一种从骨巨细胞瘤组织中纯化破骨细胞的简单方法

目的从人骨巨细胞瘤组织中分离纯化及鉴定破骨细胞。方法我们利用0．25％胰酶-EDTA和Ⅰ型胶原酶从人骨巨细胞瘤组织中纯化出大量破骨细胞，并进行表型特征的鉴定：包括抗酒石酸酸性磷酸酶染色(TRAP染色)，采用RT-PCR方法检测降钙素受体、组织蛋白酶K和破骨细胞分化因子受体(RANK)表达。结果该方法所得细胞纯度可达79．7％，具有破骨细胞表型特征。结论该方法所得破骨细胞可用于生化和分子生物学研究，是进行骨代谢研究较好的破骨细胞来源。     

骨巨细胞瘤中单核基质细胞的分离培养及其性质研究

目的对骨巨细胞瘤中单核基质细胞的性质和来源进行探讨。方法体外分离培养8例骨巨细胞瘤的单核基质细胞，用胶原酶分离方法和差速贴壁法分离纯化细胞，将分离培养的骨巨细胞瘤单核基质细胞与狗股骨薄磨片共培养，观察骨巨细胞瘤单核基质细胞的噬骨能力、TRAP染色和RT—PCR检测降钙素受体（CTR）和细胞核因子KB受体活化因子（RANK）。结果利用酶消化的方法可以获得较高纯度的单核基质细胞；TRAP染色阳性；体外培养具有噬骨能力；采用RT—PCR方法可检测到降钙素受体和细胞核因子KB受体活化因子。结论利用胶原酶-Ⅱ消化的方法结合差速贴壁方法可以获得较纯的单核基质细胞，可用于生化和分子生物学研究，是进行骨巨细胞瘤细胞学研究的细胞来源。     

软组织恶性巨细胞瘤——附四例报告

 软组织恶性巨细胞瘤(Malignant Giant Cell Tumour of Soft Part),为罕见于骨外的恶性肿瘤,其组织形态多样,但以出现大量破骨细胞样细胞为主要特征,本组四例均为本院手术切除标本,常规石蜡切片,每例切3—10片,H.E染色,VG结缔组织染色,Foot改良法网状纤维染色,2例经P.T.AM染色。四例中三例术前作针吸细胞学检查,瑞氏染色。     

图像定量分析系统(ICM)在骨巨细胞瘤DNA含量检测中的应用

 目的　研究骨巨细胞瘤DNA含量和该瘤各级别的关系。方法　对36例骨巨细胞瘤石蜡包埋标本Feulgen染色后，用Video Pro32彩色图像分析系统对它们的核DNA进行定量分析。结果　发现Jaffe氏骨巨细胞瘤的病理分级与核DNA指数及各种倍体分布类型之间无显著相关。在各级别骨巨细胞瘤中均有非整倍体例数检出，提示了该肿瘤的潜在恶性。结论　从DNA定量分析结果上看，存在有恶性巨细胞瘤。     

破骨细胞体外分离培养及其骨吸收活动的观察

目的 建立大鼠破骨细胞体外分离培养方法，为体外研究破骨细胞骨吸收机理奠定基础。方法 采用出生24h内的Wistar大鼠，从其四肢长骨中分离出破骨细胞，与盖玻片、骨磨片共同培养，观察破骨细胞的形态结构及体外骨吸收活性。结果 相差显微镜及光镜观察到分离的细胞含多个核，能够移动，胞浆有伪足样突起，这些细胞用目前公认的鉴定破骨细胞的标志—抗酒石酸酸性磷酸酶染色呈阳性反应，扫描电镜观察到这些细胞能在骨片上形成典型的骨吸收陷窝。结论 用此方法分离培养的细胞为具有骨吸收活性的破骨细胞。     

脊柱骨巨细胞瘤解整合素样-金属蛋白酶12表达与临床病理特征的相关性

目的探讨脊柱骨巨细胞瘤解整合素样-金属蛋白酶12(ADAM12)的表达与临床病理特征的相关性。方法选取2009年2月至2018年5月间榆林市第二医院收治的101例脊柱骨巨细胞瘤患者,调查患者的临床病理特征,采用免疫组化法检测ADAM12表达情况并进行相关性分析。结果脊柱骨巨细胞瘤患者瘤旁组织中ADAM12表达阳性率为9. 9%(10例),病灶组织中为66. 3%(67例),两组组织中ADAM12表达率比较,差异有统计学意义(P <0. 05)。骨巨细胞瘤组织中,ADAM12表达阳性率与Jaffe病理分级、分化情况、淋巴结转移、肺转移和肝转移均有相关性,差异均有统计学意义(均P <0. 05)。ADAM12表达阳性率为因变量,以患者临床病理特征为自变量,多元回归logistic分析分析显示,肝转移、分化程度和Jaffe病理分级为影响骨巨细胞瘤ADAM12表达阳性率的主要因素,差异均有统计学意义(均P <0. 05)。结论脊柱骨巨细胞瘤ADAM12高表达与临床病理特征相关,肝转移、分化程度和Jaffe病理分级为影响骨巨细胞瘤ADAM12表达阳性率的主要因素。     

硼替佐米对多发性骨髓瘤患者破骨细胞生成的影响及机制研究

目的:观察多发性骨髓瘤(multiple myeloma,MM)患者破骨细胞在体外诱导分化成熟过程中,硼替佐米(Bortezomib,Btzmb)对其分化和功能的影响,以及破骨细胞分化过程中上游关键信号分子肿瘤坏死因子受体相关蛋白6(tumor necrosis factor receptor-associated factor 6,TRAF6)的改变.方法:患者外周血单个核细胞在经核因子KB受体活化因子配体(receptor activator of NF-kB ligand,RANKL)及巨噬细胞集落刺激因子(macmphage colony stimulating factor,M-CSF)诱导后向破骨细胞分化过程中,采用不同剂量的Btzmb进行处理,观察抗酒石酸酸性磷酸酶(tartrateresistant acid phosphatase,TRAP)染色阳性的破骨细胞数量,检测培养液中的TRAP活性,并观察骨片上骨陷窝数量的变化.采用Westem印迹和RT-PCR法,分析Btzmb对破骨前体细胞中TRAF6蛋白和mRNA的影响.结果:2.5和5 nmol/L Btzmb组破骨细胞数量分别为(157±21)和(98±15)个,较对照组(307±25)明显减少(P均＜0.05),骨陷窝形成数量分别为对照组的(53±24)%和(29±7)%(P均＜0.05),上清液中破骨细胞活性分别为对照组的(86±24)%和(60±25)%(P均＜0.05).破骨前体细胞经Btzmb处理后观察24 h,TRAF6蛋白活性逐渐降低,TRAF6 mRNA水平也有所下降.结论:Btzmb抑制MM患者破骨细胞的分化和功能,该作用可能与TRAF6有关.     

伴破骨细胞样巨细胞的乳腺癌二例临床病理分析

目的探讨伴破骨细胞样巨细胞的乳腺癌(COGC)的临床病理特征。方法收集2016年2月江苏省泰州市人民医院和2008年5月上海市普陀区中心医院COGC患者各1例,观察其组织学形态、免疫表型及临床病理特征,并复习相关文献。结果2例患者均为女性,年龄分别为48岁及57岁,均因乳房无痛性肿块就诊。光学显微镜下主要表现为浸润性筛状癌及非特殊类型浸润性导管癌,破骨细胞样巨细胞散布在肿瘤组织内,间质内有不同程度的出血和炎症细胞浸润。免疫组织化学示癌细胞雌激素受体(ER)、孕激素受体(PR)弥漫强阳性,破骨细胞样巨细胞CD68阳性。结论COGC是一种罕见肿瘤,其内的破骨细胞样巨细胞形态特殊,需要与多种类型的多核巨细胞鉴别,其诊断及鉴别诊断需要依据形态学特征及免疫表型。     

ANGPTL4在调控骨巨细胞瘤细胞增殖、血管生成和破骨分化作用的实验研究

目的探讨人血管生成素样蛋白4(ANGPTL4)基因沉默对骨巨细胞瘤单核基质细胞(GCTSC)增殖、血管生成以及破骨分化的作用。方法体外培养GCTSC细胞系,采用ANGPTL4 shRNA、Scrambled shRNA转染作为ANGPTL4 shRNA组和阴性对照组,另设置无处理的空白对照组。采用实时荧光定量PCR(QPCR)和Western blotting法检测ANGPTL4 mRNA和蛋白表达。MTT法和流式细胞术检测细胞增殖和凋亡。将GCTSC细胞分别与人脐静脉内皮细胞(HUVECs)、小鼠骨髓单核细胞(BMM)共培养,观察HUVECs成管能力和BMM破骨分化能力。结果与空白对照组和阴性对照组比较,ANGPTL4 shRNA组ANGPTL4 mRNA(0.174±0.045)和蛋白表达量(0.098±0.020)均明显降低;而ANGPTL4 shRNA组GCTSC细胞增殖活性降低,凋亡率升高(P<0.05)。与HUVECs共培养后,ANGPTL4 shRNA组闭合管数量[(57.35±17.24)%]减少(P<0.05);与BMM共培养后,ANGPTL4 shRNA组TRAP染色阳性的破骨细胞样多核巨细胞数量[(48.36±21.79)%]减少(P<0.05)。结论沉默骨巨细胞瘤GCTSC细胞ANGPTL4基因表达后,细胞增殖活性降低,凋亡率增加,并且对肿瘤血管生成和多核巨细胞破骨分化有一定的抑制作用。     

CCN3在肺癌骨转移灶中高表达及其对破骨细胞的作用

背景与目的:肺癌易转移至骨并导致溶骨性破坏的具体机制目前还不清楚,本研究观察CCN3在肺癌骨转移灶中的表达水平以及在前体成骨细胞(MC3T3-E1)与前体破骨细胞(RAW264.7)共培养体系研究CCN3重组蛋白对前体破骨细胞分化的影响。方法:采用免疫组化检测9例肺癌骨转移转灶组织及相应癌旁组织中CCN3的表达;构建RAW264.7细胞与MC3T3-E1细胞共培养模型,向共培养体系中加或不加800 ng/mL的CCN3重组蛋白,采用Real-time PCR检测RAW264.7细胞的破骨细胞标志基因抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)、组织蛋白酶K(cathepsin K,CK)、降钙素受体(calcitonin receptor,CTR)mRNA的表达,并通过TRAP染色法对破骨细胞进行鉴定。结果:肺癌骨转移灶组织中的CCN3表达量明显高于癌旁组织;CCN3重组蛋白可显著促进共培养体系中的RAW264.7细胞的破骨细胞标志基因TRAP、CK、CTR的mRNA的表达量;TRAP染色可见对照组TRAP阳性细胞数[(6.2±1.48)个]明显低于加了CCN3重组蛋白的试验组[(21.4±3.04)个],差异具有统计学意义(P<0.05)。结论:CCN3在肺癌骨转移灶中高表达及其可诱导破骨细胞分化,CCN3可能在肺癌骨转移和溶骨性破坏中具有重要作用。     

Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient''s sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes.     

Elevated Pentraxin 3 in bone metastatic breast cancer is correlated with osteolytic function

Pentraxin 3 (PTX3), a modulator of tumor-associated inflammation, is known to be positively correlated with tumor grade and severity of malignancies, but its exact role remains unclear. This study found that PTX3 expression was up-regulated in distant bone metastases of breast cancer compared to lung, liver, and brain metastases in 64 human breast cancer patients. Elevated expression of PTX3 was correlated with poor survival in patients with breast cancer. PTX3 expression was also up-regulated in a bone metastatic breast cancer cell line and further enhanced by pro-inflammatory cytokine TNFα. Administration of PTX3 promoted the migratory potential of breast cancer cells and the mobilization of macrophages, a precursor of osteoclasts (OCs), toward breast cancer cells. In addition, elevated expression of PTX3 by TNFα led to enhanced OC formation, implying the distinct role of PTX3 in osteolytic bone metastasis of breast cancer cells. Furthermore, PTX3 silencing using siRNA-specific siRNA prevented breast cancer cell migration, macrophage Chemotaxis, and subsequent OC formation. These findings provide an important insight into the key role of PTX3 in inflammation-associated osteolytic complications of breast cancer.     

CD8+ T cells regulate bone tumor burden independent of osteoclast resorption

Blockade of osteoclast (OC) activity efficiently decreases tumor burden as well as associated bone erosion in immune-compromised animals bearing human osteolytic cancers. In this study, we showed that modulation of antitumor T-cell responses alters tumor growth in bone, regardless of OC status, by using genetic and pharmacologic models. PLCγ2(-/-) mice, with dysfunctional OCs and impaired dendritic cell (DC)-mediated T-cell activation, had increased bone tumor burden despite protection from bone loss. In contrast, Lyn(-/-) mice, with more numerous OCs and a hyperactive myeloid population leading to increased T-cell responses, had reduced tumor growth in bone despite enhanced osteolysis. The unexpected tumor/bone phenotype observed in PLCγ2(-/-) and Lyn(-/-) mice was transplantable, suggesting the involvement of an immune component. Consistent with this hypothesis, T-cell activation diminished skeletal metastasis whereas T-cell depletion enhanced it, even in the presence of zoledronic acid, a potent antiresorptive agent. Importantly, injection of antigen-specific wild-type cytotoxic CD8(+) T cells in PLCγ2(-/-) mice or CD8(+) T-cell depletion in Lyn(-/-) mice normalized tumor growth in bone. Our findings show the important contribution of CD8(+) T cells in the regulation of bone metastases regardless of OC status, thus including T cells as critical regulators of tumor growth in bone.     

骨巨细胞瘤组织中端粒酶hTERT基因表达及其意义

目的 探讨骨巨细胞瘤组织中端粒酶hTERT基因的表达及意义.方法 应用原位分子杂交技术,对4l例骨巨细胞瘤组织、17例骨质增生骨组织、11例异位骨化组织和10例正常骨组织中端粒酶hTERT基因进行检测和定位,并运用图像分析系统对hTERT基因表达水平进行研究.结果 端粒酶hTERT基因在骨巨细胞瘤中表达阳性率为41.5%(17/41),表达强度与骨巨细胞瘤的分化程度明显相关(P＜0.05):低分化肿瘤＞中分化肿瘤＞高分化肿瘤,端粒酶hTERT细胞表达水平与肿瘤细胞的分布定位一致.17例骨质增生骨组织、11例异位骨化组织和10例正常骨组织中端粒酶hTERT基因的表达均为阴性.结论 原位分子杂交是检测端粒酶亚单位hTERT的有效方法,在骨巨细胞瘤细胞水平检测端粒酶hTERT基因的定位诊断具有重要意义.在骨巨细胞瘤发展和维持中端粒酶可能起到重要的作用,同时还可能存在端粒酶以外的机制导致肿瘤细胞永生化.     

CC-chemokine ligand 20/macrophage inflammatory protein-3α and CC-chemokine receptor 6 are overexpressed in myeloma microenvironment related to osteolytic bone lesions

The expression of the chemokine CC-chemokine ligand 20 (CCL20)/macrophage inflammatory protein (MIP)-3alpha and its receptor CC-chemokine receptor 6 (CCR6) by multiple myeloma (MM) and microenvironment cells and their potential relationship with osteoclast (OC) formation and osteolytic bone lesions in MM patients was investigated in this study. First, we found that MM cells rarely produce CCL20/MIP-3alpha but up-regulate its production by bone marrow (BM) osteoprogenitor cells and osteoblasts in coculture with the involvement of soluble factors as interleukin-1beta and tumor necrosis factor alpha. MM cells also stimulate both CCL20/MIP-3alpha and CCR6 expression by OCs in coculture. Thereafter, we showed that CCL20/MIP-3alpha significantly increases both the number of multinucleated tartrate-resistant acid phosphatase-positive OCs and receptor activator of nuclear factor-kappaB-positive OC progenitor cells similar to CCL3/MIP-1alpha. Finally, we found that blocking anti-CCL20/MIP-3alpha and anti-CCR6 antibodies significantly inhibits MM-induced OC formation. In vitro data were further expanded in vivo analyzing a total number of 64 MM patients. Significantly higher CCL20/MIP-3alpha levels were detected in MM patients versus monoclonal gammopathy of uncertain significance (MGUS) subjects and in MM osteolytic patients versus nonosteolytic ones. Moreover, a significant increase of CCL20/MIP-3alpha-positive osteoblasts in osteolytic MM patients compared with nonosteolytic ones was observed. Interestingly, no significant difference in BM CCL20/MIP-3alpha expression and level was observed between MGUS and nonosteolytic MM patients. Our data indicate that CCL20/MIP-3alpha and its receptor CCR6 are up-regulated in the bone microenvironment by MM cells and contribute to OC formation and osteolytic bone lesions in MM patients.     

The multinucleate cells in giant cell granulomas of the jaw are osteoclasts

The giant cell granuloma of jaw is a well-vascularised lesion comprising a mononuclear cell infiltrate with a large number of giant cells. It has been suggested that the lesion is reparative in nature, rather than neoplastic, and that the giant cells are phagocytes accumulating in chronic reparative granulation tissue. However, the nature of the multinucleate giant cells never has been established. One possibility is that the constituent giant cells are osteoclasts. The authors assessed expression by the giant cells of several osteoclast-specific characteristics: excavation of bone; motility inhibition by calcitonin (CT); and binding of osteoclast specific monoclonal antibodies. Two tumors were disaggregated and incubated on slices of cortical bone in the presence and absence of CT. Both tumors were found to excavate bone, a function unique to osteoclasts. The giant cells also were responsive to CT, resulting in cytoplasmic quiescence and inhibition of bone resorption. Two osteoclast-specific monoclonal antibodies bound all the giant cells in one central and six peripheral tumors examined immunohistochemically. These results provide strong evidence for the osteoclastic nature of the giant cells. The presence of alkaline phosphatase-positive cells forming woven bone in giant cell granulomas suggests that osteoblasts are present in the lesion. As cells of osteoblastic lineage are known to regulate osteoclastic function, it may be that osteoblasts account for the characteristic infiltration of osteoclasts into giant cell granulomas of jaws, either as part of a reparative response by reactive osteoblasts or as an infiltrate induced by osteoblasts of aberrant function, as suggested for giant cell tumors of bone.     

p63蛋白在软骨母细胞瘤中的表达及其价值探讨

目的探讨p63蛋白在软骨母细胞瘤中的表达水平及其诊断价值。方法 采用免疫组织化学法(Maxvision)技术对25例软骨母细胞瘤、52例骨巨细胞瘤、10例动脉瘤样骨囊肿和20例腱鞘巨细胞瘤中p63和S-100p蛋白的表达水平进行分析。结果 p63蛋白在软骨母细胞瘤中呈单核细胞的胞核阳性,阳性率为88.0%(22/25)。而骨巨细胞瘤中除呈单核细胞的胞核阳性,少数多核巨细胞的胞核亦呈阳性,阳性表达率为92.3%(48/52)。p63蛋白在软骨母细胞瘤和骨巨细胞瘤中的表达两者比较差异无统计学意义(P>0.05),而S-100p在软骨母细胞瘤和骨巨细胞瘤中阳性表达率分别为88.0%(22/25)和1.9%(1/52),两者差异有统计学意义(P<0.05)。p63蛋白和S-100p在动脉瘤样骨囊肿和腱鞘巨细胞瘤中均不表达。结论 p63蛋白在软骨母细胞瘤和骨巨细胞瘤中均呈高表达,并有相似的免疫表型,提示两者在发生分化上可能均来自相同的起源细胞或肿瘤干细胞。p63蛋白结合S-100p联合应用对鉴别软骨母细胞瘤及其相关性病变具有一定辅助诊断意义。     

骨巨细胞瘤中 nm23 和 ras 基因的表达及临床意义

目的 :探讨nm2 3和ras基因在骨巨细胞瘤 (GCT)的表达及与GCT病理分级和复发的关系。方法 :应用SP免疫组织化学方法检测nm2 3和ras在 52例GCT (GCT按Jaffe分级 :Ⅰ级 15例、Ⅱ级2 5例、Ⅲ级 12例 )中的表达。结果 :2 1例nm2 3表达阳性 ,阳性率 4 0 .4 % ,7例ras表达呈阳性 ,阳性率13.5%。其阳性表达与病理分级均无显著相关性 (P >0 .0 5)。nm2 3和ras在复发和无复发的病例中 ,阳性表达率分别为 6 9.2 %、30 .8%和 2 3.1%、10 .3%。nm2 3过表达与GCT复发有高度相关性 (P <0 0 5)。而ras基因表达无相关性。结论 :nm2 3和ras表达均与GCT病理分级无关。nm2 3表达与肿瘤复发有关 ,而ras表达与复发未见相关性     

骨肿瘤组织C-erbB-2原癌基因产物表达及临床意义

 应用S-P法观察了67例骨肿瘤标本C-erbB-2癌基因产物的表达。结果显示：20例良性骨软骨瘤呈阴。性反应，而侵袭性骨巨细胞瘤与骨肉瘤分别有48%、65%的阳性表达（P<0.001）。提示C-erbB-2原癌基因产物的表达与骨肿瘤的细胞生物学行为有关，随骨肿瘤的恶性程度增高，阳性表达频率显著性增加。进一步研究表明：C-erbB-2的阳性表达随骨巨细胞瘤病理分级提高，软组织受累和局部复发而明显增高；肿瘤＞5cm的骨肉瘤患者C－erbB－2的阳性率越高。C-erbB-2阳性表达的骨肉瘤患者预后较差。     

葡萄糖转运蛋白-1在不同骨肿瘤中的表达

目的研究葡萄糖转运蛋白-1（GLUT-1）在正常骨组织及不同骨肿瘤标本中的表达情况。方法收集我院临床骨肉瘤标本30例，骨巨细胞瘤标本15例，骨样骨瘤标本10例及正常骨组织标本10例，采用免疫组化染色检测GLUT-1的分布。体外培养骨肉瘤细胞系MG63、临床骨肉瘤细胞、临床骨巨细胞瘤细胞及成骨细胞，RT-PCR检测GLUT-1基因的表达。结果免疫组化显示，骨肉瘤标本GLUT-1染色强阳性占总例数的53％；骨巨细胞瘤切片GLUT-1染色，阳性占总例数的60％；骨样骨瘤标本多为阴性，阴性占总例数的80％；正常骨组织中未见GLUT-1阳性表达。骨肉瘤明显高于与其余3组标本（χ^2=1．622，P=0．009；χ^2=34．667，P〈0．001，χ^2=40．000，P〈0．001）。RT-PCR显示，GLUT-1基因表达存在于MG63细胞、临床骨肉瘤细胞及骨巨细胞瘤中，而正常成骨细胞内并无GLUT-1表达。结论骨肿瘤中存在GLUT-1表达，且GLUT—1与骨肿瘤的恶性程度有较密切的关系。GLUT-1有可能作为判断骨肿瘤恶性程度参考指标及骨肿瘤治疗的新靶点。