脊索细胞遗迹与脊索瘤发生的相关性研究
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摘要
第一部分:胎儿、成人髓核和脊索瘤的组织形态学研究
目的:对胎儿、成人髓核和脊索瘤组织的细胞组成和组织学行比较研究,寻找脊索瘤起源于胚胎残余脊索细胞的组织学依据。
方法:取12-40周胎儿髓核30例,成人退变髓核组织26例,每个标本分成两部分,一部分10%的中性福尔马林固定后石蜡包埋,另一部分冰冻液氮保存。并与同时有腊块和冰冻保存的21例脊索瘤标本共同研究。
石蜡包埋的标本通过切片、脱蜡、水化、消除内源性过氧化酶反应,抗原修复,滴加vimentin、galectin-3和connexin-43工作液50ul,置于4℃冰箱过夜,蒸馏水及PBS冲洗后滴加生物素标记的二抗显色、复染、分化、脱水,切片用中性树胶封片,干燥后显微镜下观察。同时取出液氮冰冻组织块切片,滴加特异性抗体vimentin、galectin-3,connexin-43,加入FITC标记的二抗,37℃孵育,PBS冲洗后加PI染细胞核,避光室温下保存10分钟,PBS冲洗、90%酒精脱水、荧光抗淬灭封片剂封片,以备激光共聚焦显微镜下观察,半定量。
结果:显微镜下观察胎儿髓核标本,发现脊索细胞和软骨样髓核细胞在细胞大小上有明显的区别,前者直径是后者的三倍左右。脊索细胞的数目与胎儿孕龄成负相关.从细胞组成上分析,22周前脊索细胞为主要成分,占细胞总数的60%以上;28周后脊索细胞数目显著减少至40%以下,进而软骨样髓核细胞成为主要细胞类型。脊索细胞主要呈集落形式存在,随着年龄增长,脊索细胞集落逐渐减少。免疫荧光标记的蛋白vimentin ,galectin-3,connexin-43在胎儿髓核脊索细胞中亦与孕龄呈现明显的负相关,孕龄越小,表达强度越大。胎儿脊索细胞与脊索瘤细胞相比较:两者细胞大小无显著差异;vimentin平均荧光强度胎儿脊索细胞明显高于脊索瘤细胞,存在显著差异;galectin-3在胎儿脊索细胞中等程度表达,而在脊索瘤细胞呈强表达;connexin-43在胎儿脊索细胞表达,而在脊索瘤细胞中无表达。胎儿软骨样髓核细胞和成人退变髓核细胞的比较:两种细胞大小直径无显著差异;vimentin表达亦无显著差异;galectin-3在胎儿软骨样髓核细胞中不表达,而在成人退变髓核细胞中呈现强表达;connexin-43在两种组织中均不表达。
结论:组织学研究发现随着孕龄的增加,胎儿髓核内脊索细胞的数目逐渐减少,并逐渐被软骨样髓核细胞所取代。通过对胎儿和成人退变髓核以及脊索瘤标本的免疫组化和共聚焦显微镜荧光强度的测量,vimentin ,galectin-3,connexin-43在脊索细胞中的表达随着孕龄增加逐渐减弱。脊索细胞特征性的标志物vimentin,galectin-3在胎儿脊索细胞,脊索瘤细胞中均表达。作为细胞缝隙连接的特异性标志物connexin-43,在胎儿脊索细胞中由于细胞集落的存在,其表达阳性;脊索瘤中由于细胞间失去连接而呈无序生长,因而其表达变为阴性。胎儿脊索细胞和脊索瘤细胞在形态学上有相似之处,但脊索瘤细胞同时表现出肿瘤细胞的特性。
第二部分:各种脊索瘤标志物的比较研究
目的:分析比较各种脊索瘤标记物的敏感性。
方法:收集1990年1月-2009年5月临床及病理资料均完整的脊索瘤标本46例,腊块4um厚连续切片,60oC烤箱烤片30分钟,脱蜡、水化、消除内源性过氧化物酶,抗原修复,滴加一抗(包括EMA,AE1/3,CAM5.2,Vimentin,S-100,Brachyury,Galectin-3),冰箱过夜;滴加生物素标记的二抗,新鲜配置DAB显色,以切片出现较强的棕黄色后双蒸水终止染色,中性树胶封片,干燥后显微镜下观察结果。表达部位:EMA浆和膜表达;Cytokeratin AE1/3和vimentin浆表达;galectin-3和S-100核/浆达;brachyury核表达。免疫组化的评分根据阳性反应细胞的百分比评估:1+=<50%,2+=50%-80%,3+=81%-100%。在400倍镜下随机取5个视野后平均。
结果:HE染色普通脊索瘤可见肿瘤细胞被纤维组织分隔成小叶状的结构,瘤细胞常有大的异型性核,嗜酸性胞浆,含有大小不一的空泡,具有低度恶性肿瘤的形态学特点。软骨样脊索瘤镜下特点除普通脊索瘤所见外,尚含有多少不等的透明软骨样区域。未分化脊索瘤细胞增殖活跃,粘液含量显著减少并可见到核分裂象,胞浆内空泡较少或无空泡,胞核形态轻度异形。
免疫组织化学:所有脊索瘤均对传统上皮性和间叶性脊索瘤肿瘤标记物具有良好的敏感性,上皮膜抗原EMA,AE1/AE3,CAM5.2,Vimentin,S-100 protein等阳性率均为91.3-100%。新的脊索瘤标志物brachyury和galectin-3的阳性率均为100%。结论:脊索瘤虽源于间叶组织,但上皮性标记物为阳性。传统脊索瘤标志物显示出良好的敏感性;Brachyury是第一个被证实的独特联系胚胎结构和肿瘤的特殊分子,对脊索瘤亦显示出良好的敏感性;传统的脊索细胞标志物galectin-3在本课题46例脊索瘤中阳性表达率为100%。Brachyury和galectin-3可作为传统脊索瘤标志物的有益补充,但其特异性尚需与其它肿瘤一起作大样本进一步研究。
第三部分:胎儿脊索细胞遗迹和脊索瘤的组织学比较研究
目的:寻找脊索瘤中共存的脊索细胞遗迹存在的几率,并将其与胎儿脊索细胞遗迹,脊索瘤组织作比较组织学研究,寻找脊索瘤起源于脊索细胞遗迹的可能证据。
方法:收集脊索瘤标本46例,石蜡切片HE染色后光镜下寻找脊索细胞遗迹。脊索瘤标本和30例胎儿髓核腊块切片,脱蜡至水、消除内源性过氧化物酶的活性,正常山羊血清封闭,滴加稀释后的一抗工作液(EMA,AE1/3,CAM5.2,vimentin,S-100,brachyury,galectin-3,connexin-43)4℃过夜;滴加生物素标记二抗,新鲜配置DAB显色,以切片出现较强的棕黄色后双蒸水终止染色,用中性树胶封片,干燥后显微镜下观察结果。阳性表达为清晰的棕黄色颗粒,connexin-43定位在细胞膜细胞间连接处,其余抗体定位同第二部分。免疫组化的评分根据阳性反应细胞的百分比评估:1+=<50%,2+=50%-80%,3+=81%-100%。在400倍镜下随机取5个视野后平均。
结果:本研究通过光学显微镜分析脊索瘤的组织病理学,在46例脊索瘤标本中发现发现6例脊索细胞遗迹:由片状空泡细胞组成,核位于中心或周围,呈圆形;核通常小,温和,无有丝分裂像;缺乏细胞外粘液样基质。传统脊索瘤标志物EMA,AE/AE3,CAM5.2,vimentin,S-100在脊索瘤和脊索细胞遗迹中表达率均较高,两者无显著差异。而brachyury在脊索瘤中阳性表达率为100%,在脊索瘤内共存的脊索细胞遗迹全部阴性,可作为脊索瘤的特异性标志。脊索瘤细胞brachyury核表达强阳性,而共存的脊索细胞遗迹完全阴性,两种成分共存形成强烈对比,中间被纤维结缔组织间隔分割。galectin-3在脊索瘤中阳性表达率为100%,在共存的6例脊索细胞遗迹中1例表达,可作为脊索瘤与共存的脊索细胞遗迹鉴别的辅助分子。胎儿脊索细胞遗迹和脊索瘤中共存的脊索细胞遗迹之间的比较:Connexin-43阳性表达率在两者之间无显著差异;vimentin在两者均强表达; brachyury在两者均不表达;galectin-3在胎儿脊索细胞遗迹中的阳性率为66.7%,而在脊索瘤内共存的脊索细胞遗迹中其阳性率为16.7%,两者相比有显著差异。
结论:本实验通过回顾性研究,在46例脊索瘤标本中发现6例共存的脊索细胞遗迹。比较脊索瘤和共存的脊索细胞遗迹,brachyury可作为鉴别两者的特异性分子,galectin-3可作为辅助鉴别的分子。比较胎儿脊索细胞遗迹和脊索瘤内共存的脊索细胞遗迹,brachyury在两者均不表达,而在脊索瘤中却全部强表达,提示brachyury活化可能参与脊索瘤的形成。
PartⅠMorphological study on fetal , adult nucleus pulposus and Chordoma
Objectives: To evaluate the cell and tissure composition of fetal , adult nucleus pulposus and chordoma, providing the morphological evidence that chordoma originate from notochordal cell rests
Methods: Taking 12-40 weeks fetal and adult nucleus pulposus ,each specimen was divided into two parts, one part was put into 10% neutral formalin, paraffin-embedded ; another part was frozen cryopreserved. The chordoma specimens both paraffin-embedded and frozen cryopreserved were studied together.
By immunohistochemistry method ,all specimens were sectioned, dewaxed, hydrated, eliminated endogenous peroxidase enzyme reaction. Microwave antigen retrieval solution were used to take antigen repair, dropping vimentin, galectin-3 and connexin-43 working fluid 50ul on the sections, then placed in 4℃refrigerator overnight, distilled water and PBS washed ,dropping biotinylated secondary antibody ,DAB colour, neutral gum Fengpian slice, observed under the microscope after drying. At the same time removed the liquid nitrogen frozen tissue block, sliced, dropping specific antibody vimentin, galectin-3,connexin-43, added FITC labeled secondary antibodies ,incubated at 37℃, PI stained nucleus, darkly stored at room temperature for10min, PBS washed , 90% alcohol dehydrated, anti-fluorescence quenching Fengpian to prepare for laser confocal microscope observation, semi-quantitative analysis.
Results: fetal nucleus pulposus specimens were observed under the microscope and found that the diameter of notochordal cells is three folds of chondrocyte-like cells.The amount of notochordal cells negatively correlated with the gestation weeks. The notochordal cells accounted for the main cell population, more than 60% before 22th gestation weeks, then the number of notochordal cells were significantly reduced to 40% or less after 28th gestation weeks, chondrocyte-like nucleus pulposus cells became the main cell type. Notochordal cells existed in cluster. The number of clusters decreased continuously with gestational weeks. The mean fluorescent intensity of vimentin, galectin-3 and connexin-43 in the notochordsl cells showed a negative correlation with gestational age.There was no significant difference in cell size between notochordal cells and chordoma tumor cells.the mean fluorescent intensity of vimentin in notochordal cells was greater than in chordoma tumor cells.the expression of galectin-3 showed positive in fetal notochodal cells and strong in chordoma tumor cells. Connexin-43 showed positive in fetal notochordal cells and negative in chordoma tumor cells.there was no significant difference in cell size and vimentin expression between fetal chondrocyte-like cells and adult nucleus pulposus cells. Galectin-3 showed negative in fetal chondrocyte-like cells and stong expression in adult nucleus pulposus cells. No galectin-3 expression was detected in both fetal and adult chondrocyte-like cells.
Conclusions: In this study, using immunohistochemical methods, the amount of notochordal cells in the fetal nucleus pulposus decreased continuously with the increasement of gestational weeks. Through the immunohistochemistry and the measurement of fluorescent intensity in CLSM, the expression of vimentin, galectin-3 and connexin-43 continously decreased with gestational weeks increasement. Vimentin and galectin-3 also was expressed in chordoma tumor cells. connexin-43 was positive in fetal nucleus pulposus and negative in chordoma . The notochordal cells in fetal nucleus pulposus demonstrated some similarity with chordoma tumor cells ,but the latter showed tumor cell characteristics simultaneously.
PartⅡComparative Study of various markers of chordoma
Objectives: To compare the sensitivity of various chordoma markers.
Methods: Collect 46 cases of chordoma specimens from January 1990 to May 2009, 4um thick serial sections, 60oC baking sheet for 30 minutes, dewaxed, hydrated, eliminated endogenous peroxidase enzymes , antigen repair, dropping first antibodies (including:EMA,AE1/3,CAM5.2,vimentin,S-100,brachyury,galectin-3);following dropping biotinylated secondary antibody, fresh configuration DAB color to slices untill appearing strong brown staining, terminating staining with double-distilled water, using the Fengpian of gum . Interpretation of protein expression in tumor cells was as follows: cytoplasmic and membranous expression for EMA; cytoplasmic expression for cytokeratin AE1/3 and vimentin;nuclear and cytoplasmic expression for galectin-3 and S-100 protein; nuclear expression for brachyury, Immunoreactivity was assessed for percent of positive expression cells, 1+=<50%,2+=50%-80%,3+=81%-100%.
Results: The classic chordoma consists of cords and strands of atypical physaliphorous cells set within abundant myxoid matrix, The tumor cells show irregular-shaped, hyperchromatic nuclei–demonstrating the morphological characteristics of low-grade malignant tumor. Chondroid chordoma , besides the classic chordoma appearance, contained hyaline cartilage-like domain. Anaplastic chordoma cells showed active proliferation and little myxoid matrix, sometimes mitotic figures can be seen, little or no intracytoplasmic vacuoles .
Immunohistochemistry: All traditional epithelial and mesenchymal tumor marker for chordoma has a good sensitivity, epithelial membrane antigen EMA, AE1/AE3, CAM5.2, vimentin, S-100 protein showed positive-cell expression rates of 91.3-100%. The new markers ,brachyury and galectin-3,showed 100% positive-cell expression rate.
Conclusions: Although chordoma derived from mesenchymal tissue, but showed positive for epithelial markers. Traditional chordoma markers showed good sensitivity. Brachyury is the first specific molecular linking embryo structure with tumor ,it showed a good sensitivity for chordoma in this study. Galectin-3,as a traditional notochordal cell marker ,showed 100% of positive expression rate in total 46 cases chordoma. Brachyury and galectin-3 can be used as a supplement to traditional chordoma markers, but their specificity to chordoma awaits further study together with other large number of various tumors.
PartⅢThe comparative histological study of fetal notochordal cell rest and chordoma.
Objectives: To detect the probability of notochordal cell rests coexisting with chordoma, and further compare it with fetal notochordal cell rests and chordoma in order to find the possible evidence that chordoma originate from notochordal cell rests.
Methods: Collected 46 cases of chordoma specimens , paraffin section and HE staining ,then observed to find notochordal cell rests coexsting. Chordoma and fetal nuclesus pulposus specimens were dewaxed and operated with immunohistochemistryl staining. The first antibodies ( EMA,AE1/3,CAM5.2, vimentin, S-100, brachyury, galectin-3, connexin-43)were added ,further dropped with diluted biotinylated secondary antibody, at 37℃incubated for 10 to 30 minutes, then dropped with diluted horseradish HRP-streptavidin . DAB colored and Fengpian gum sealed.
Results: We found six cases of notochordal cell rests coexist with chodoma specimens..They were composed of physaliphorous cells with bland nuclei and large intracytoplasmic vacuoles, no extracellular mucous matrix existing. All the traditional chordoma markers including EMA, AE/AE3, CAM5.2, vimentin, s-100 demonstrated high positive expression rate in chordoma and coexisting notochordal cell rests. Brachyury was expressed in 100% of chordoma and not expressed in notohordal cell rests,it can be used as specific markers for chordoma.Galectin-3 was expressed in total chordomas and 1 of 6 cases coexisting notochordal cell rests, this molecular can be used as an auxillary tool in differentiating chordoma with coexisting notochordal cell rests.
Conclusions: By retrospective study, we found 6 cases of notochordal cell rests coexisting with chordoma specimens. Brachyury can be used as a specific molecular in differentiating chordoma with notochordal cell rests, galectin-3 can be used as an auxillary tool. Due to no expression of brachyury in fetal notochordal cell rests and coexisting notochordal cell rests in chordoma , yet strong expression in chordoma ,it suggested that brachyury activation may be involved in chordoma tumorigenesis.
目的:对胎儿、成人髓核和脊索瘤组织的细胞组成和组织学行比较研究,寻找脊索瘤起源于胚胎残余脊索细胞的组织学依据。
方法:取12-40周胎儿髓核30例,成人退变髓核组织26例,每个标本分成两部分,一部分10%的中性福尔马林固定后石蜡包埋,另一部分冰冻液氮保存。并与同时有腊块和冰冻保存的21例脊索瘤标本共同研究。
石蜡包埋的标本通过切片、脱蜡、水化、消除内源性过氧化酶反应,抗原修复,滴加vimentin、galectin-3和connexin-43工作液50ul,置于4℃冰箱过夜,蒸馏水及PBS冲洗后滴加生物素标记的二抗显色、复染、分化、脱水,切片用中性树胶封片,干燥后显微镜下观察。同时取出液氮冰冻组织块切片,滴加特异性抗体vimentin、galectin-3,connexin-43,加入FITC标记的二抗,37℃孵育,PBS冲洗后加PI染细胞核,避光室温下保存10分钟,PBS冲洗、90%酒精脱水、荧光抗淬灭封片剂封片,以备激光共聚焦显微镜下观察,半定量。
结果:显微镜下观察胎儿髓核标本,发现脊索细胞和软骨样髓核细胞在细胞大小上有明显的区别,前者直径是后者的三倍左右。脊索细胞的数目与胎儿孕龄成负相关.从细胞组成上分析,22周前脊索细胞为主要成分,占细胞总数的60%以上;28周后脊索细胞数目显著减少至40%以下,进而软骨样髓核细胞成为主要细胞类型。脊索细胞主要呈集落形式存在,随着年龄增长,脊索细胞集落逐渐减少。免疫荧光标记的蛋白vimentin ,galectin-3,connexin-43在胎儿髓核脊索细胞中亦与孕龄呈现明显的负相关,孕龄越小,表达强度越大。胎儿脊索细胞与脊索瘤细胞相比较:两者细胞大小无显著差异;vimentin平均荧光强度胎儿脊索细胞明显高于脊索瘤细胞,存在显著差异;galectin-3在胎儿脊索细胞中等程度表达,而在脊索瘤细胞呈强表达;connexin-43在胎儿脊索细胞表达,而在脊索瘤细胞中无表达。胎儿软骨样髓核细胞和成人退变髓核细胞的比较:两种细胞大小直径无显著差异;vimentin表达亦无显著差异;galectin-3在胎儿软骨样髓核细胞中不表达,而在成人退变髓核细胞中呈现强表达;connexin-43在两种组织中均不表达。
结论:组织学研究发现随着孕龄的增加,胎儿髓核内脊索细胞的数目逐渐减少,并逐渐被软骨样髓核细胞所取代。通过对胎儿和成人退变髓核以及脊索瘤标本的免疫组化和共聚焦显微镜荧光强度的测量,vimentin ,galectin-3,connexin-43在脊索细胞中的表达随着孕龄增加逐渐减弱。脊索细胞特征性的标志物vimentin,galectin-3在胎儿脊索细胞,脊索瘤细胞中均表达。作为细胞缝隙连接的特异性标志物connexin-43,在胎儿脊索细胞中由于细胞集落的存在,其表达阳性;脊索瘤中由于细胞间失去连接而呈无序生长,因而其表达变为阴性。胎儿脊索细胞和脊索瘤细胞在形态学上有相似之处,但脊索瘤细胞同时表现出肿瘤细胞的特性。
第二部分:各种脊索瘤标志物的比较研究
目的:分析比较各种脊索瘤标记物的敏感性。
方法:收集1990年1月-2009年5月临床及病理资料均完整的脊索瘤标本46例,腊块4um厚连续切片,60oC烤箱烤片30分钟,脱蜡、水化、消除内源性过氧化物酶,抗原修复,滴加一抗(包括EMA,AE1/3,CAM5.2,Vimentin,S-100,Brachyury,Galectin-3),冰箱过夜;滴加生物素标记的二抗,新鲜配置DAB显色,以切片出现较强的棕黄色后双蒸水终止染色,中性树胶封片,干燥后显微镜下观察结果。表达部位:EMA浆和膜表达;Cytokeratin AE1/3和vimentin浆表达;galectin-3和S-100核/浆达;brachyury核表达。免疫组化的评分根据阳性反应细胞的百分比评估:1+=<50%,2+=50%-80%,3+=81%-100%。在400倍镜下随机取5个视野后平均。
结果:HE染色普通脊索瘤可见肿瘤细胞被纤维组织分隔成小叶状的结构,瘤细胞常有大的异型性核,嗜酸性胞浆,含有大小不一的空泡,具有低度恶性肿瘤的形态学特点。软骨样脊索瘤镜下特点除普通脊索瘤所见外,尚含有多少不等的透明软骨样区域。未分化脊索瘤细胞增殖活跃,粘液含量显著减少并可见到核分裂象,胞浆内空泡较少或无空泡,胞核形态轻度异形。
免疫组织化学:所有脊索瘤均对传统上皮性和间叶性脊索瘤肿瘤标记物具有良好的敏感性,上皮膜抗原EMA,AE1/AE3,CAM5.2,Vimentin,S-100 protein等阳性率均为91.3-100%。新的脊索瘤标志物brachyury和galectin-3的阳性率均为100%。结论:脊索瘤虽源于间叶组织,但上皮性标记物为阳性。传统脊索瘤标志物显示出良好的敏感性;Brachyury是第一个被证实的独特联系胚胎结构和肿瘤的特殊分子,对脊索瘤亦显示出良好的敏感性;传统的脊索细胞标志物galectin-3在本课题46例脊索瘤中阳性表达率为100%。Brachyury和galectin-3可作为传统脊索瘤标志物的有益补充,但其特异性尚需与其它肿瘤一起作大样本进一步研究。
第三部分:胎儿脊索细胞遗迹和脊索瘤的组织学比较研究
目的:寻找脊索瘤中共存的脊索细胞遗迹存在的几率,并将其与胎儿脊索细胞遗迹,脊索瘤组织作比较组织学研究,寻找脊索瘤起源于脊索细胞遗迹的可能证据。
方法:收集脊索瘤标本46例,石蜡切片HE染色后光镜下寻找脊索细胞遗迹。脊索瘤标本和30例胎儿髓核腊块切片,脱蜡至水、消除内源性过氧化物酶的活性,正常山羊血清封闭,滴加稀释后的一抗工作液(EMA,AE1/3,CAM5.2,vimentin,S-100,brachyury,galectin-3,connexin-43)4℃过夜;滴加生物素标记二抗,新鲜配置DAB显色,以切片出现较强的棕黄色后双蒸水终止染色,用中性树胶封片,干燥后显微镜下观察结果。阳性表达为清晰的棕黄色颗粒,connexin-43定位在细胞膜细胞间连接处,其余抗体定位同第二部分。免疫组化的评分根据阳性反应细胞的百分比评估:1+=<50%,2+=50%-80%,3+=81%-100%。在400倍镜下随机取5个视野后平均。
结果:本研究通过光学显微镜分析脊索瘤的组织病理学,在46例脊索瘤标本中发现发现6例脊索细胞遗迹:由片状空泡细胞组成,核位于中心或周围,呈圆形;核通常小,温和,无有丝分裂像;缺乏细胞外粘液样基质。传统脊索瘤标志物EMA,AE/AE3,CAM5.2,vimentin,S-100在脊索瘤和脊索细胞遗迹中表达率均较高,两者无显著差异。而brachyury在脊索瘤中阳性表达率为100%,在脊索瘤内共存的脊索细胞遗迹全部阴性,可作为脊索瘤的特异性标志。脊索瘤细胞brachyury核表达强阳性,而共存的脊索细胞遗迹完全阴性,两种成分共存形成强烈对比,中间被纤维结缔组织间隔分割。galectin-3在脊索瘤中阳性表达率为100%,在共存的6例脊索细胞遗迹中1例表达,可作为脊索瘤与共存的脊索细胞遗迹鉴别的辅助分子。胎儿脊索细胞遗迹和脊索瘤中共存的脊索细胞遗迹之间的比较:Connexin-43阳性表达率在两者之间无显著差异;vimentin在两者均强表达; brachyury在两者均不表达;galectin-3在胎儿脊索细胞遗迹中的阳性率为66.7%,而在脊索瘤内共存的脊索细胞遗迹中其阳性率为16.7%,两者相比有显著差异。
结论:本实验通过回顾性研究,在46例脊索瘤标本中发现6例共存的脊索细胞遗迹。比较脊索瘤和共存的脊索细胞遗迹,brachyury可作为鉴别两者的特异性分子,galectin-3可作为辅助鉴别的分子。比较胎儿脊索细胞遗迹和脊索瘤内共存的脊索细胞遗迹,brachyury在两者均不表达,而在脊索瘤中却全部强表达,提示brachyury活化可能参与脊索瘤的形成。
PartⅠMorphological study on fetal , adult nucleus pulposus and Chordoma
Objectives: To evaluate the cell and tissure composition of fetal , adult nucleus pulposus and chordoma, providing the morphological evidence that chordoma originate from notochordal cell rests
Methods: Taking 12-40 weeks fetal and adult nucleus pulposus ,each specimen was divided into two parts, one part was put into 10% neutral formalin, paraffin-embedded ; another part was frozen cryopreserved. The chordoma specimens both paraffin-embedded and frozen cryopreserved were studied together.
By immunohistochemistry method ,all specimens were sectioned, dewaxed, hydrated, eliminated endogenous peroxidase enzyme reaction. Microwave antigen retrieval solution were used to take antigen repair, dropping vimentin, galectin-3 and connexin-43 working fluid 50ul on the sections, then placed in 4℃refrigerator overnight, distilled water and PBS washed ,dropping biotinylated secondary antibody ,DAB colour, neutral gum Fengpian slice, observed under the microscope after drying. At the same time removed the liquid nitrogen frozen tissue block, sliced, dropping specific antibody vimentin, galectin-3,connexin-43, added FITC labeled secondary antibodies ,incubated at 37℃, PI stained nucleus, darkly stored at room temperature for10min, PBS washed , 90% alcohol dehydrated, anti-fluorescence quenching Fengpian to prepare for laser confocal microscope observation, semi-quantitative analysis.
Results: fetal nucleus pulposus specimens were observed under the microscope and found that the diameter of notochordal cells is three folds of chondrocyte-like cells.The amount of notochordal cells negatively correlated with the gestation weeks. The notochordal cells accounted for the main cell population, more than 60% before 22th gestation weeks, then the number of notochordal cells were significantly reduced to 40% or less after 28th gestation weeks, chondrocyte-like nucleus pulposus cells became the main cell type. Notochordal cells existed in cluster. The number of clusters decreased continuously with gestational weeks. The mean fluorescent intensity of vimentin, galectin-3 and connexin-43 in the notochordsl cells showed a negative correlation with gestational age.There was no significant difference in cell size between notochordal cells and chordoma tumor cells.the mean fluorescent intensity of vimentin in notochordal cells was greater than in chordoma tumor cells.the expression of galectin-3 showed positive in fetal notochodal cells and strong in chordoma tumor cells. Connexin-43 showed positive in fetal notochordal cells and negative in chordoma tumor cells.there was no significant difference in cell size and vimentin expression between fetal chondrocyte-like cells and adult nucleus pulposus cells. Galectin-3 showed negative in fetal chondrocyte-like cells and stong expression in adult nucleus pulposus cells. No galectin-3 expression was detected in both fetal and adult chondrocyte-like cells.
Conclusions: In this study, using immunohistochemical methods, the amount of notochordal cells in the fetal nucleus pulposus decreased continuously with the increasement of gestational weeks. Through the immunohistochemistry and the measurement of fluorescent intensity in CLSM, the expression of vimentin, galectin-3 and connexin-43 continously decreased with gestational weeks increasement. Vimentin and galectin-3 also was expressed in chordoma tumor cells. connexin-43 was positive in fetal nucleus pulposus and negative in chordoma . The notochordal cells in fetal nucleus pulposus demonstrated some similarity with chordoma tumor cells ,but the latter showed tumor cell characteristics simultaneously.
PartⅡComparative Study of various markers of chordoma
Objectives: To compare the sensitivity of various chordoma markers.
Methods: Collect 46 cases of chordoma specimens from January 1990 to May 2009, 4um thick serial sections, 60oC baking sheet for 30 minutes, dewaxed, hydrated, eliminated endogenous peroxidase enzymes , antigen repair, dropping first antibodies (including:EMA,AE1/3,CAM5.2,vimentin,S-100,brachyury,galectin-3);following dropping biotinylated secondary antibody, fresh configuration DAB color to slices untill appearing strong brown staining, terminating staining with double-distilled water, using the Fengpian of gum . Interpretation of protein expression in tumor cells was as follows: cytoplasmic and membranous expression for EMA; cytoplasmic expression for cytokeratin AE1/3 and vimentin;nuclear and cytoplasmic expression for galectin-3 and S-100 protein; nuclear expression for brachyury, Immunoreactivity was assessed for percent of positive expression cells, 1+=<50%,2+=50%-80%,3+=81%-100%.
Results: The classic chordoma consists of cords and strands of atypical physaliphorous cells set within abundant myxoid matrix, The tumor cells show irregular-shaped, hyperchromatic nuclei–demonstrating the morphological characteristics of low-grade malignant tumor. Chondroid chordoma , besides the classic chordoma appearance, contained hyaline cartilage-like domain. Anaplastic chordoma cells showed active proliferation and little myxoid matrix, sometimes mitotic figures can be seen, little or no intracytoplasmic vacuoles .
Immunohistochemistry: All traditional epithelial and mesenchymal tumor marker for chordoma has a good sensitivity, epithelial membrane antigen EMA, AE1/AE3, CAM5.2, vimentin, S-100 protein showed positive-cell expression rates of 91.3-100%. The new markers ,brachyury and galectin-3,showed 100% positive-cell expression rate.
Conclusions: Although chordoma derived from mesenchymal tissue, but showed positive for epithelial markers. Traditional chordoma markers showed good sensitivity. Brachyury is the first specific molecular linking embryo structure with tumor ,it showed a good sensitivity for chordoma in this study. Galectin-3,as a traditional notochordal cell marker ,showed 100% of positive expression rate in total 46 cases chordoma. Brachyury and galectin-3 can be used as a supplement to traditional chordoma markers, but their specificity to chordoma awaits further study together with other large number of various tumors.
PartⅢThe comparative histological study of fetal notochordal cell rest and chordoma.
Objectives: To detect the probability of notochordal cell rests coexisting with chordoma, and further compare it with fetal notochordal cell rests and chordoma in order to find the possible evidence that chordoma originate from notochordal cell rests.
Methods: Collected 46 cases of chordoma specimens , paraffin section and HE staining ,then observed to find notochordal cell rests coexsting. Chordoma and fetal nuclesus pulposus specimens were dewaxed and operated with immunohistochemistryl staining. The first antibodies ( EMA,AE1/3,CAM5.2, vimentin, S-100, brachyury, galectin-3, connexin-43)were added ,further dropped with diluted biotinylated secondary antibody, at 37℃incubated for 10 to 30 minutes, then dropped with diluted horseradish HRP-streptavidin . DAB colored and Fengpian gum sealed.
Results: We found six cases of notochordal cell rests coexist with chodoma specimens..They were composed of physaliphorous cells with bland nuclei and large intracytoplasmic vacuoles, no extracellular mucous matrix existing. All the traditional chordoma markers including EMA, AE/AE3, CAM5.2, vimentin, s-100 demonstrated high positive expression rate in chordoma and coexisting notochordal cell rests. Brachyury was expressed in 100% of chordoma and not expressed in notohordal cell rests,it can be used as specific markers for chordoma.Galectin-3 was expressed in total chordomas and 1 of 6 cases coexisting notochordal cell rests, this molecular can be used as an auxillary tool in differentiating chordoma with coexisting notochordal cell rests.
Conclusions: By retrospective study, we found 6 cases of notochordal cell rests coexisting with chordoma specimens. Brachyury can be used as a specific molecular in differentiating chordoma with notochordal cell rests, galectin-3 can be used as an auxillary tool. Due to no expression of brachyury in fetal notochordal cell rests and coexisting notochordal cell rests in chordoma , yet strong expression in chordoma ,it suggested that brachyury activation may be involved in chordoma tumorigenesis.
引文
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[18] Choi KS, Cohn MJ, Harfe BD. Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: implications for disk degeneration and chordoma formation. Dev Dyn. 2008. 237(12): 3953-8.
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