双膦酸盐对骨巨细胞瘤细胞超微结构的影响及细胞组织学变化

目的：探讨双膦酸盐对骨巨细胞瘤细胞超微结构的影响及细胞组织学变化。方法：取我院收治的7例双膦酸盐（唑来膦酸）治疗前后的患者肿瘤组织切片进行了透视电镜观察和TUNEL凋亡染色及图像分析。结果：双膦酸盐（唑来膦酸）治疗后的肿瘤组织切片中透视电镜下多核巨细胞和基质细胞出现了明显的凋亡表现,细胞质变化的特点是大量扩张紊乱的粗面内质网及分散于细胞质中包含于囊泡中央的高电子密度核。线粒体水肿或空泡化。巨细胞核变化的特点是形成致密的染色质材料分散于细胞核中,核膜增厚或分离,部分核碎裂和形成凋亡小体;双膦酸盐（唑来膦酸）治疗后的肿瘤组织切片中TUNEL染色强烈阳性,基质细胞凋亡率从治疗前的1.31%至治疗后的33.42%,差异有显著性意义（P=0.018）,多核巨细胞凋亡率从治疗前的8.41%至治疗后的56.83%,差异有显著性意义（P=0.018）。结论：双膦酸盐可以诱导骨巨细胞瘤基质细胞和多核巨细胞的凋亡。从细胞和分子生物学水平证实双膦酸盐作为骨巨细胞瘤辅助治疗方法的可行性。     

双膦酸盐诱导骨巨细胞瘤细胞凋亡的实验研究

目的　探讨双膦酸盐（唑来膦酸）对骨巨细胞瘤多核巨细胞和基质细胞的诱导凋亡作用。方法　对我院收治的７例唑来膦酸治疗前后患者的肿瘤组织切片进行透射电镜观察和ＴＵＮＥＬ凋亡染色及图像分析。另１０例未予唑来膦酸治疗的患者,取术中切除的骨巨细胞瘤新鲜组织进行细胞原代培养,分别给予５、３０、１５０μｍｏｌ／Ｌ唑来膦酸诱导肿瘤细胞凋亡４８ｈ,倒置相差显微镜观察细胞形态,Ａｎｎｅｘｉｎ-ＶＦＩＴＣ凋亡染色及流式细胞仪定量分析。结果　唑来膦酸治疗后的肿瘤组织切片透射电镜观察到多核巨细胞和基质细胞呈明显的凋亡表现;ＴＵＮＥＬ染色呈强阳性;基质细胞凋亡率治疗前为１.３１％,治疗后３３.４２％（Ｐ＝０.０１８）;多核巨细胞凋亡率治疗前为８.４１％,治疗后５６.８３％（Ｐ＝０.０１８）。在原代培养的骨巨细胞瘤基质细胞中予不同浓度的唑来膦酸诱导凋亡４８ｈ后,细胞数量减少,部分细胞形态有显著变化;Ａｎｎｅｘｉｎ-ＶＦＩＴＣ凋亡染色及流式细胞仪定量分析结果显示,对照组以及５、３０、１５０μｍｏｌ／Ｌ唑来膦酸诱导组的凋亡率分别为（１０.６７±２.１５）％、（２０.４７±３.７７）％、（４４.２１±８.３４）％和（５６.９６±９.６８）％,各诱导组显著高于对照组（Ｐ＜０.０５）,且凋亡率呈剂量依赖性（ｒ＝０.７７５,Ｐ＝０.０００）。结论　双膦酸盐能够诱导骨巨细胞瘤基质细胞和多核巨细胞的凋亡,且骨巨细胞瘤基质细胞的凋亡率呈剂量依赖性,因此,双膦酸盐可以作为骨巨细胞瘤辅助治疗的方法之一。     

唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的实验研究

目的　探讨唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的可行性。方法　取我院收治的７例唑来膦酸治疗前后的患者肿瘤组织切片行ＨＥ染色、茜素红染色、碱性磷酸酶（ＡＬＰ）染色以及成骨前因子Ⅰ型胶原和骨唾液蛋白（ＢＳＰ）的免疫组化染色。取１０例未予唑来膦酸治疗的患者术中切除的骨巨细胞瘤新鲜组织进行原代培养,待骨巨细胞瘤基质细胞贴壁生长后分别给予１μｍｏｌ／Ｌ唑来膦酸诱导１２天后行ＡＬＰ染色、免疫组化法检测Ⅰ型胶原和ＲＴ-ＰＣＲ检测Ｃｂｆａ-１的表达。结果　（１）唑来膦酸治疗前,肿瘤组织切片中骨巨细胞瘤基质细胞ＢＳＰ染色阴性或低至中度阳性、Ⅰ型胶原染色阴性或轻度阳性,茜素红和ＡＬＰ染色均为阴性;治疗后,茜素红染色阳性,ＡＬＰ染色弱阳性,ＢＳＰ和Ⅰ型胶原染色均转为强阳性,３例患者组织切片中ＨＥ染色发现类骨质或骨矿化的增加。（２）原代培养的骨巨细胞瘤基质细胞中予１μｍｏｌ／Ｌ唑来膦酸诱导１２天,空白对照组ＡＬＰ和Ⅰ型胶原染色均为弱阳性表达,唑来膦酸诱导组呈阳性表达。ＲＴ-ＰＣＲ检测发现唑来膦酸诱导组骨巨细胞癌基质细胞中Ｃｂｆａ-１表达,而空白对照组未见Ｃｂｆａ-１表达。结论　体内外研究初步证实,骨巨细胞瘤基质细胞具有向成骨细胞分化的潜能并在唑来膦酸的诱导下向成骨细胞分化,这从细胞和分子生物学水平部分解释了临床上长期应用唑来膦酸治疗后肿瘤病灶和术后残留囊壁中的明显骨生成的现象。     

双膦酸盐辅助治疗骨巨细胞瘤的临床研究

目的：探讨双膦酸盐作为辅助治疗方法降低四肢骨巨细胞瘤复发率的可行性。方法：２００１年２月～２００７年８月我院及香港中文大学威尔士亲王医院收治的３９例四肢骨巨细胞瘤患者,男性２８例,女性１１例,年龄１８～４５岁。肿瘤影像学分级按Ｃａｍｐａｎａｃｃｉ分级标准,其中Ⅱ级１８例,Ⅲ级２１例。所有患者治疗前均经过病理活检诊断为骨巨细胞瘤。分为治疗组和对照组。对照组２０例患者,未予双膦酸盐治疗。治疗组１９例患者,于手术前给予１～３疗程的双膦酸盐治疗（唑来膦酸４ｍｇ,２～３周１次）,术后继续给予３个疗程以上的双膦酸盐治疗。所有患者均接受了手术治疗,手术方式包括囊内切除＋骨水泥或骨移植重建,瘤段切除＋骨移植或人工关节重建。结果：随访１９～３４个月。双膦酸盐治疗后患者疼痛减轻,ＶＡＳ疼痛评分由８.１９±１.３３减轻至２.４２±１.０１,差异有显著性意义（ｔ＝１５.３０４Ｐ＝０.０００）。７８.９％的患者肿胀减轻,６３.２％的患者在唑来膦酸治疗２～３疗程后Ｘ线片上肿瘤内出现明显的骨矿化增加或骨生成。治疗组的复发率为５.３％,低于对照组３５％,差异有显著性意义（χ２＝５.２８４,Ｐ＝０.０２２）。３级骨巨细胞瘤对照组的复发率为５０.０％,治疗组为９.１％,差异有显著性意义（χ２＝４.２９５,Ｐ＝０.０３８）。结论：双膦酸盐作为辅助治疗能够显著降低四肢骨巨细胞瘤的复发率。用药后症状与体征的改善,影像学中骨矿化或骨生成都可作为双膦酸盐疗效的评价方法。     

TNF—α、M—CSF及IL—1在骨巨细胞瘤中的表达及其意义

目的：探讨细胞因子与骨巨细胞瘤局部溶骨的关系。方法：采用ELISA法、Western blot分析和免疫组化双染检测了10例骨巨细胞瘤、5例骨肉瘤和5例正常人血清中TNF－α、含量和M－CSF、IL－1的表达,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。TNF－α、由骨巨细胞的部分单核基因细胞和多核巨细胞分泌;而－CSF及IL－1由部分单核基质细胞份泌,多核巨细胞则不表达。结论骨巨细胞瘤中各种细胞份泌不同的细胞因子可能与该肿瘤的局部溶骨过程。     

唑来膦酸在乳腺癌中抗肿瘤作用的研究现状

唑来膦酸作为第3代双膦酸盐药物已广泛用于乳腺癌骨转移相关事件的防治。唑来膦酸在乳腺癌中具有抗肿瘤治疗作用。唑来膦酸通过抑制乳腺癌细胞的增殖和诱导细胞凋亡,降低肿瘤细胞的迁移、侵袭能力,防止有利于肿瘤生长的骨微环境的形成,抑制肿瘤新生血管形成,调节免疫等多种途径直接或间接发挥其抗肿瘤作用,与放化疗及内分泌等辅助治疗有序贯协同增效作用。唑来膦酸在乳腺癌患者辅助治疗中的协同抗肿瘤作用也在几项相关的前瞻性临床试验中进行。     

地舒单抗在骨巨细胞瘤治疗中的地位

正骨巨细胞瘤是原发交界性骨肿瘤,具有较高局部侵袭性和少见的远处转移性,主要好发于长骨的骨端,也会发生于较难治疗的隐匿部位如骨盆、骶骨和脊柱。骨巨细胞瘤的病理基础是由分泌表达RANKL的梭形基质细胞诱导和促进表达RANK的类破骨前体细胞融合形成多核巨细胞发挥溶骨作用,从而导致病理性骨质破坏。作为局部侵袭性肿瘤,局部的控制率成为骨巨细胞瘤治疗最重要的疗效指标,根据肿瘤的部位,侵     

唑来膦酸对骨肉瘤细胞增殖凋亡和VEGF表达的影响

背景与目的：双膦酸盐是一类强有力的骨吸收抑制剂,被广泛的应用于治疗骨质疏松症、变形性骨炎和恶性肿瘤引起的高钙血症。越来越多实验表明双膦酸盐具有直接的抗肿瘤作用。本研究探讨第三代双膦酸盐：唑来膦酸诱导骨肉瘤LM8细胞凋亡的机制和对VEGF表达的影响。方法：CCK-8法、Hoechst 33258染色形态学观察及Annexin V-FITC/PI双染法证实细胞凋亡,流式细胞仪检测活化caspase-3的表达,免疫组化SABC法检测VEGF的表达。结果：唑来膦酸对骨肉瘤细胞株LM8的增殖抑制呈浓度依赖性,50μmol/L唑来膦酸作用48 h能明显抑制LM8细胞增殖,抑制率为（54±3）%,形态学观察可见明显胞核凝聚、固缩;流式细胞仪检测可见细胞凋亡率为（31.7±2.9）%和caspase-3活化;免疫组化SABC显示VEGF的表达明显被抑制。结论：唑来膦酸可能通过激活caspase-3诱导LM8细胞凋亡,并抑制LM8细胞VEGF的表达。     

常见骨肿瘤及瘤样病变中的巨噬细胞和多核巨细胞观察

本文采用细胞涂片法,利用非特异性酯酶(ANAE)和酸性磷酸酶(ACP)反应。在35例中常见骨肿瘤和瘤样病变中显示巨噬细胞和多核巨细胞。结果表明:所有骨肿瘤和瘤样病变都含有数量不等的巨噬细胞和多核巨细胞。含巨噬细胞较多的病变,多核巨细胞也较多。巨噬细胞与多核巨细胞的ANAE和ACP反应相似,呈弥漫强阳性。这说明两者是有密切的关系。另一方面,从有些多核巨细胞的形态,也支持多核巨细胞是由许多单核巨噬细胞互相融合而成的观点。巨噬细胞和多核巨细胞无异型性,也未见核分裂,因而它们不可能是恶性细胞,而是机体对肿瘤的反应性细胞。基于这个观点,就不难理解本文所有骨病变都存在有巨噬细胞和多核细胞。经统计学分析,各类骨肿瘤和瘤样病变的巨噬细胞数有显著性差异。Ⅰ级骨巨细胞瘤的巨噬细胞数比Ⅱ级骨巨细胞瘤多。骨巨细胞瘤中的巨噬细胞样间质细胞可能是浸润到肿瘤中的巨噬细胞。     

PCNA在骨巨细胞瘤的表达及与病理分级和复发的关系

应用免疫组织化学技术，对32便骨巨细胞瘤进行增殖细胞核抗元原（PCNA）的检测分析，探讨PCNA在骨巨细胞瘤的表达分布及与Jaffe病理分级和复发的关系。结果显示，PCNA的阳性反应只在骨巨细胞瘤波形蛋白阳性的单核基质细胞的胞核中出现，而多核巨细胞均无阳性表达；PCNA在骨巨细胞瘤中的增殖指数虽随Jaffe病理分级增高有呈递增趋势，但各级相互之间在交叉重叠现象；PCNA增殖指数低即低度增生的骨巨细胞瘤复发率低，PCNA增殖指数高即中度和重度增生者复发率明显增高。本文结果进一步支持单核基质细胞是骨巨细胞瘤的主要肿瘤细胞成份的观点，并提示PCNA的表达与Jaffe病理分级不完全一致，但PCNA是判断骨巨细胞瘤预后的一个重要参考指标。     

Expression of c-Src and comparison of cytologic features in cherubism, central giant cell granuloma and giant cell tumors

Cherubism (CBM) and central giant cell granuloma (CGCG) of the jaw and giant cell tumor (GCT) of the long bone are clinically different diseases. Histologically, they are all multinucleated giant cell (MGC)-containing lesions. This study aims to evaluate the expression of c-Src and cytologic features in CBM, CGCG and GCT and to clarify whether there is a common mechanism underlying the formation of multi-nucleated giant cells (MGCs) in these lesions. Specimens and paraffin blocks were collected from patients with CBM (12 cases), CGCG (24 cases) and GCT (37 cases). Histomorpho-metric differences in MGCs were compared among the three types of lesions. The expression of c-Src by immunohistochemistry and in situ hybridization and the expression of TRAP by enzyme histochemical staining were examined. Expression of c-Src mRNA and protein, as well as TRAP staining, was detected in both MGCs and a fraction of mononuclear cells in all investigated lesions. There are no quantitative differences for cytologic features and c-Src expression among the lesions. The results suggested that CBM, CGCG and GCT have overlapping cytological features at the histological level, and c-Src may be involved in the formation of MGCs in the three different diseases.     

Osteoclastoma-like giant cell tumor of the lung

The main components of an unusual form of lung tumor were osteoclast-like multinucleated giant cells and mononuclear stromal cells. Besides, scattered islands of moderately differentiated squamous cells also appeared. Both the mononuclear and the osteoclast-like giant cells reacted with antibodies against CD68 and vimentin, but did not react with antibodies against cytokeratin, EMA and CEA, or lysozyme and α-1-antitrypsin. The p53 and PCNA antigens were positive only in mononuclear cells and not the osteoclast-like giant cells, suggesting that mononuclear cells represent proliferating elements with histiocytic differentiation while osteoclast-like giant cells are stromal, presumably reactive components of the tumor.     

Relationship between binuclear and multinuclear cells in giant cell tumor of bone

Giant cell tumor of bone (GCT) consists of stromal and multinuclear type tumor cells. Although most people believe that the stromal cells are mononuclear, we recently found the existence of many binuclear cells among stromal cells using DNA cytofluorometric examination. This study, using 18 tumors of GCT was conducted to elucidate the cell biological significance of the binuclear cell, especially its relationship to multinuclear cell formation or tumor cell proliferation. The investigation was carried out by means of DNA-RNA cytofluorometry with acridine orange (AO) and histological method. Using fluorescence microscopic observation, we counted the numbers of both mononuclear and binuclear cells and calculated the index of % BNC, which expresses the frequency (percentage) of binuclear cells in a population of mononuclear and binuclear cells. The index of % S-G2 obtained by DNA-RNA cytofluorometry showed the frequency (percentage) of mononuclear cells in the S and G2 phases of the cell cycle. In the histological study, we counted the numbers of multinuclear giant cells with more than 3 nuclei in the cytoplasm and stromal cells including mononuclear and binuclear cells and calculated MNS/SC, which showed the percentage of multinuclear cells in the stromal cells in the microscopic field. Eight tumors showed a value of % BNC greater than 10% and 2 had a value of 40%. The index of % BNC significantly correlated with the average value of MNC/SC in all tumors. There was no significant correlation between % BNC and the average value of % S-G2, in 18 tumors although 4 tumors having a % BNC value greater than 20% showed a % S-G2 value greater than 12% in 18 tumors. These results revealed the presence of many binuclear cells among stromal cells of GCT and suggested that these binuclear cells might be formed in association with the active proliferation of mononuclear cells and closely relate to the formation of multinuclear giant cells.     

Silencing of the UCHL1 gene in giant cell tumors of bone

Giant cell tumors are heterogeneous tumors consisting of multinucleated giant cells, fibroblast‐like stromal cells and mononuclear histiocytes. The stromal cells have been identified as the neoplastic cell population, which promotes the recruitment of histiocytes and the formation of giant cells. Strong evidence exists that these cells develop from mesenchymal stem cells (MSCs) but little is known about the molecular mechanisms involved in GCT tumorigenesis. The aim of our study was the identification of cancer‐related genes differentially expressed in GCTs compared to MSCs in order to identify possible targets for aberrant promoter methylation, which may contribute to MSC transformation and GCT development. Gene expression of 440 cancer‐related genes was analyzed by DNA microarrays in GCT stromal cells and bone marrow‐derived MSCs (BMSCs) isolated from the same patient (n = 3) to avoid interindividual variations. Differential expression was identified for 14 genes, which could be confirmed by quantitative PCR in further 21 GCT and 10 BMSC samples. The most pronounced difference in gene expression was detected for UCHL1, an important regulator of the ubiquitin proteasome pathway. Methylation‐specific PCR and bisulfite sequencing revealed a strong methylation of the CpG island covering the UCHL1 promoter in GCT stromal cells, whereas methylation was completely absent in BMSCs. UCHL1 expression in stromal cells could be restored by the methylation inhibitor 5‐aza‐dC. These data demonstrate that the UCHL1 gene is inactivated in GCTs but not in MSCs, suggesting a possible role of UCHL1 in MSC transformation and GCT development.     

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。     

两例颈部淋巴结转移性低分化癌伴Langhans样巨细胞反应针吸细胞学误诊分析

目的 探讨颈部肿块细针吸取细胞学(Fine Needle Aspiration Cytology,FNAC)误诊原因,提高FNAC诊断水平。方法 回顾性分析两例颈部淋巴结转移性低分化癌伴Langhans样巨细胞反应的临床表现、影像学检查、细胞学特征、病理形态和免疫组织化学(EnVision法)染色观察。结果 两例患者颈部肿块穿刺,镜下均以淋巴细胞为主,少许类上皮细胞和多核巨细胞,以及少许坏死组织,偶见少许成团或散在的椭圆形细胞,个别细胞核大,意义不明确。误诊为淋巴结肉芽肿性病变,倾向结核。手术标本,见结节多枚,包膜完整,切面灰白、质中,可见灶性坏死。组织学特点:淋巴结组织中见多灶成巢分布小圆形或椭圆形细胞,在癌巢周围可见成片或散在的Langhans样巨细胞,癌巢中可见多灶性凝固性坏死。免疫组织化学标记结果:瘤细胞均示CKpan+++,p63+++,1例CK5/6+++,另1例CK8/18++;病理诊断为淋巴结转移性低分化鳞状细胞癌,伴Langhans样巨细胞反应。结论 淋巴结针吸细胞学发现多核巨细胞并坏死组织时易误诊为肉芽肿性病变或结核,需仔细寻找有无肿瘤细胞,必要时需经组织学活检,并辅以免疫组织化学确诊。