应用单抗GCF-5观察骨巨细胞瘤细胞成分

采用免疫细胞化学和免疫电镜方法,以我室制备的抗骨巨细胞瘤(GCT)肿瘤细胞的单克隆抗体GCF-5,对41例GCT及其它肿瘤细胞进行观察,结果表明:41例中的35例GCT标本与GCF-5结合呈阳性反应;除1例骨肉瘤细胞系(OS-732)细胞为阳性反应外,其它骨肿瘤均为阴性反应。经免疫金染色后电镜下,在阳性细胞表面可见金颗粒,证明GCF-5抗体是抗细胞表面抗原的单克隆抗体,GCF-5与GCT中部分基质细胞(STC)结合,除与一些双核细胞和核数少的多核巨细胞(MGC)结合外,与绝大多数的MGC不发生反应,但能与GCT体外培养、多次传代后的MGC发生反应。均支持本作者以前的观点,即GCT中的MGC与STC各自包含两种截然不同的细胞成分:肿瘤细胞和与肿瘤免疫有关的细胞,仅肿瘤细胞成分能在体外培养中生长、增殖。     

CELLULAR ELEMENTS IN GIANT CELL TUMOR OF BONE FEULGEN-DNA QUANTITATION, 3H_TdR INCORPORATION AND ULTRASTRUCTURE OBSERVATION

Anti-erythrocyte rosette assay was used to sub divide stromal cells (StCs) of cultured giant cell tumor (GCT) of bone into two groups, the rosette forming cells (RFCs) and non-rosette forming cells (NRFCs). Characteristics of Feulgen-DNA content, 3H.TdR uptake and ultrastructure of different cellular elemcnts in GCT were then investigated by micro- spectrophotometry, autoradiography and both TEM and SEM. Two types of StCs and multinucleated giant cells (MGCs) showed 2C DNA stem line, which contrasted strikingly with aneuploid DNA in osteosarcoma cells. Autoradiography revealed that the labelling index of NRFCs increased with the time of 3H.TdR incorporation in vitro, while RFCs and MGCs were scarcely tagged. The two types of StCs were distinctly different in both surface and intra- cellular structures. The phagocytic function and sur- face appearance of RFCs resembled those of ma crophages, and no polymorphism was found in RFCs. These facts suggest that NRFCs are the neoplastic element in GCT, whereas RFCs are the end-stage cells and possibly the macrophages related to tumor immunity.     

Effect of cytokines on in vitro bone resorption by cells isolated from giant cell tumor of bone

ＯｂｊｅｃｔｉｖｅＴｏｉｎｖｅｓｔｉｇａｔｅｔｈｅｅｆｅｃｔｏｆｃｙｔｏｋｉｎｅｓｏｎｉｎｖｉｔｒｏｂｏｎｅｒｅｓｏｒｐｔｉｏｎｂｙｃｅｌｓｉｓｏｌａｔｅｄｆｒｏｍｇｉａｎｔｃｅｌｔｕｍｏｒｏｆｂｏｎｅ．ＭｅｔｈｏｄｓＭｏｎｏｎｕｃｌｅａｒｓｔｒｏｍ...     

CYTOLOGIC OBSERVATION ON GIANT CELL TUMOR OF BONE

By combined immunologic, histochemical and other approaches, we studied the macro- phage content in 20 giant cell tumors of bone (GCT) as compared with that in 26 benign and malignant lesions of osseous and soft tissues. We found that many macrophages existed in GCvF. In the cell suspensions prepared by en- zymatic disaggregation, the percentage of macro- phge was apparently higher than in co.ntrol le- sions. After serial passage, macropha.ges and mult.inucleated giant cells (MGC) gradually de- creased in number, whereas spindle-shaped cells, which were negative for Fc, Cq recepitor and nonspecific esterase (NSE), maintained their growth. KeeIoin.g the cuture media uncha.nged for 7 t0 31 da.ys, cells containing Z t0 8 nuclei could be found in primary culture of macropha- ges isolated from GCT. Although Fc, C3 recep- tors and phagocytosis were not demonstrated in almost all MGC, some of them showed NSE ac- tivity and weak res.isitance to trypsin. It is worth noting that a few bi-, tri- and tetra-nucleated cells form.ed EA rosettes and phagocytosed E.A. The place of macrophages in GCT, their inter- relationship, ancl MGC are discussed.     

骨巨细胞瘤内多核巨细胞的超微结构观察

本文观察了六例骨巨细胞瘤,探讨了多核巨细胞的超微結构改变,其组织发生有迹象表明由单核基质细胞融合形成的。根据形态特征、恒定存在和生物学行为综合分析,认为多核巨细胞也是骨巨细胞瘤的实质瘤细胞。     

uPA-uPAR系统对骨巨细胞瘤细胞p44(MAPK)信号转导的影响

目的研究uPA／uPAR系统对骨巨细胞瘤细胞MAPK信号转导的影响。方法分离并培养骨巨细胞瘤细胞，用免疫组化检测骨巨细胞瘤组织中uPAR的表达水平，用免疫共沉淀方法检测外源激活或阻断uPA—uPAR对骨巨细胞瘤细胞信号转导通路的p44（MAPK）蛋白磷酸化水平的影响。结果仅有单核基质细胞可以在体外培养中长期生存；在骨巨细胞瘤组织中uPAR主要表达在部分单核基质细胞和一些多核巨细胞的胞膜上；将uPA—ATF加入培养的骨巨细胞瘤细胞后，细胞信号通路上的p44蛋白磷酸化水平明显增高。用uPAR抗体处理后，细胞p44蛋白磷酸化水平明显降低。说明uPA—ATF具有细胞信号转导活性，该活性受uPAR拮抗剂的影响。结论本实验检测到uPA—uPAR系统是通过p44（MAPK）信号转导通路转导信息，从而调节骨巨细胞瘤细胞增殖、分化及其他生物学行为。     

CYTOLOGICAL CHARACTERISTICS OF MAIN CELLULAR ELEMENTS IN BFNIGN AND MALIGNANT GIANT CELL TUMOR OF BONE

Cytological characterist;cs of three main cellular elements iii benign and malignant giant cell tumor of bone were investigated by cytochemistry, tissue ccilture. electron microscopy and microspectropho. toinetry. The macrophage (MO), as identified by EA rosette assay, and the multinucleated giant celf (MGC) had similar enzyme activities, which differed from those of the non rosette-forming cell (NRFC). MO and MGC of malignant giant cell tumor of bone (MCCT) did not proliferate in vitro while ;ts NRFC could maintain seri81 growtfi with concomi tant formation of some multinuclear cells which are clifferent from MCC in the light and eleccron of atypical MCCs were certain aspects. Under both microscope a small number found in MGCT. The;r nii clei varied in size and shape. The NRFC in MGCT showed more prominent ritypical ultrastructural features. Part of the NRFCs had aneuploid DNA content above 4C (-4C), which is generally consi dered as the evidence of rnalignancy. Some nuclei i" the MGCs of MGCT contained }40 DNA con tent, but not those in MGCs of benign giant cell tumor of bone. Usually, no aneupaoidy was detected in MO of all the samples with the exception of one MGCT sample. in which l O% M¢ contains :4C DNA content. Iri the in virro labelling, :II-TdR labelled NRFC were in varying percentage, but labelled MO and MGC were infrequently found. The circumstan tial evidences suggest a close relationship exists be. tween M¢ and MGC: but NRFC presents neoplastic featurea. The atypical giant cells in MGCT might be of tumor origin.     

HISTOMORPHOMETRICAL STUDY OF MULTINUCLEATED GIANT CELLS OF GIANT CELL TUMOR OF BON BY AUTOMATIC IMAGE ANALYSIS SYSTEM

The histomorphometry of ten cases of giant cell tumor of bone was studied using an automatic image analysis system and eight criteria of measurements were analysed. The study showed that this system provided a new technique in medical research and that similar multinucleated giant cells in various lesions of bone may be distinguished with bone histomorphometry. This method will form the basis for the reconstruction of three demensional structure of cells.     

IN VITRO TISSUE CUL,TURE STUDY ON GIANT CELL TUMOR OF BONE

In vitro tissue culture of 22 giant cell tumors (GCTs) of the bone in 19 cases was undertaken. Primary growth was successful in all. 11 were Grade I tumors, 10 Grade n and l Grade III. The average survival of the 3 grades of GCTs was in order 167.3, 219 and 625 days. Of these, 6 were postoperative recurrent GCTs, their life span ranging from 198 t0 6.25 days. It is apparent that the higher the pathologic grade of GCT, the longer the survival of cultured cells. Recurring tumors also manifested more active growth po- tential. This lends support to the view that the existing pathologic grading remains valuable for clinical reference. Multinucleated giant cell usually survived 3 weeks, occasionally 3 months. Growth of the stromal cells maint.ained from 5 weeks t0 8 months. The circumstantial evidence indicates that the stromal cells, not the giant cells, should be considered as the offending neoplastic invader.     

骨巨细胞瘤的p16蛋白表达特点

目的 :探讨p16蛋白表达与骨巨细胞瘤发生的关系。方法 :采用免疫组织化学S P法检测 4 2例骨巨细胞瘤的p16蛋白表达。结果 :骨巨细胞瘤的p16蛋白表达缺失率为 6 6 .7% ,p16蛋白表达随骨巨细胞瘤恶性程度的上升而降低。骨巨细胞瘤的多核巨细胞有 3种染色状态。结论 :p16蛋白表达降低与骨巨细胞瘤的发生有关 ,骨巨细胞瘤的多核巨细胞可能存在不同种类。     

Ezrin在骨巨细胞瘤中的表达及意义

目的：探讨骨巨细胞瘤（GCT）中埃兹（Ezrin）蛋白的表达及其与GCT临床病理参数的关系。方法：采用SP免疫组化法检测75例GCT中Ezrin蛋白的表达,并分析其与GCT临床病理参数的关系。结果：Ezrin在GCT中的阳性表达率按临床影像学分级分别为Ⅰ级13.3%、Ⅱ级33.3%、Ⅲ级80.0%。GCT中Ezrin的阳性表达率与患者的年龄、性别以及病理分期无关（P＞0.05）,与GCT的临床影像学分级有关（P＜0.01）。结论：Ezrin可作为判断GCT局部浸润的指标,从而为GCT的临床治疗提供帮助。     

TNF-α对骨巨细胞瘤u-PA系统表达的调节及意义

目的探讨TNF-α对骨巨细胞瘤u-PA系统mRNA表达的调节及意义。方法骨巨细胞瘤原代培养及传代,加入外源性TNF-α,观察加入TNF-α前后细胞生长特性的变化,流式细胞分析仪检测细胞的增殖指数,RTPCR检测u-PA系统mRNA的表达。结果原代培养过程中可见到三种细胞:多核巨细胞(MGC)、巨噬细胞样单核细胞(MC)及纤维母细胞样单核细胞(FC),传代几次以后只有FC及MC;RT-PCR检测细胞中有u-PA、uPAR及PAI-1mRNA表达,加入TNF-α后mRNA的表达较加入前增高(P<0.05);流式细胞分析仪检测细胞的增殖指数(PI),加入TNF-α后PI值较加入前增高(P<0.05)。结论外源性TNF-α促进骨巨细胞瘤原代培养细胞u-PA系统mRNA的表达及细胞增殖。     

Apoptosis in giant cell tumors of bone

Although giant cell tumor of bone (GCT) is characterized by the extensive multinucleated giant cells among mononuclear stromal cells, proliferation of these cells and multinucleation are not without limit in certain cases. Few studies on oncogenesis of GCT have focused on the negative growth control, including growth arrest and apoptosis. The purpose of this study was to investigate the mechanism of cell death in multinucleated giant cells and stromal cells of GCT. In this study, we have demonstrated that GCT cells can undergo apoptosis. The cells in surgical specimen were positively stained in situ nick end labeling methods, and electron micrographs showed the morphological changes associated with apoptosis in some of stromal cells and multinucleated giant cells. A candidate responsible for this apoptosis was then examined using cultured GCT cells. We focused on Fas that is a major trigger of apoptosis. Cultured GCT cells expressed detectable amount of Fas on their surface. Although GCT cells did a little undergo apoptosis following treatment with anti-Fas alone, combination treatment with cyclohexamide led to an increase in apoptosis of the GCT cells. These data suggested that the sensitizing activity of cyclohexamide on anti-Fas mediated cytotoxicity could happen in vitro.     

骨巨细胞瘤p~（53）蛋白和PCNA的表达及其意义

目的探讨抑癌基因ｐ~（５３）蛋白和增殖细胞核抗原（ＰＣＮＡ）在骨巨细胞瘤（ＧＣＴ）中的表达特征及其临床病理意义。方法应用免疫组化Ｓ－Ｐ法检测３９例ＧＣＴ组织中ｐ~（５３）和ＰＣＮＡ的表达与定位分布。结果ｐ~（５３）和ＰＣＮＡ阳性反应主要定位于单核基质细胞核上，少数多核巨细胞呈阳性反应，多数多核巨细胞呈阴性反应。ｐ~（５３）在ＧＣＴⅢ级中阳性率为６０％，显著高于Ｉ，Ⅱ级（Ｐ＜０，０５）。ＰＣＮＡ的阳性分级和ｐ~（５３）表达两者间呈显著正相关（Ｐ＜０．０５）。结论ｐ~（５３）和ＰＣＮＡ的表达可作为评估ＧＣＴ增殖活性和恶性程度的指标。     

骨巨细胞瘤体外培养及其生物学特性的观察

１９８３～１９９２年作者通过组织培养，组化及免疫组化、电镜、细胞遗传学，动物接种等方法，对骨巨细胞瘤（ＧＣＴ）的生物学特性进行研究，认为ＧＣＴ的基质细胞有两种，即间叶性基质细胞及巨噬细胞性基质细胞，巨噬细胞性基质细胞可能为许多种不同的巨噬细胞混合体，这两种类型细胞在瘤组织内互相依赖存活。在体外培养见巨噬细胞性基质细胞逐渐减少而消亡，间叶性基质细胞则在没有巨噬细胞性基质细胞的情况下也容易老化及消亡，故目前尚未有本瘤的细胞株建立。多核巨细胞是一反应性及终末性细胞，可能由于基质细胞分泌目前尚末清楚的细胞因子，吸引血中破骨细胞前身的单核巨噬细胞到瘤组织内，并促进其分化成破骨细胞样多核巨细胞。     

骨巨细胞瘤中血管内皮生长因子及微血管密度的表达

目的 观察血管内皮生长因子(vasclar endohelial growth factor,VEGF)在骨巨细胞瘤(giant cell tumorof bone,GCT)中的免疫组织化学表达和肿瘤组织的微血管密度(microvessel density,MVD),并探讨VEGF和MVD与骨巨细胞瘤组织学分级(Jeffe分级)及肿瘤预后等临床资料之间的关系。方法 对30例骨巨细胞瘤患者的病理及临床资料进行回顾性研究。应用免疫组织化学方法(Envision System二步法)检测肿瘤组织中VEGF的表达和MVD,并将上述资料与Jeffe分级和患者预后等临床资料进行统计学分析。结果 GCT中VEGF的阳性表达与MVD显著相关(P<0.001)。GCT术后复发、转移等不良预后与较高的VEGF阳性表达率(P=0.015)和MVD(P=0.015)显著相关。结论 VEGF阳性表达的GCT组织中的MVD显著高于VEGF阴性表达的肿瘤组织。VEGF的阳性表达和高MVD预示GCT预后不良,其可能作为评价GCT预后的参考指标。     

骨巨细胞瘤中PCNA、p16、Rb与bcl-2基因表达及凋亡的检测研究

目的：探讨骨巨细胞瘤（ＧＣＴ）的细胞学性质、肿瘤发生以及生物学行为。方法：用免疫组化法检测了５１例ＧＣＴ中ＰＣＮＡ、ｐ１６、Ｒｂ和ｂｃｌ－２蛋白的表达及用ＴＵＮＥＬ法检测其凋亡细胞。结果：全部标本ＰＣＮＡ阳性表达，其中典型多核巨细胞未见着色；ｐ１６、Ｒｂ和ｂｃｌ－２在ＧＣＴ中的阳性率分别为９２２％、９６１％和３３３％。Ｊａｆｅ分级间ＰＣＮＡ的表达具有显著性差异；ｐ１６和Ｒｂ在ＧＣＴ中的表达具有一定的负相关；ｂｃｌ－２在Ⅰ与Ⅲ级及Ⅰ与Ⅱ＋Ⅲ级间表达差异有显著性。ＧＣＴ的凋亡率（ＡＩ）为０８６％，多核巨细胞凋亡偶见。ＧＣＴ的ＡＩ与ｂｃｌ－２的表达呈显著负相关，ｂｃｌ－２表达与否的ＡＩ之间有显著的差异。结论：Ｊａｆｅ分级可以反映出ＧＣＴ内的增殖状态；多核巨细胞是一种静息细胞；ｂｃｌ－２在ＧＣＴ发展中可能起一定的作用；联合检测ＰＣＮＡ和ｂｃｌ－２可以作为判断其生物学行为的指标。     

血管内皮生长因子与骨巨细胞瘤预后的关系

目的:研究血管内皮生长因子(VEGF)及微血管密度(MVD)对估计人骨巨细胞瘤预后的价值。方法:通过对69例骨巨细胞瘤患者的随访,将他们分为无复发和无转移组及复发或转移组。采用免疫组织化学SABC法检测骨巨细胞瘤中VEGF、CD34的表达,用MVD值衡量CD34的表达。结果:无复发组42例的中位阳性细胞率为28%,复发转移组27例的中位阳性细胞率为48%,两组间差别有极显著性意义(P<0.001)。无复发组MVD值为33.55±10.82,复发转移组MVD值为56.78±16.17,两组间MVD值的差别有极显著性意义(P<0.001)。VEGF阳性细胞率与MVD值的关系经Spearman等级相关检验,两者呈显著正相关(r=0.845,P<0.01)。结论:VEGF和MVD是估计骨巨细胞瘤预后的新指标,VEGF是重要的促血管生成因子。     

Quantitative pathological study of aggressiveness of giant cell tumor of bone.

Clinical behavior of giant cell tumor of bone (GCT) may vary remarkably from latent to very aggressive. Quantitative pathological methods were used to evaluate the aggressiveness of GCT. Fifteen cytometric parameters were measured and computed on routine sections of 40 GCTs which had been treated with curettage. The surgery factor (with or without additional procedures like bone cement packing or freezing after the curettage) was also taken into account and computed. Nineteen of the 40 GCTs were cured and 21 recurred. While no single one from the 15 parameters measured showed significant differences between the cured and recurrent groups according to the Mann-Whitney's U test or Student's t test, a 4-variable function was established with a stepwise discriminant analysis which could correctly identify 70.8% of the predicted cases as cured or recurrent (jackknife procedure). The function also suggested that in addition to the surgery factor, which no doubt had close relation with the prognosis, the most important risk factor in histological parameters was SA40, i.e. the percent of cells with nucleus larger than 40 square microns. Single cells extracted from paraffin-embedded blocks of 38 GCTs were analyzed by DNA-image cytometry. The 2c deviation index (2cDI) showed wide heterogeneity ranging from those consistent with benign tumors to those with apparent malignant ones, which may account for its diversity in clinical behaviour. Sixteen of the 38 cases had been treated with curettage, 8 of them were cured and followed up for at least 3 years and the remaining 8 recurred. The significant difference of 2cDI between the two groups suggested that these DNA parameters are useful in evaluating the aggressiveness of GCT for the selection of an adequate treatment. Quantitative approaches appeared to be more objective and more sensitive in evaluating the aggressiveness and predicting the prognosis of GCT than the subjective grading system used before.

     

鼻咽涂片中巨大裸核细胞的性质和形成的探讨

26例光镜电镜对照观察,证明鼻咽涂片中的巨大裸核癌细胞具有恶性肿瘤和鼻咽癌胞核的超微结构特点。DNA Feulgen 反应阳性,显示其为胞核成分,并非粘液或异物。透射电镜可见组织表面巨核癌细胞胞浆脱失,并有向巨大裸核癌细胞逐步过渡的形态改变,说明巨大裸核癌细胞系由巨核癌细胞胞浆脱失而来。