| IntroductionOsteosarcoma is currently considered the most prevalent malig-nant bone tumor with high malignancy and poor prognosis. The definite mechanism of it is still unclear. Studies show that telomerase regulatory system may play an important role in the malignant formation, but its definite role in the formation of osteosarcoma remain unclear. We de-tected the expression of hTERT, c - myc gene in osteosarcoma using immunochemistry to study the relationship between the expression of these two genes and tumorigenesis of osteosarcoma.Materials MethodsCollect the samples of osteosarcoma and chondrosarcoma in path-ologic department of CMU, including 78 cases of osteosarcoma as the experimental group and 18 cases of chondrosarcoma as the control group. 5ujn slide was used and confirmed by experts before experi-ment.MethodsStreptomycin - biotin - peroxidase ( S - P) method was used.Rabbit anti - human hTERT polyclonal antibody was from Santa Cruz CO. and mouse anti- human c- myc monoclonal antibody from Maxim CO. The specific procedure was as follows:dewaxing and watering, in-cubation with hydrogen peroxide to block endogenous peroxidase activ-ity, antigen repair, incubation with normal rabbit serum, incubation with rabbit or mouse anti - human antibody, treatment with biotinylat-ed anti - rabbit or mouse immunoglobulin, incubation with S - P fol-lowed by DAB, counterstaining with hematoxylin,blockage with neu-tral gum. PBS(PH 7.4) was used among every step. Negative con-trols were established by replacing the primary antibody with PBS and positive controls by known positive section.If cytoplasm or nuclear of cells was stained brown - color,the cell would be called positive cell. We categorized the tumor cells as nega-tive or positive according to whether the tumor cells were stained. Posi-tive expression of tumor was subdivided into normal expression and re-duced expression according to staining intensity and the percentage of positive cells. 6-8 representative HPs were selected and analyzed by microscopic imaging analytical system. Mean gray value was calculated and expression intensity was evaluated according to it. Statistical sig-nificance was calculated by T test and Pearson correlation analysis with SPSS software.ResultsThe positive expression of hTERT and c - myc protein are both expressed in cytoplasm and nuclears of osteosarcoma and in nuclears of chondrosarcoma. Statistical analysis showed that there was a signifi-cant difference in hTERT and c - myc protein expression between os-teosarcoma and chondrosarcoma (P <0.01) , which indicated a higher expression of hTERT and c - myc in osteosarcoma than those in chon-drosarcoma. There is no relationship between expression of hTERT and c - myc protein in osteosarcoma.DiscussionThe telomerase activity was initially discovered in Hela cells. Lat-er scholars found that the same activity existed in ovarian carcinoma, malignant hematologic diseases and giant cell tumour of bone using i-sotope label method. Kim adopted amplification of telomerase repeat segment to analyze its activity and found that positive reactivity existed in 94 cell lines and 90 out of 101 malignant samples. The following studies indicated that telomerase expression existed in almost all of the malignant tumors and the intensity of expression was correlated with malignant degree and prognosis. Today, It is generally accepted that there is a close correlation between telomerase expression and tumori-genesis.The studies about Telomerase regulatory system further demon-strated its activation mechanism. The telomerase associated protein Est2P,P123 are considered telomerase catalytical subunit of some spe-cies. Later the telomerase catalytical subunit cDNA was cloned and named hTERT,which is restrictive factor of telomerase. Wu cloned the promoter region of hTERT and found two binding sites of c-myc gene there. TERT was the direct target gene of c - myc transcription, which meant that c - myc could directly activate TERT transcription to pro-m... |
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