脊柱骨巨细胞瘤的病理研究

本文对7例脊柱原发性骨巨细胞瘤(GCT)进行了光镜(LM),组化和免疫组化研究,其中2例进行了电镜(EM)观察。LM和EM显示GCT由单个核细胞(MC)和多核巨细胞(MGC)组成。MC包括成纤维细胞(FB)、组织细胞(HC)、原始间叶细胞、中间型细胞、黄色瘤细胞和肌纤维母细胞(MFB),并见原始间叶细胞过渡为不同分化的FB和HC。MGC由MC融合而成,多数为HC型,少数为兼有FB和HC的混合型。我们认为GCT起源于原始间叶细胞;HC、FB和MFB属肿瘤性细胞,MGC属反应性细胞。     

抗人肝癌细胞单克隆抗体的研制及鉴定

利用杂交瘤技术,以人肝癌细胞株HC_(108)免疫Balb/c小鼠,4周后取其脾与小鼠骨髓瘤细胞SP2/0融合,经细胞抗原ELISA和间接酶桥法筛选,获得一株能稳定分泌抗人肝癌单克隆抗体的杂交瘤株,其染色体众数为96～110个,DNA指数为3.0。此单抗为IgM型,轻链为k链。免疫组化证实此单抗与2例胚胎肝脏组织、胚胎结肠组织呈现较弱的阳性反应;除与3例宫颈癌细胞有很弱的着色反应外,与5例肝癌组织和肝癌细胞株均呈明确的阳性反应,而与其他肿瘤细胞,胚胎组织,正常成人肝脏、心脏、肾脏、肺脏、结肠、胰腺等均未见反应。琼脂糖免疫双扩散和单抗吸收试验证实此肿瘤抗原与甲胎蛋白(AFP)、癌胚抗原(CEA)、铁蛋白(Ferritin)、癌糖蛋白(CA_(19-9))无免疫相关性。免疫酶电镜染色显示此肝癌相关抗原定位于细胞膜,Western Blot显示其分子量为38kD。是一株特异性良好,阳性反应率较高的抗人肝癌单克隆抗体。     

人胰岛提取物与大鼠β肿瘤细胞具有共同抗原决定簇

鉴定抗大鼠β肿瘤细胞系Rin5F细胞膜单克隆抗体所结合的胰岛特异性抗原。采用匀浆后超速离心的方法提取动物胰腺及胰岛细胞膜蛋白。经SDS-PAGE、电转移后,分别与大鼠β肿瘤细胞系Rin5F细胞膜单克隆抗体1A2(第一抗体)和羊抗鼠(第二抗体)反应。用放射自显影、Dot blot、western blot方法显示结果。除单抗1C7以外的其他6株抗大鼠β肿瘤细胞系Rin5F细胞膜单克隆抗体能与正常大鼠胰腺提取物不同成分结合。其中单抗1A2能与大鼠、狗、猪、猴、人的胰腺提取物及人、猪胰岛提取物不同分子量的抗原结合。大鼠β肿瘤细胞系Rin5F细胞与正常大鼠胰腺有共同的抗原决定簇。单抗1A2所结合抗原在不同动物胰腺,尤其是在人和猪胰岛上具有共同抗原决定簇。为1型糖尿病自身免疫发病机制及临床前早期诊断带来了新的希望。     

抗人膀胱癌双功能抗体的制备与鉴定

目的:制备抗人膀胱癌双功能抗体(BfAb)并进行体外实验与鉴定.方法:提取膀胱癌抗原,应用膀胱癌抗原通过杂交瘤技术制备抗人膀胱癌单克隆抗体,后者经胃蛋白酶水解后制备抗人膀胱癌单克隆抗体的F(ab′)2片段.然后将该片段通过二次杂交制备杂交瘤,用其免疫新西兰白兔,血清经凝胶纯化后获抗人膀胱癌BfAb,应用ELISA和免疫组化方法进行鉴别.结果:抗人膀胱癌BfAb与抗人膀胱癌单克隆抗体呈阳性反应,与BALB/C小鼠γ球蛋白呈阴性反应;BfAb可以同时结合靶细胞的肿瘤抗原和效应细胞表面的触发分子,引发正常细胞对肿瘤细胞的防御机制.结论:BfAb既能竞争性地与膀胱移行细胞癌抗原结合,又具有激活淋巴细胞的功能,从而特异性的直接杀伤癌细胞并诱发一系列的免疫应答反应.     

人胰岛提取物与大鼠β肿瘤细胞具有共同抗原决定簇

鉴定抗大鼠β肿瘤细胞系Rin5F细胞膜单克隆抗体所结合的胰岛特异性抗原。采用匀浆后超速离心的方法提取动物胰腺及胰岛细胞膜蛋白。经SDS-PAGE、电转移后，分别与大鼠β肿瘤细胞系Rin5F细胞膜单克隆抗体1A2(第一抗体)和羊抗鼠(第二抗体)反应。用放射自显影、Dot blot、western blot方法显示结果。除单抗1C7以外的其他6株抗大鼠β肿瘤细胞系Rin5F细胞膜单克隆抗体能与正常大鼠胰腺提取物不同成分结合。其中单抗1A2能与大鼠、狗、猪、猴、人的胰腺提取物及人、猪胰岛提取物不同分子量的抗原结合。大鼠β肿瘤细胞系Rin5F细胞与正常大鼠胰腺有共同的抗原决定簇。单抗1A2所结合抗原在不同动物胰腺，尤其是在人和猪胰岛上具有共同抗原决定簇。为1型糖尿病自身免疫发病机制及临床前早期诊断带来了新的希望。     

骨巨细胞瘤中多核巨细胞与其它类型多核细胞的对比观察

通过电镜观察、细胞化学、间接免疫荧光和免疫组织化学方法,对12例骨巨细胞瘤中多核巨细胞(MGC)进行观察,并与破骨细胞(OC)、炎性(异物)巨细胞对比。结果表明四种细胞极为相似,均具有酸性磷酸酶、过氧化物酶、α-1-抗胰蛋白酶、α-1-1抗糜蛋白酶等,这些物质被认为是巨噬细胞标志,提示MGC起源与巨噬细胞有关,并为MGC与OC来自同一祖先提供一定证据。     

体外培养骨巨细胞瘤的细胞DNA合成观察

<正> 骨巨细胞瘤(以下简称 GCT)是由梭形或圆形单核基质细胞及多核巨细胞(以下简称(MGC)构成的一种多细胞成份的半恶性肿瘤。因含有大量 MGC 而得名。我们在过去几年中,对此种肿瘤曾进行了病理学、超微结构、体外组织培养,组织化学、异种动物的接种以及体外培养细胞的定格显微摄影等方面的研究与观察。本文是上述研究工作的继续。采用 ~3H 胸腺嘧啶核苷(~3H—TdR)掺入,利用放射自显影技术,观察 GCT 在体外培养中各种细胞成份的 DNA 合成情况。     

死亡受体5在肿瘤细胞系表面的表达

目的利用抗DR5单克隆抗体(mAb),检测死亡受体5(DeathRecepter5)在肿瘤细胞系表面的表达。方法sDR5免疫BALB/c小鼠,小鼠脾细胞与SP2/0-Ag14细胞在50%PEG条件下融合,获得能够分泌抗DR5单克隆抗体的杂交瘤细胞株,利用杂交瘤细胞株分泌的抗DR5特异性单克隆抗体,采用流式细胞仪技术测定抗DR5mAb结合至细胞表面的量来分析死亡受体在肿瘤细胞系的表达情况。结果不同肿瘤细胞表面DR5的表达水平分别为Jurkat细胞91.61%、NS-10.66%。结论抗DR5mAb能够特异性与DR5结合。DR5在Jurkat细胞系高水平表达,NS-1细胞系几乎不表达。该特异性mAb在研究TRAIL受体的生物功能上是一种有用的工具,对研究DR5在组织和细胞系表达十分有价值。     

泰泽病原体单克隆抗体的制备和鉴定

目的　制备多种抗泰泽病原体的单克隆抗体 ,用于该菌隐性感染的检测。方法　用含泰泽病原体的大鼠肝匀浆免疫BALB c小鼠 ,采用杂交瘤技术和间接免疫荧光方法筛选抗该菌的单克隆抗体 ;运用免疫电镜、Westernblotting和免疫双向扩散试验确定单克隆抗体的抗原定位和相对分子质量及IgG亚类型。结果　共筛选出 5种单克隆抗体 (M2、M3、M4、M5和M7)。免疫电镜表明 :抗体M2、M4、M5、和M7与泰泽病原体的细胞壁结合 ,而M3为抗菌毛的单克隆抗体 ;Westernblotting结果表明 :抗体M2、M3和M4与相对分子质量约为 6 4× 10 3的抗原有特异性结合 ,M5与M7与相对分子质量约为 4 1× 10 3的抗原特异性结合 ;M2、M5、M7为IgG1 ,M3为IgG2b,M4为IgG2a。对模拟自然感染大鼠考的松激发试验阳性为 2 10 ,而单克隆抗体为诊断抗体的IFA则为 8 10。除M7外 ,4种单抗不与清洁级以上大鼠回盲部细菌发生反应。结论　筛选的杂交瘤细胞能稳定分泌抗体 ,其IFA效价达 1∶10 2 4 ;表明这些抗体具有高效价、较高特异性和敏感性。     

大疱性皮肤病自身抗体靶抗原定位研究

目的　进一步确定存在分歧的靶抗原在细胞间或皮肤基底膜带中的位置。验证有关大疱性皮肤病自身抗体靶抗原定位的不同研究结果。方法　采用包埋后金标记直接和间接免疫电镜技术,结合免疫印迹技术分别对5例天疱疮(寻常型4型、落叶型1例)皮损组织中IgG的沉积部位、8例天疱疮(寻常型6例、落叶型2例)患者血清中自身抗体IgG结合正常人皮肤的部位进行超微水平的研究;采用同样方法对5例大疱性类天疱疮皮损组织中IgG的沉积部位及8例大疱性类天疱疮患者血清中自身抗体IgG结合正常人皮肤组织的部位进行定位研究;对3例线形IgA大疱病的皮损组织和血清中自身抗体IgA结合正常人皮肤组织的位置进行定位研究。结果　5例天疱疮患者皮损组织的直接免疫电镜见金颗粒沉积在桥粒上,非桥粒部位的角朊细胞间未见金颗粒的沉积,8例天疱疮血清的间接免疫电镜见金颗粒沉积在桥粒上,寻常型和落叶型的金颗粒沉积部位无区别。5例大疱性类天疱疮皮损组织的直接免疫电镜见有2例金颗粒沉积在半桥粒上,3例金颗粒沉积在半桥粒上及其下方的透明层中;间接免疫电镜技术对8例大疱性类天疱疮血清进行金标记研究,发现有2例金颗粒沉积在半桥粒部位(免疫印迹显示其血清抗体识别BP230)、4例在半桥粒下方的透明层内见金颗粒的沉积(其血清抗体分别识别     

CONTRAST STUDY ON MULTINUCLEATED GIANT C,FLLS IN GIANT CELL TUMORS OF BONE AND OTHER MULTINUCLEATED CELLS

By means of tissue culture, electron microscopy, cytochemistry, indirect immunofluorescence and immunohistochemistry, the multinucleated giant cells(MGCs) in 12 giant cell tumors of bone (GCT) were studied in contrast with osteoclasts (OCs), foreign body giant cells (FGCs) and inflammatory giant cells (IGCs). The findings in the majority of MGCs were identical with those in OCs, suggesting that they most probably derived from the same precursor. Continuous in vitro culture revealed two kinds of MGCs, which were designated preliminarily as short-lived MGCs and long-lived MGCs for their difference in morphology and in several biological features, which suggests two kinds of MGCs exist in GCT. We conclude that the short-lived conform to the typical MGCs known generally to the pathologists, while the long-lived are deemed to be closely related to the neoplastic elements of the tumor.     

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

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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.     

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。     

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.     

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.

     

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。     

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.     

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。