而生长板前方肥大软骨细胞层则表现为不规则增厚，此外HE染色还显示骨松质密度增加导致ADAM10△EC小鼠股骨髓腔较对照小鼠明显变窄，生后42天出现整个生长板区多个部位显著中断；ADAM10△EC小鼠胫骨生长板于生后14天开始出现不同于股骨生长板改变的中央型肥大软骨细胞层明显增厚，一直持续至监测到的生后42天，并导致生长板下骨缺损。番红精-固绿软骨染色显示生后21天ADAM10△EC小鼠股骨及胫骨生长板增生软骨细胞层开始出现软骨合成减少。茜素红-阿辛蓝全组织染色显示ADAM10△EC小鼠生后42天股骨远端骨骺线出现早闭。
3.小鼠内皮细胞中缺失ADAM10导致骨骺区血管发育紊乱。免疫荧光染色显示ADAM10△EC小鼠股骨骨骺区于生后14天出现endomucin荧光抗体显著增强，分析显示骨骺区血管密度显著增加，生后21天生长板中断处证实有血管侵入，至28天生长板被贯穿处上下侧均发现有血管侵入，胫骨骨骺区则出现襻样血管团，或于增厚的生长板下方出现血管缺失；相较ADAM10△EC小鼠而言，对照小鼠骨骺区血管分布规则、均匀，且endomucin荧光染色较浅；甲基丙烯酸酯血管铸型解剖显微镜下显示对照小鼠股骨血管为单个大的中央动脉延伸至骨骺区分支为逐渐增多的规则、平行、细小的血管环，而ADAM10△EC小鼠股骨血管铸型则显示骨骺区血管分布杂乱、交错，以及多处小的血管球样扩张。
4. ADAM10△EC小鼠长骨发育障碍由骨骺区破骨细胞数目明显减少引起。TRAP染色显示在ADAM10△EC小鼠股骨骨骺区，生后7天及14天破骨细胞数目及分布较对照小鼠无明显改变，生后21天破骨细胞数目开始出现显著减少直至28天，胫骨骨骺区破骨细胞变化与股骨类似。
5. ADAM10内皮细胞缺失引起的骨骺区破骨细胞数目减少与血管发育紊乱密切相关。Endomucin-TRAP双重染色显示，生后14天及28天股骨骨骺区TRAP+破骨细胞与endomucin+内皮细胞在空间位置上紧密联系，14天血管密度明显增加而破骨细胞数目未见明显减少；28天同样可见相似的血管密度增加，但股骨骨骺区破骨细胞数目则显著减少。
6.内皮细胞缺失ADAM10导致小鼠体外破骨细胞分化能力下降，但RANKL/OPG信号通路未受影响。体外破骨细胞细胞分化实验表明ADAM10△EC小鼠来源于股骨及胫骨的骨髓细胞破骨细胞生成较对照小鼠稍延迟，导致TRAP+破骨细胞数目出现少量但统计学显著的减少，而来源于小鼠上下肢骨骺区组织的定量PCR结果显示ADAM10△EC小鼠与对照小鼠RANKL/OPG比值无显著性差异。
第二部分：ADAM10在骨肉瘤进展中的作用
材料与方法
1.骨肉瘤样本。主要样本来源于购买的组织芯片，每张包含40例人骨肉瘤组织块(多聚甲醛处理，石蜡包埋)病理分期从IA至IIB期，组织类型包括：骨母细胞型骨肉瘤、软骨母细胞型骨肉瘤、纤维母细胞型骨肉瘤、以及富含巨细胞骨肉瘤；部分石蜡包埋组织来源于本院病理科。
2.常规HE染色。确定肿瘤组织来源、形态、及病理类型。
3.免疫荧光染色。抗CD31、抗胞内ADAM10、抗胞内段/活化Notch1(Notch1intracellular domain, NICD)，DAPI(胞核染色)，免疫荧光染色骨肉瘤组织切片，并计算CD31、NICD表达强度，血管分支数目及ADAM10阳性细胞数目与肿瘤分期及组织类型的相关性；双重免疫荧光染色(CD31/ADAM10, CD31/NICD)研究ADAM10/Notch1表达与肿瘤血管内皮细胞之间的关系。
4. TRAP染色。确定破骨细胞在骨肉瘤组织中的表达情况，并明确富含巨细胞骨肉瘤中的破骨细胞样多核巨细胞是否为破骨细胞。
结果
1. ADAM10在肿瘤细胞中的表达与骨肉瘤的进展呈正相关。在所有骨肉瘤分期及病例中均发现有ADAM10局部聚集表达于骨肉瘤细胞中，随着骨肉瘤从IA期进展为IIB期，ADAM10+肿瘤细胞数目也显著增加，IIA和IIB期的ADAM10+肿瘤细胞数目平均值较IA期显著升高；此外骨母细胞型骨肉瘤中ADAM10+肿瘤细胞也显著多于软骨母细胞型骨肉瘤及纤维母细胞型骨肉瘤。
2. IA/IB期骨肉瘤存在一类血管原性肿瘤细胞，可能通过“胞质内陷”方式形成血管管腔。HE染色发现IA/IB期骨肉瘤中存在一类嗜碱性圆形肿瘤细胞，CD31染色呈阳性，证明其为血管原性，其中部分肿瘤细胞出现不同阶段的“胞质内陷”，可能为形成血管管腔的一种机制，同时ADAM10也均一表达于这类细胞胞质中。
3.富含巨细胞骨肉瘤中的破骨细胞样多核巨细胞并非破骨细胞，而是血管原性肿瘤细胞，涉及ADAM10/Notch1信号活化。TRAP/DAPI双重染色证明富含巨细胞骨肉瘤中的多核巨细胞TRAP染色阴性，而骨母细胞型骨肉瘤、软骨母细胞型骨肉瘤及纤维母细胞型骨肉瘤中均含TRAP+肿瘤细胞。CD31以中度水平均一表达于巨细胞胞质中，ADAM10和活化的Notch1也共同表达于巨细胞中，其表达模式与CD31类似。
4.部分骨肉瘤血管结构内皮缺失。CD31染色发现IA期-IIB期部分骨肉瘤血管结构中红细胞未被血管内皮包绕，但血管内皮CD31染色阳性，证明其中并非由于血管内皮不表达CD31而是因为血管内皮缺失造成，其形成可能与多核巨细胞相关。
结论
1.生长板区血管在调节破骨细胞仅在诸如股骨、肱骨、胫骨、尺骨、以及桡骨这些较大长骨的发育后期显现出作用。
2.内皮细胞中ADAM10对于长骨中特化的血管在软骨内骨化中的正常发育及功能是必需的。
3. ADAM10△EC小鼠长骨骨骺区破骨细胞数目的减少很有可能是由ADAM10缺乏导致Notch信号异常，引起血管内皮细胞分化障碍，导致血管结构异常造成的，而不是由ADAM10缺乏导致血管内皮释放RANKL等可溶性因子缺陷造成的。
4. ADAM10在肿瘤细胞中的表达与骨肉瘤进展正相关。
5. IA/IB期骨肉瘤中存在一类血管原性肿瘤细胞，可能通过“胞质内陷”形成血管管腔，ADAM10参与其中。
6.富含巨细胞骨肉瘤中破骨细胞样多核巨细胞并非破骨细胞，而是一种血管原性细胞，其中涉及ADAM10/Notch1信号活化；该细胞可能与骨肉瘤血管内皮缺失有关。
The chondro-osseus junction (COJ) is the site of dynamic interactions between severaldifferent cell types that drive bone growth during endochondral ossification andosteosarcoma tumorigenesis and progression. The most prominent cell types at the growthplate are the hypertrophic chondrocytes on one side of the COJ, and invading osteoclastsand endothelial cells as well as osteoblasts on the other side. A considerable amount ofinformation is available on the regulation of chondrocytes, osteoclasts and osteoblasts at theCOJ during endochondral ossification, but only little is known about the role of endothelialcells in this process. We have previously identified a defect in long bone growth in micelacking the cell surface metalloprotease ADAM10(a disintegrin and metalloprotease10) inendothelial cells, raising questions about the underlying cause of this growth defect,additionally, whether ADAM10is involved in osteosarcoma is still unknown. ADAM10is aprincipal regulator of Notch signaling, and mice lacking ADAM10in endothelial cells(ADAM10△EC mice) display a characteristic increase in vascular branching in thedeveloping retinal vasculature that is a hallmark for defects in Notch signaling. MostADAM10△EC mice survive for many months, providing a unique opportunity to study howthe lack of ADAM10in endothelial cells affects the growth of long bones. On the otherhand, recent studies have shown that Notch signaling contribute to osteosarcomatumorigenesis and invasion, providing clues for the involvement of ADAM10as a majorsheddase of Notch in osteosarcoma progression through angiogenesis. This study was toexamine the long bones of ADAM10△EC mice at different times of development comparedto controls, with an emphasis on identifying possible abnormalities in the appearance anddistribution of endothelial cells, chondrocytes and osteoclasts at the COJ. In addition,ADAM10on the progression of human osteosarcoma and its angiogenesis were alsoinvestigated.
Part1. ADAM10in mouse long bone growth defectMaterials and methods
1. Establishment of ADAM10△EC mice. ADAM10△EC mice were generated bymating ADAM10lox/loxmice with transgenic mice expressing the endothelial-specificTie2-Cre transgene, to yield endothelial cell depleted ADAM10mice (ADAM10△EC,genotype as ADAM10lox/loxTie2-Cre+/-) and normal control mice (Control, genotype asADAM10lox/loxTie2-Cre-/-). To identify these mice, DNA was extracted from the tail andPCR was done to examine the amplification products of loxP and Tie2in order todistinguish whether it was from ADAM10△EC or control mice.
2. Dynamic alterations of long bones growth defect in ADAM10△EC mice. FaxitronX-ray scanner was used to monitor the dynamic alterations of long bone growth defect bycomparing ADAM10△EC mice and control mice, trabecular bone density was alsocalculated. The overall alterations of bone and cartilage of ADAM10△EC mice wereexamined by Alizarin Red&Alcian Blue whole-mount staining. The alteration of thegrowth plate and its cartilage of femurs and tibiae from tissue sections of mouse knee jointwas examined by H&E staining and Safranin O&Fast Green cartilage staining, and theeffect of the chondrocyte growth by ADAM10deletion was also calculated.
3. Vascular development at COJ affected by ADAM10deletion in endothelial cells.Mouse endothelial cell specific marker-endomucin antibody was used to detect the vascualrmorphological changes at the COJ region after deletion of ADAM10in endothelial cells byimmunofluorescent staining of the knee joint sections. In addition, methacrylate resin wasused as a casting material to establish the3D replica of the femoral vascular structure at theCOJ.
4. The effect of ADAM10depletion to osteoclast at COJ and the relationship betweenthe alteration of osteoclast and the abnormalities of the vasculature at the COJ. TRAPstaining was performed to examine the alterations of the osteoclast number at the COJ ofthe femur after deletion of ADAM10in endothelial cells, and endomucin-TRAP doublestaining was performed to elucidate the correlation between the vascular abnormality andthe osteoclast alteration.
5. The effect of ADAM10deletion in endothelial cells to osteoclastogenesis. Bonemarrow cells from the femurs and tibiae of both ADAM10△EC mice and control mice were isolated and cultured in vitro by stimulating with M-CSF to differentiate intomacrophages, which were continued stimulated with/without RANKL to differentiate intoosteoclast, as examined by TRAP staining to obtain the numerical and morphologicalalterations.
6. RANKL/OPG signaling in ADAM10△EC mice. RNA was extracted in the COJregion from the forelimbs and hindlimbs of ADAM10△EC and control mice. Real timequantitative RT-PCR was performed to detect the changes of the RANKL/OPG ratio toaddress the effect of RANKL/OPG pathway on abnormal osteoclastogenesis in ADAM10△EC mice.
Results
1. Deletion of ADAM10in mouse endothelial cells caused multiple long bone growthdefect. Faxitron data analysis revealed that ADAM10△EC femurs were slightly shorter thanthose of littermate controls at P7, and that this growth defect persisted at all time points, butwas most severe at6months of age. Measurements of the length of the tibiae by faxitronanalysis showed that there was a significant growth defect at all stages from P7to P42inADAM10△EC mice compared to littermate controls.The length of the humerus, ulna andradius were slightly, but significantly shorter in ADAM10△EC mice compared to controllittermates starting at P14and at all later stages until P42. The length of the metacarpals ofADAM10△EC mice was comparable to that of controls at all stages of development. Theincreased density of the trabecular bone in ADAM10△EC femurs was also visible onfaxitron images at P28and P42. The tibia growth plates also displayed an increase in thedensity of trabecular bone at P21and later stages of development.
2. Deletion of ADAM10led to structural abnormalities of long bone growth plate. Thehistopathological analysis of the growth plate at P7and P14did not uncover any evidentabnormalities in ADAM10△EC femurs at these early time points compared to controls. AtP21, the posterior part of the ADAM10△EC distal femur growth plate had developed adiscontinuity in three out of four samples. By P28, the discontinuity of the posterior aspectof the distal femur growth plate had progressed to a point where the entire growth plate wasbisected in ADAM10△EC mice, whereas the anterior aspects of the growth plate hadirregular enlargements of the zone of hypertrophic chondrocytes compared to controls. Inaddition, ADAM10△EC femurs showed an increase in the density of trabecular bone under the growth plate at P28, which was more pronounced towards the cortical aspects of thebone, and resulted in narrowing of the central bone marrow cavity compared to wild typecontrols. By P42, several large discontinuities spanning the entire zone of chondrocytes haddeveloped, and there was an overall increase in the density of trabecular bone. When asimilar analysis of tibiae was performed by H&E staining at different stages ofdevelopment, the growth plate in ADAM10△EC mice appeared normal at P7, but had anincreased size of the central part of the zone of hypertrophic chondrocytes starting at P14and persists until P42that was monitored, which caused subchondral bone deficiencytherein. However, unlike at the distal femur growth plate, there were no discontinuities ofthe proximal tibial growth plate. In addition, whole-mount Alizarin Red&Alcian Bluestaining revealed an early closure of the epiphyseal line of the femur of ADAM10△ECmice at P42. Safranin O&Fast Green cartilage staining also showed that the cartilagesynthesis in the proliferating zone of the femoral and tibial growth plate of ADAM10△ECmice markedly decreased starting at P21.
3. Loss of ADAM10in mouse endothelial cells led to vascular developmentabnormality at COJ. The endomucin staining at the COJ at P14was stronger in samplesfrom ADAM10△EC mice compared to controls, and that there was an increased density ofendomucin-positive cells at the COJ. A quantification of the vessel density at the COJ atP14confirmed the increase in ADAM10△EC femurs at P14compared to controls. Bloodvessel invasion was evident at the discontinuity site at P21, and this blood vessel invasionwas found both at the superior and inferior of the bisected part of the growth plate. In tibia,bulb-like vessels were found at the COJ, and loss of blood vessels was underneath theenlarged hypertrophic growth plate, while compared with ADAM10△EC mice, the bloodvessel at the COJ of control mice shows a regular and even distribution pattern and theendomucin staining was less intensed. Examination of the vascular casts of the femur undera stereo microscope showed a large central artery with increased branching towards thegrowth plates that ended in regularly distributed fine vascular loop structures in controlmice. By comparison, the vascular casts from ADAM10△EC mice showed that the largecentral artery branched out in vascular loops that were less parallel and less well organizedand had numerous small bulb-like expansions.
4. Long bone growth defect of ADAM10△EC mice was caused by the decrease of the osteoclast number at COJ. We found similar numbers and distribution of TRAP stainedcells in the growth plates of ADAM10△EC femurs compared to controls at P7and P14.However, at P21, the distal femoral growth plate in ADAM10△EC mice had a significantreduction in the number of TRAP-stained cells at the COJ compared to controls, and thisdifference persisted at P28. Similar observations were made in an analysis of the proximalgrowth plate of the tibia, where the number of TRAP+cells was comparable at P7and P14in ADAM10△EC mice and controls, but was significantly reduced in ADAM10△EC micefrom P21onward.
5. The decrease of osteoclast number at COJ caused by ADAM10deletion inendothelial cells colsely related with disorganized vascular development at COJ.Co-staining of these samples for TRAP followed by merging of the images revealed a tightassociation of TRAP+cells with endothelial cells at the COJ at P14in ADAM10△EC miceand controls. At P28, there was a similar increase in the endomucin-positive cells at theCOJ in femurs from ADAM10△EC mice, but very few TRAP+cells could be detected atthe COJ.
6. Loss of ADAM10in endothelial cells led to a decrease of osteoclastogenesis in vitro,while the RANKL/OPG pathway was not affected. When we cultured osteoclast precursorsfrom ADAM10△EC mice, we found that the osteoclastogenesis was slightly delayedcompared to the control mice, resulting in a small but significant reduction in the number ofTRAP+multinucleated osteoclasts. However, there was no significant difference in theRANKL/OPG ratio as determined by qPCR in samples extracted from the zone ofhypertrophic and terminally differentiation/COJ zones in the hindlimbs and forelimbs ofADAM10△EC or control mice.
Part2. ADAM10in osteosarcoma progressionMaterials and methods
1. Human osteosarcoma samples. Main samples were purchased osteosarcoma tissuechip slides, each includes40cases of human osteosarcoma tissue spots treated with PFAand embedded in paraffin. The pathological stages were from IA to IIB and the histologicaldiagnosis include ostoeblastic osteosarcoma, chondroblastic osteosarcoma, fibroblasticosteosarcoma, and giant cell rich osteosarcoma. Some samples were from the Departmentof Pathology of our Hospital.
2. H&E staining. To address the origin, histological morphology, and pathologicaltypes of osteosarcoma tissues.
3. Immunofluorescent staining. Osteosarcoma tissue sections were stained withanti-CD31, anti-cytoplasmic ADAM10, anti-activated Notch1(NICD, Notch1intracellulardomain) antibodies，and DAPI (to stain the neuclei), the expression density of CD31andNICD and the ADAM10+tumor cells were calculated and correlated with tumor stagingand histological types. In addition, CD31/ADAM10, CD31/NICD doubleimmunofluorescent stainings were done to investigate the correlation of ADAM10/Notch1with osteosarcoma vascular endothelial cells.
4. TRAP staining. To investigate the expression of osteoclast in osteosarcoma tissuesand address whether osteoclast-like multinucleated giant cell is an authentic osteoclast.
Results
1. ADAM10expression positively correlated with osteosarcoma progression. Apolarized and condensed expression pattern of cytoplasmic ADAM10was observed in allcases and stages of osteosarcoma. ADM10+tumor cells increase dramatically asosteosarcoma advances from IA to IIB, the average ADAM10+tumor cell numbers of stageIIA and IIB were significantly higher than that of stage IA. In addition, the number ofADAM10+tumor cells was significantly larger in osteoblastic osteosarcoma than that inchondroblastic and fibroblastic osteosarcoma.
2. A cluster of angiogenic tumor cells were identified in stage IA/IB osteosarcoma,which might form vascular lumens through “cytoplasmic invagination”. H&E stainingidentified a cluster of basophilic round tumor cells, its positivity for CD31staining suggestsits angiogenic property, some of these cells underwent different stages of “cytoplasmicinvagination”, which might be a mechanism utilized by osteosarcoma to form vascularlumen. Additionally, cytoplasmic ADAM10was homogeneously expressed in theseangiogenic tumor cells.
3. Osteoclast-like multinucleated giant cell is not an osteoclast but an angiogenictumor cell, where ADAM10/Notch1signaling was activated. TRAP/DAPI double stainingshows that multinucleated giant cell was negative for TRAP staining in giant cell-richosteosarcoma, while osteoblastic, chondroblastic, and fibroblastic osteosarcoma all hadTRAP+tumor cells respectively. CD31was expressed at intermediate level in the cytoplasm of ginat cells, ADAM10and activated Notch1were also expressed in giant cellsin a similar pattern as CD31.
4. The vasculature of partial osteosarcoma had endothelium deficiency. CD31stainingrevealed red blood cells in some osteosarcoma from stage IA to IIB osteosarcomavasculature were not surrounded by endothelium, which was stained positive for CD31anddisproved the reason as CD31was not expressed in endothelium of osteosarcoma but ratheras endothelium deficiency, which might be related with multinucleated giant cells.
Conclusions
1. The role of bone vessels at the growth plate in regulating the function of osteoclastsonly manifests itself in the large long bones such as the femur, humerus, tibia, ulna andradius at the later stages of their growth.
2. ADAM10in endothelial cells is required for the proper development and function ofthe specialized vasculature in the bone during the process of endochondral ossification.
3. The reduced number of osteoclasts at the COJ in joints of long bones of ADAM10△EC mice are likely the consequence of alterations in Notch-dependent differentiation ofendothelial cells in the absence of ADAM10, instead of defects in release of a soluble factorfrom endothelial cells by ADAM10.
4. The expression of ADAM10in tumor cells positively correlates with osteosarcomaprogression.
5. ADAM10participated in the cytoplasmic invagination of angiogenic tumor cells ofstage IA/IB osteosarcoma, which might contribute to vascular lumen formation.
6. ADAM10/Notch1signaling was activated in multinucleated giant cell, which wasnot osteoclast, but rather was angiogenic and might be related with endothelium deficiencyin partial osteosarcoma vasculature.
第一部分：ADAM10在小鼠长骨发育障碍中的作用
材料与方法
1.内皮细胞ADAM10敲除(ADAM10△EC)模型小鼠的建立及鉴定。通过将ADAM10lox/lox小鼠与表达内皮细胞特异性Tie2-Cre转基因小鼠杂交，建立ADAM10内皮细胞敲除小鼠(ADAM10△EC，基因型为ADAM10lox/loxTie2-Cre+/-)及正常对照小鼠(Control,基因型为ADAM10lox/loxTie2-Cre-/-)；提取小鼠尾部DNA，PCR法通过检测loxP及Tie2产物鉴定小鼠型别(ADAM10△EC小鼠或对照小鼠)。
2. ADAM10△EC小鼠长骨发育缺陷的动态改变。采用Faxitron X射线仪监测ADAM10△EC/对照小鼠不同发育时间点多种长骨发育缺陷的动态变化，并计算松质骨密度改变；茜素红-阿辛蓝大体骨-软骨染色确定敲除ADAM10对长骨的骨及软骨的大体影响；小鼠膝关节骨组织切片HE染色、番红精-固绿软骨染色，确定股骨及胫骨生长板区软骨代谢及软骨细胞改变，并计算内皮细胞敲除ADAM10后对生长板内软骨细胞增生的影响。
3.内皮细胞敲除ADAM10对小鼠长骨骨骺区血管发育的影响。利用小鼠内皮细胞特异性endomucin抗体行膝关节骨组织切片免疫荧光染色，对比检测内皮细胞敲除ADAM10后对股骨骨骺区血管形态的改变；通过甲基丙烯酸酯血管铸型构建股骨骨骺区血管三维模型，研究内皮细胞敲除ADAM10对小鼠长骨骨骺区血管发育的三维改变。
4. ADAM10在小鼠内皮细胞缺失后对骨骺区破骨细胞的影响及与血管异常的联系。小鼠膝关节骨组织切片TRAP染色，确定内皮细胞缺失ADAM10后骨骺区破骨细胞数目的改变；endomucin-TRAP双重染色，研究ADAM10内皮细胞敲除后血管发育异常与破骨细胞改变的相关性。
5.敲除小鼠内皮细胞ADAM10对破骨细胞分化的影响。分离ADAM10△EC/对照小鼠股骨及胫骨骨髓细胞，体外用巨噬细胞-击落刺激因子(M-CSF)分化培养为巨噬细胞，加入/不加入RANKL继续分化为破骨细胞，TRAP染色确定破骨细胞数目及形态改变。
6.小鼠内皮细胞敲除ADAM10对骨骺区RANKL/OPG信号通路的影响。分离ADAM10△EC/对照小鼠上下肢骨骺区软骨并提取RNA，实时定量逆转录PCR(real timequantitative RT-PCR)mRNA水平检测RANKL/OPG比值改变，以确定RANKL/OPG信号通路对ADAM10内皮细胞敲除引起的破骨细胞改变的影响。
结果
1. ADAM10内皮细胞敲除导致小鼠多个长骨发育障碍。Faxitron X线摄片长度测量分析显示，小鼠内皮细胞缺失ADAM10导致股骨及胫骨最早于生后7天明显短缩(P7)，其中股骨短缩随时间进展明显加重，肱骨、尺骨、桡骨于生后14天开始明显短缩，而上肢掌骨在所有监测时间点(P7-P183)均无明显短缩；骨密度分析显示ADAM10△EC小鼠股骨及胫骨生长板下骨松质密度均显著增高，分别始于P28及P21。
2. ADAM10敲除导致长骨生长板结构异常。小鼠膝关节骨组织切片HE染色显示，与对照小鼠相比ADAM10△EC小鼠股骨生长板生后7天及14天未见明显异常，生长板后方在生后21天则开始出现不连续，至生后28天生长板后方进展为局部中断，而生长板前方肥大软骨细胞层则表现为不规则增厚，此外HE染色还显示骨松质密度增加导致ADAM10△EC小鼠股骨髓腔较对照小鼠明显变窄，生后42天出现整个生长板区多个部位显著中断；ADAM10△EC小鼠胫骨生长板于生后14天开始出现不同于股骨生长板改变的中央型肥大软骨细胞层明显增厚，一直持续至监测到的生后42天，并导致生长板下骨缺损。番红精-固绿软骨染色显示生后21天ADAM10△EC小鼠股骨及胫骨生长板增生软骨细胞层开始出现软骨合成减少。茜素红-阿辛蓝全组织染色显示ADAM10△EC小鼠生后42天股骨远端骨骺线出现早闭。
3.小鼠内皮细胞中缺失ADAM10导致骨骺区血管发育紊乱。免疫荧光染色显示ADAM10△EC小鼠股骨骨骺区于生后14天出现endomucin荧光抗体显著增强，分析显示骨骺区血管密度显著增加，生后21天生长板中断处证实有血管侵入，至28天生长板被贯穿处上下侧均发现有血管侵入，胫骨骨骺区则出现襻样血管团，或于增厚的生长板下方出现血管缺失；相较ADAM10△EC小鼠而言，对照小鼠骨骺区血管分布规则、均匀，且endomucin荧光染色较浅；甲基丙烯酸酯血管铸型解剖显微镜下显示对照小鼠股骨血管为单个大的中央动脉延伸至骨骺区分支为逐渐增多的规则、平行、细小的血管环，而ADAM10△EC小鼠股骨血管铸型则显示骨骺区血管分布杂乱、交错，以及多处小的血管球样扩张。
4. ADAM10△EC小鼠长骨发育障碍由骨骺区破骨细胞数目明显减少引起。TRAP染色显示在ADAM10△EC小鼠股骨骨骺区，生后7天及14天破骨细胞数目及分布较对照小鼠无明显改变，生后21天破骨细胞数目开始出现显著减少直至28天，胫骨骨骺区破骨细胞变化与股骨类似。
5. ADAM10内皮细胞缺失引起的骨骺区破骨细胞数目减少与血管发育紊乱密切相关。Endomucin-TRAP双重染色显示，生后14天及28天股骨骨骺区TRAP+破骨细胞与endomucin+内皮细胞在空间位置上紧密联系，14天血管密度明显增加而破骨细胞数目未见明显减少；28天同样可见相似的血管密度增加，但股骨骨骺区破骨细胞数目则显著减少。
6.内皮细胞缺失ADAM10导致小鼠体外破骨细胞分化能力下降，但RANKL/OPG信号通路未受影响。体外破骨细胞细胞分化实验表明ADAM10△EC小鼠来源于股骨及胫骨的骨髓细胞破骨细胞生成较对照小鼠稍延迟，导致TRAP+破骨细胞数目出现少量但统计学显著的减少，而来源于小鼠上下肢骨骺区组织的定量PCR结果显示ADAM10△EC小鼠与对照小鼠RANKL/OPG比值无显著性差异。
第二部分：ADAM10在骨肉瘤进展中的作用
材料与方法
1.骨肉瘤样本。主要样本来源于购买的组织芯片，每张包含40例人骨肉瘤组织块(多聚甲醛处理，石蜡包埋)病理分期从IA至IIB期，组织类型包括：骨母细胞型骨肉瘤、软骨母细胞型骨肉瘤、纤维母细胞型骨肉瘤、以及富含巨细胞骨肉瘤；部分石蜡包埋组织来源于本院病理科。
2.常规HE染色。确定肿瘤组织来源、形态、及病理类型。
3.免疫荧光染色。抗CD31、抗胞内ADAM10、抗胞内段/活化Notch1(Notch1intracellular domain, NICD)，DAPI(胞核染色)，免疫荧光染色骨肉瘤组织切片，并计算CD31、NICD表达强度，血管分支数目及ADAM10阳性细胞数目与肿瘤分期及组织类型的相关性；双重免疫荧光染色(CD31/ADAM10, CD31/NICD)研究ADAM10/Notch1表达与肿瘤血管内皮细胞之间的关系。
4. TRAP染色。确定破骨细胞在骨肉瘤组织中的表达情况，并明确富含巨细胞骨肉瘤中的破骨细胞样多核巨细胞是否为破骨细胞。
结果
1. ADAM10在肿瘤细胞中的表达与骨肉瘤的进展呈正相关。在所有骨肉瘤分期及病例中均发现有ADAM10局部聚集表达于骨肉瘤细胞中，随着骨肉瘤从IA期进展为IIB期，ADAM10+肿瘤细胞数目也显著增加，IIA和IIB期的ADAM10+肿瘤细胞数目平均值较IA期显著升高；此外骨母细胞型骨肉瘤中ADAM10+肿瘤细胞也显著多于软骨母细胞型骨肉瘤及纤维母细胞型骨肉瘤。
2. IA/IB期骨肉瘤存在一类血管原性肿瘤细胞，可能通过“胞质内陷”方式形成血管管腔。HE染色发现IA/IB期骨肉瘤中存在一类嗜碱性圆形肿瘤细胞，CD31染色呈阳性，证明其为血管原性，其中部分肿瘤细胞出现不同阶段的“胞质内陷”，可能为形成血管管腔的一种机制，同时ADAM10也均一表达于这类细胞胞质中。
3.富含巨细胞骨肉瘤中的破骨细胞样多核巨细胞并非破骨细胞，而是血管原性肿瘤细胞，涉及ADAM10/Notch1信号活化。TRAP/DAPI双重染色证明富含巨细胞骨肉瘤中的多核巨细胞TRAP染色阴性，而骨母细胞型骨肉瘤、软骨母细胞型骨肉瘤及纤维母细胞型骨肉瘤中均含TRAP+肿瘤细胞。CD31以中度水平均一表达于巨细胞胞质中，ADAM10和活化的Notch1也共同表达于巨细胞中，其表达模式与CD31类似。
4.部分骨肉瘤血管结构内皮缺失。CD31染色发现IA期-IIB期部分骨肉瘤血管结构中红细胞未被血管内皮包绕，但血管内皮CD31染色阳性，证明其中并非由于血管内皮不表达CD31而是因为血管内皮缺失造成，其形成可能与多核巨细胞相关。
结论
1.生长板区血管在调节破骨细胞仅在诸如股骨、肱骨、胫骨、尺骨、以及桡骨这些较大长骨的发育后期显现出作用。
2.内皮细胞中ADAM10对于长骨中特化的血管在软骨内骨化中的正常发育及功能是必需的。
3. ADAM10△EC小鼠长骨骨骺区破骨细胞数目的减少很有可能是由ADAM10缺乏导致Notch信号异常，引起血管内皮细胞分化障碍，导致血管结构异常造成的，而不是由ADAM10缺乏导致血管内皮释放RANKL等可溶性因子缺陷造成的。
4. ADAM10在肿瘤细胞中的表达与骨肉瘤进展正相关。
5. IA/IB期骨肉瘤中存在一类血管原性肿瘤细胞，可能通过“胞质内陷”形成血管管腔，ADAM10参与其中。
6.富含巨细胞骨肉瘤中破骨细胞样多核巨细胞并非破骨细胞，而是一种血管原性细胞，其中涉及ADAM10/Notch1信号活化；该细胞可能与骨肉瘤血管内皮缺失有关。
The chondro-osseus junction (COJ) is the site of dynamic interactions between severaldifferent cell types that drive bone growth during endochondral ossification andosteosarcoma tumorigenesis and progression. The most prominent cell types at the growthplate are the hypertrophic chondrocytes on one side of the COJ, and invading osteoclastsand endothelial cells as well as osteoblasts on the other side. A considerable amount ofinformation is available on the regulation of chondrocytes, osteoclasts and osteoblasts at theCOJ during endochondral ossification, but only little is known about the role of endothelialcells in this process. We have previously identified a defect in long bone growth in micelacking the cell surface metalloprotease ADAM10(a disintegrin and metalloprotease10) inendothelial cells, raising questions about the underlying cause of this growth defect,additionally, whether ADAM10is involved in osteosarcoma is still unknown. ADAM10is aprincipal regulator of Notch signaling, and mice lacking ADAM10in endothelial cells(ADAM10△EC mice) display a characteristic increase in vascular branching in thedeveloping retinal vasculature that is a hallmark for defects in Notch signaling. MostADAM10△EC mice survive for many months, providing a unique opportunity to study howthe lack of ADAM10in endothelial cells affects the growth of long bones. On the otherhand, recent studies have shown that Notch signaling contribute to osteosarcomatumorigenesis and invasion, providing clues for the involvement of ADAM10as a majorsheddase of Notch in osteosarcoma progression through angiogenesis. This study was toexamine the long bones of ADAM10△EC mice at different times of development comparedto controls, with an emphasis on identifying possible abnormalities in the appearance anddistribution of endothelial cells, chondrocytes and osteoclasts at the COJ. In addition,ADAM10on the progression of human osteosarcoma and its angiogenesis were alsoinvestigated.
Part1. ADAM10in mouse long bone growth defectMaterials and methods
1. Establishment of ADAM10△EC mice. ADAM10△EC mice were generated bymating ADAM10lox/loxmice with transgenic mice expressing the endothelial-specificTie2-Cre transgene, to yield endothelial cell depleted ADAM10mice (ADAM10△EC,genotype as ADAM10lox/loxTie2-Cre+/-) and normal control mice (Control, genotype asADAM10lox/loxTie2-Cre-/-). To identify these mice, DNA was extracted from the tail andPCR was done to examine the amplification products of loxP and Tie2in order todistinguish whether it was from ADAM10△EC or control mice.
2. Dynamic alterations of long bones growth defect in ADAM10△EC mice. FaxitronX-ray scanner was used to monitor the dynamic alterations of long bone growth defect bycomparing ADAM10△EC mice and control mice, trabecular bone density was alsocalculated. The overall alterations of bone and cartilage of ADAM10△EC mice wereexamined by Alizarin Red&Alcian Blue whole-mount staining. The alteration of thegrowth plate and its cartilage of femurs and tibiae from tissue sections of mouse knee jointwas examined by H&E staining and Safranin O&Fast Green cartilage staining, and theeffect of the chondrocyte growth by ADAM10deletion was also calculated.
3. Vascular development at COJ affected by ADAM10deletion in endothelial cells.Mouse endothelial cell specific marker-endomucin antibody was used to detect the vascualrmorphological changes at the COJ region after deletion of ADAM10in endothelial cells byimmunofluorescent staining of the knee joint sections. In addition, methacrylate resin wasused as a casting material to establish the3D replica of the femoral vascular structure at theCOJ.
4. The effect of ADAM10depletion to osteoclast at COJ and the relationship betweenthe alteration of osteoclast and the abnormalities of the vasculature at the COJ. TRAPstaining was performed to examine the alterations of the osteoclast number at the COJ ofthe femur after deletion of ADAM10in endothelial cells, and endomucin-TRAP doublestaining was performed to elucidate the correlation between the vascular abnormality andthe osteoclast alteration.
5. The effect of ADAM10deletion in endothelial cells to osteoclastogenesis. Bonemarrow cells from the femurs and tibiae of both ADAM10△EC mice and control mice were isolated and cultured in vitro by stimulating with M-CSF to differentiate intomacrophages, which were continued stimulated with/without RANKL to differentiate intoosteoclast, as examined by TRAP staining to obtain the numerical and morphologicalalterations.
6. RANKL/OPG signaling in ADAM10△EC mice. RNA was extracted in the COJregion from the forelimbs and hindlimbs of ADAM10△EC and control mice. Real timequantitative RT-PCR was performed to detect the changes of the RANKL/OPG ratio toaddress the effect of RANKL/OPG pathway on abnormal osteoclastogenesis in ADAM10△EC mice.
Results
1. Deletion of ADAM10in mouse endothelial cells caused multiple long bone growthdefect. Faxitron data analysis revealed that ADAM10△EC femurs were slightly shorter thanthose of littermate controls at P7, and that this growth defect persisted at all time points, butwas most severe at6months of age. Measurements of the length of the tibiae by faxitronanalysis showed that there was a significant growth defect at all stages from P7to P42inADAM10△EC mice compared to littermate controls.The length of the humerus, ulna andradius were slightly, but significantly shorter in ADAM10△EC mice compared to controllittermates starting at P14and at all later stages until P42. The length of the metacarpals ofADAM10△EC mice was comparable to that of controls at all stages of development. Theincreased density of the trabecular bone in ADAM10△EC femurs was also visible onfaxitron images at P28and P42. The tibia growth plates also displayed an increase in thedensity of trabecular bone at P21and later stages of development.
2. Deletion of ADAM10led to structural abnormalities of long bone growth plate. Thehistopathological analysis of the growth plate at P7and P14did not uncover any evidentabnormalities in ADAM10△EC femurs at these early time points compared to controls. AtP21, the posterior part of the ADAM10△EC distal femur growth plate had developed adiscontinuity in three out of four samples. By P28, the discontinuity of the posterior aspectof the distal femur growth plate had progressed to a point where the entire growth plate wasbisected in ADAM10△EC mice, whereas the anterior aspects of the growth plate hadirregular enlargements of the zone of hypertrophic chondrocytes compared to controls. Inaddition, ADAM10△EC femurs showed an increase in the density of trabecular bone under the growth plate at P28, which was more pronounced towards the cortical aspects of thebone, and resulted in narrowing of the central bone marrow cavity compared to wild typecontrols. By P42, several large discontinuities spanning the entire zone of chondrocytes haddeveloped, and there was an overall increase in the density of trabecular bone. When asimilar analysis of tibiae was performed by H&E staining at different stages ofdevelopment, the growth plate in ADAM10△EC mice appeared normal at P7, but had anincreased size of the central part of the zone of hypertrophic chondrocytes starting at P14and persists until P42that was monitored, which caused subchondral bone deficiencytherein. However, unlike at the distal femur growth plate, there were no discontinuities ofthe proximal tibial growth plate. In addition, whole-mount Alizarin Red&Alcian Bluestaining revealed an early closure of the epiphyseal line of the femur of ADAM10△ECmice at P42. Safranin O&Fast Green cartilage staining also showed that the cartilagesynthesis in the proliferating zone of the femoral and tibial growth plate of ADAM10△ECmice markedly decreased starting at P21.
3. Loss of ADAM10in mouse endothelial cells led to vascular developmentabnormality at COJ. The endomucin staining at the COJ at P14was stronger in samplesfrom ADAM10△EC mice compared to controls, and that there was an increased density ofendomucin-positive cells at the COJ. A quantification of the vessel density at the COJ atP14confirmed the increase in ADAM10△EC femurs at P14compared to controls. Bloodvessel invasion was evident at the discontinuity site at P21, and this blood vessel invasionwas found both at the superior and inferior of the bisected part of the growth plate. In tibia,bulb-like vessels were found at the COJ, and loss of blood vessels was underneath theenlarged hypertrophic growth plate, while compared with ADAM10△EC mice, the bloodvessel at the COJ of control mice shows a regular and even distribution pattern and theendomucin staining was less intensed. Examination of the vascular casts of the femur undera stereo microscope showed a large central artery with increased branching towards thegrowth plates that ended in regularly distributed fine vascular loop structures in controlmice. By comparison, the vascular casts from ADAM10△EC mice showed that the largecentral artery branched out in vascular loops that were less parallel and less well organizedand had numerous small bulb-like expansions.
4. Long bone growth defect of ADAM10△EC mice was caused by the decrease of the osteoclast number at COJ. We found similar numbers and distribution of TRAP stainedcells in the growth plates of ADAM10△EC femurs compared to controls at P7and P14.However, at P21, the distal femoral growth plate in ADAM10△EC mice had a significantreduction in the number of TRAP-stained cells at the COJ compared to controls, and thisdifference persisted at P28. Similar observations were made in an analysis of the proximalgrowth plate of the tibia, where the number of TRAP+cells was comparable at P7and P14in ADAM10△EC mice and controls, but was significantly reduced in ADAM10△EC micefrom P21onward.
5. The decrease of osteoclast number at COJ caused by ADAM10deletion inendothelial cells colsely related with disorganized vascular development at COJ.Co-staining of these samples for TRAP followed by merging of the images revealed a tightassociation of TRAP+cells with endothelial cells at the COJ at P14in ADAM10△EC miceand controls. At P28, there was a similar increase in the endomucin-positive cells at theCOJ in femurs from ADAM10△EC mice, but very few TRAP+cells could be detected atthe COJ.
6. Loss of ADAM10in endothelial cells led to a decrease of osteoclastogenesis in vitro,while the RANKL/OPG pathway was not affected. When we cultured osteoclast precursorsfrom ADAM10△EC mice, we found that the osteoclastogenesis was slightly delayedcompared to the control mice, resulting in a small but significant reduction in the number ofTRAP+multinucleated osteoclasts. However, there was no significant difference in theRANKL/OPG ratio as determined by qPCR in samples extracted from the zone ofhypertrophic and terminally differentiation/COJ zones in the hindlimbs and forelimbs ofADAM10△EC or control mice.
Part2. ADAM10in osteosarcoma progressionMaterials and methods
1. Human osteosarcoma samples. Main samples were purchased osteosarcoma tissuechip slides, each includes40cases of human osteosarcoma tissue spots treated with PFAand embedded in paraffin. The pathological stages were from IA to IIB and the histologicaldiagnosis include ostoeblastic osteosarcoma, chondroblastic osteosarcoma, fibroblasticosteosarcoma, and giant cell rich osteosarcoma. Some samples were from the Departmentof Pathology of our Hospital.
2. H&E staining. To address the origin, histological morphology, and pathologicaltypes of osteosarcoma tissues.
3. Immunofluorescent staining. Osteosarcoma tissue sections were stained withanti-CD31, anti-cytoplasmic ADAM10, anti-activated Notch1(NICD, Notch1intracellulardomain) antibodies，and DAPI (to stain the neuclei), the expression density of CD31andNICD and the ADAM10+tumor cells were calculated and correlated with tumor stagingand histological types. In addition, CD31/ADAM10, CD31/NICD doubleimmunofluorescent stainings were done to investigate the correlation of ADAM10/Notch1with osteosarcoma vascular endothelial cells.
4. TRAP staining. To investigate the expression of osteoclast in osteosarcoma tissuesand address whether osteoclast-like multinucleated giant cell is an authentic osteoclast.
Results
1. ADAM10expression positively correlated with osteosarcoma progression. Apolarized and condensed expression pattern of cytoplasmic ADAM10was observed in allcases and stages of osteosarcoma. ADM10+tumor cells increase dramatically asosteosarcoma advances from IA to IIB, the average ADAM10+tumor cell numbers of stageIIA and IIB were significantly higher than that of stage IA. In addition, the number ofADAM10+tumor cells was significantly larger in osteoblastic osteosarcoma than that inchondroblastic and fibroblastic osteosarcoma.
2. A cluster of angiogenic tumor cells were identified in stage IA/IB osteosarcoma,which might form vascular lumens through “cytoplasmic invagination”. H&E stainingidentified a cluster of basophilic round tumor cells, its positivity for CD31staining suggestsits angiogenic property, some of these cells underwent different stages of “cytoplasmicinvagination”, which might be a mechanism utilized by osteosarcoma to form vascularlumen. Additionally, cytoplasmic ADAM10was homogeneously expressed in theseangiogenic tumor cells.
3. Osteoclast-like multinucleated giant cell is not an osteoclast but an angiogenictumor cell, where ADAM10/Notch1signaling was activated. TRAP/DAPI double stainingshows that multinucleated giant cell was negative for TRAP staining in giant cell-richosteosarcoma, while osteoblastic, chondroblastic, and fibroblastic osteosarcoma all hadTRAP+tumor cells respectively. CD31was expressed at intermediate level in the cytoplasm of ginat cells, ADAM10and activated Notch1were also expressed in giant cellsin a similar pattern as CD31.
4. The vasculature of partial osteosarcoma had endothelium deficiency. CD31stainingrevealed red blood cells in some osteosarcoma from stage IA to IIB osteosarcomavasculature were not surrounded by endothelium, which was stained positive for CD31anddisproved the reason as CD31was not expressed in endothelium of osteosarcoma but ratheras endothelium deficiency, which might be related with multinucleated giant cells.
Conclusions
1. The role of bone vessels at the growth plate in regulating the function of osteoclastsonly manifests itself in the large long bones such as the femur, humerus, tibia, ulna andradius at the later stages of their growth.
2. ADAM10in endothelial cells is required for the proper development and function ofthe specialized vasculature in the bone during the process of endochondral ossification.
3. The reduced number of osteoclasts at the COJ in joints of long bones of ADAM10△EC mice are likely the consequence of alterations in Notch-dependent differentiation ofendothelial cells in the absence of ADAM10, instead of defects in release of a soluble factorfrom endothelial cells by ADAM10.
4. The expression of ADAM10in tumor cells positively correlates with osteosarcomaprogression.
5. ADAM10participated in the cytoplasmic invagination of angiogenic tumor cells ofstage IA/IB osteosarcoma, which might contribute to vascular lumen formation.
6. ADAM10/Notch1signaling was activated in multinucleated giant cell, which wasnot osteoclast, but rather was angiogenic and might be related with endothelium deficiencyin partial osteosarcoma vasculature.
引文
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Krebs LT, Shutter JR, Tanigaki K, et al.2004. Haplo insufficient lethality and formation of arteriovenousmalformations in Notch pathway mutants. Genes Dev.18(20):2469-2473.
Kopan R, Ilagan MX.2009. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell.137(2):216-233
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Glomski K, Monette S, Manova K, et al.2011. Deletion of Adam10in endothelial cells leads to defects inorgan-specific vascular structures. Blood118:1163–1174.
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