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。     

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.     

骨巨细胞瘤瘤组织中M-CSF、TNF_(-α)的检测及其意义

目的：探讨细胞因子与骨巨细胞瘤局部溶骨的关系。方法：通过ＥＬＩＳＡ法、Ｗｅｓｔｅｎｂｌｏｔ法和免疫组化法对１０例骨巨细胞瘤、５例骨肉瘤和５例正常人血清进行了ＴＮＦ－α和Ｍ－ＣＳＦ表达及定位检测。结果：骨巨细胞瘤组织ＴＮＦ－α含量和Ｍ－ＣＳＦ表达率明显高于骨肉瘤和正常人血清。ＴＮＦ－α由骨巨细胞瘤的部分单核基质细胞和多核巨细胞分泌;而Ｍ－ＣＳＦ由部分单核基质细胞分泌,多核巨细胞则不表达。结论：骨巨细胞瘤中各种细胞分泌不同的细胞因子可能参与该肿瘤的局部骨质吸收。     

骨巨细胞瘤中多核巨细胞的纯化及其性质的探讨

目的 :利用酶消化方法对骨巨细胞瘤中多核巨细胞进行纯化 ,以弥补破骨细胞获取量少的问题 ,为骨质疏松的体外研究提供丰富的细胞来源。并用免疫组化的方法对骨巨细胞瘤中多核巨细胞的性质和来源进行探讨。方法 :体外分离培养 8例骨巨细胞瘤的多核巨细胞 ,在培养 2 0h后 ,用 0 .5 g·L-1胰蛋白酶 / 0 .2 g·L-1EDTA联合消化的方法去除贴壁能力较弱的单核基质细胞。将分离培养的骨巨细胞瘤多核巨细胞与牙磨片共培养 ,观察骨巨细胞中多核巨细胞的噬骨能力。并用破骨细胞特异表达的空泡型质子泵、Ⅱ型碳酸酐酶、组织蛋白酶K、基质金属蛋白酶 9对多核巨细胞进行免疫组化染色和TRAP染色。结果 :利用酶联合消化的方法可以获得较高纯度的多核巨细胞 ,纯化率达 85 %。骨巨细胞瘤中的多核巨细胞对破骨细胞所特异表达的空泡型质子泵、Ⅱ型碳酸酐酶、组织蛋白酶K、基质金属蛋白酶 9呈强阳性表达 ,TRAP染色阳性 ,体外培养具有噬骨能力。结论 :利用 0 .5 g·L-1胰蛋白酶 / 0 .2g·L-1EDTA消化的方法可获得较高纯度的多核巨细胞 ,可为破骨细胞体外研究提供丰富的细胞来源。骨巨细胞瘤中的多核巨细胞在功能表达上与破骨细胞相似 ,它可能来源于病变中圆形单核基质细胞的融合     

人骨巨细胞瘤细胞系GCT-0404的建立和生物学特性

目的:研究骨巨细胞瘤细胞体外培养的生物学特性,并建立人骨巨细胞瘤细胞系. 方法: 采用原代组织块培养法培养骨巨细胞瘤手术标本,对存活细胞进行形态学观察、免疫组织化学染色、细胞周期检查、核型分析、裸鼠移植. 结果: 建立人骨巨细胞瘤细胞系GCT-0404,其形态学表现、免疫组织化学染色均符合骨巨细胞瘤纤维母细胞样基质细胞的特征. 经过近1 a的体外培养,现已传代100次,细胞倍增时间39.7 h,细胞周期测定G1期为67.5%,G2期为8.9%,S期为23.6%. 染色体具有三倍体核型. 裸鼠移植成瘤率100%,无支原体污染. 结论: 人骨巨细胞瘤细胞系GCT-0404可以用于对骨巨细胞瘤的研究.     

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。     

骨巨细胞瘤中成纤维样基质细胞体外钙化能力的研究

目的：探讨骨巨细胞瘤的组织来源。方法：体外培养。Ｇｏｍｏｒｉ法碱性磷酸酶染色,生化检测细胞培养上清液中的碱性磷酸酶含量。免疫组织化学染色检测骨钙素在成纤维样基质细胞中的表达。使用含Ｂ甘油磷酸钠和ＣａＣｌ２的培养液,观察成纤维样基质细胞体外钙化能力。结果：成纤维样基质细胞中碱性磷酸酶和骨钙素呈阳性表达,在体外具有钙化能力。在细胞培养上清液中有碱性磷酸酶。结论：骨巨细胞瘤中的成纤维样基质细胞具有某些成骨细胞的特点,为进一步探讨其组织来源提供了实验依据。     

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。     

M-CSF的表达与骨巨细胞瘤病理分级及生物学行为的关系

目的:探讨巨噬细胞集落刺激因子(M-CSF)在骨巨细胞瘤中的表达与肿瘤病理学分级及生物学行为的相关性。方法:对58例骨巨细胞瘤进行Jaffe分级;采用组织学方法观察出血坏死、侵袭血管或周围组织,病例随访6月-6年确认是否复发;免疫组化SP法检测M-CSF在骨巨细胞瘤中的表达,半定量分析它们与肿瘤病理Jaffe分级及生物学行为之间的相关性。结果:Jaffe分级与肿瘤生物学行为无明显相关性;M-CSF表达与肿瘤的出血坏死、侵袭存在一定的相关性,而与肿瘤的复发无明显相关性;M-CSF表达强度与病理Jaffe分级呈负性相关关系。结论:通过检测骨巨细胞瘤中M-CSF的表达,可以协助进行肿瘤病理Jaffe分级,推断肿瘤的生物学行为并判断预后。     

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.     

肿瘤坏死因子α对小鼠破骨细胞分化的影响

目的:在诱导破骨细胞分化的体外骨髓细胞培养系统中,研究破骨细胞分化因子(RANKL)存在和不存在的情况下,肿瘤坏死因子α(TNF-α)对破骨细胞分化的影响.方法:选用小鼠巨噬细胞集落刺激因子(M-CSF)依赖性非附着性骨髓细胞,在含有25μg/L M-CSF和0,l,10,100μg/L TNF-α的α-MEM培养液中培养5 d后,观察抗酒石酸酸性磷酸酶染色(TRAP)阳性多核细胞的形成;细胞在含有25 μg/L M-CSF和30μg/L sRANKL的α-MEM培养液中进行培养,比较加入和不加人10μg/L TNF-α培养4、5、6和9 d后,所形成的TRAP( )多核细胞的数目和骨吸收面积.结果:TNF-α在没有RANKL的情况下,不能诱导小鼠骨髓细胞形成破骨细胞.在RANKL存在的情况下,TNF-α可促进破骨样细胞的形成和骨吸收,但对破骨细胞分化的促进作用仅表现在培养的早期.结论:在RANKL存在的情况下TNF-α可促进破骨细胞的分化,但不能取代RANKL.TNF-α加速破骨细胞的形成,却并不延长其生存时间.     

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。     

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.     

In vitro osteoclast-suppressing effect of sodium ibandronate

Background  Bisphosphonates (BPs) have been reported to reduce local recurrence in giant cell tumor (GCT) of bone because of their osteoclast-suppressing effect; however, the optimal mode of delivery and the dose and duration of treatment of BPs remain to be established. To address these issues, it is first necessary to clarify the manner of action of BPs on osteoclasts. We herein evaluated the osteoclast-suppressing effect of sodium ibandronate in vitro.

Methods  Mouse osteoclasts (OCLs) were generated in vitro using mouse bone marrow mononuclear cells. First, various concentrations of sodium ibandronate and equal amounts of phosphate-buffered saline were added to cell culture media. The number of multinucleated cells (over three nuclei) was recorded in each group, OCL formation was compared, and the most effective concentration of sodium ibandronate was determined. Then, high concentrations of sodium ibandronate were added to the experimental cell culture media; no ibandronate was given in the control group. Comparisons were made between the two groups in terms of OCL adhesion, migration, and bone resorption.

Results  OCL formation was suppressed by sodium ibandronate in vitro; the most pronounced effect was observed at the concentration of 10^-5 mol/L. OCL migration and bone resorption were significantly suppressed at this concentration, though there was no effect on OCL adhesion.

Conclusions  Sodium ibandronate was effective in suppressing OCLs and decreasing resorption in GCT. The strong anti-OCL effectiveness at a high concentration in vitro indicates a topical mode of application.

     

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.     

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。     

多种细胞因子对人骨髓基质细胞生长的影响

目的:探讨多种细胞因子对骨髓基质细胞生长的影响。方法:分离人骨髓单个核细胞,经培养获得骨髓基质细胞(BMSC),分别用不同的细胞因子组合作用于BMSC,MTT法测细胞增殖。采用Mini MACs系统阳性分离纯化脐血CD34+细胞,用BMSC结合不同的细胞因子组合培养14天后计数CFU-Mix集落数。结果r:hGM-CSF结合FL、SCFI﹑L-6对BMSC生长的增殖作用最明显;BMSC联合SCFI、L-3、FL、G-CSF、EPO对促进CD34+细胞体外集落形成的作用最明显。结论:细胞因子作用下的BMSC扩增有可能应用于造血损伤修复。     

骨巨细胞瘤的诊断与治疗

骨巨细胞瘤(GCT)是一种高侵袭性良性骨肿瘤,主要发生于年轻人中,骨端发病典型的影像学是骨端完全溶骨的膨胀性改变,多不伴有骨膜反应及软组织包块;CT、磁共振可以很好地显示肿瘤的侵及范围;X线平片最具诊断意义。圆形及卵圆形基质细胞及多核巨细胞是GCT的基本结构成分。刮除术是治疗GCT的基本外科手段,但其复发率较高。行刮除术的同时应用高速磨钻处理髓腔或用液氮冷冻、石碳酸烧灼、50%氯化锌液体浸泡以及放疗等辅助方法处理刮除后的病灶,可明显降低复发率。     

骨巨细胞瘤体外原代培养及特性

目的观察骨巨细胞瘤体外培养细胞的形态生长特性及DNA倍体含量。方法取手术切取的骨巨细胞瘤新鲜标本8例,以组织培养法进行原代培养,观察细胞形态、生长曲线,FCM分析其倍体含量。结果骨巨细胞瘤原代培养种可见3种细胞;多核巨细胞可以长期培养。FCM分析显示:5例复发及2例原发病例是二倍体或亚二倍体,1例原发病例是非整倍体。结论单核梭形细胞是骨巨细胞瘤的主要肿瘤性成分;FCM无助于骨巨细胞瘤的预后判断。     

人骨巨细胞瘤细胞DNA含量的研究

将体外培养的人骨巨细胞瘤第四代和第五代瘤细胞,进行~3H—TdR放射自显影术和Feulgen反应,先计算出银粒标记细胞(S,G_2)的增殖比率,再用显微分光光度法测定未被标记的细胞(G_0和G_1期)的DNA含量。结果,第四代单核瘤细胞DNA含量为亚四倍体值((?)=87.36,DI=1.83),第五代单核瘤细胞DNA含量为超四倍体值((?)=118.13,DI=2.47).两代瘤细胞DNA含量经统计学处理,有显著差异(P<0.01),证实了骨巨细胞瘤细胞长期传代培养可以恶化。