Study On The Relationship Of Notochordal Cell Rest And The Tumorigenesis Of Chordoma

PartⅠMorphological study on fetal , adult nucleus pulposus and ChordomaObjectives: To evaluate the cell and tissure composition of fetal , adult nucleus pulposus and chordoma, providing the morphological evidence that chordoma originate from notochordal cell restsMethods: 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 chordomaObjectives: 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.