Expression Of Tenascin-C And Its Role In Osteosarcoma

Osteosarcoma (OS) is a kind of malignacies and the tumor cells can form malignant bone-like or bone tissues. OS is the most seen within primary bone malignant neoplasm and takes 1/3 of all the bone-origined malignancies. OS occurrs more in male patients than the female and it is distributed in various age stages clinically. Most of the cases are among 11-25 years old. The morbidity is decreased when age is increased. OS appears on bone parts with high development status and is with high malignancy. Tenascin-C (TN-C) is main components of extracellular matrix and plays important roles in tumor development. Therefore, this study was aimed to explore TN-C distribution and role in OS.Aim: To explore the distribution and role of TN-C in OS. To prepare its specific monoclonal antibodies. To discuss TN-C distribution in OS patients and the modification of OS-related cytokines EGF,IGF-1 and mechanical stress on TN-C, which may supply new potential and effective therapy for OS. Methods: TN-C cDNA was cloned with RT-PCR and inserted into GST-tagged plasmid. The recombinant protein was induced and purified. Mice were immunized with the recombinant protein and the corresponding monoclonal antibodies were prepared and characterized by indirect ELISA, western blot and immunohistochemistry (IHC). A sandwich ELISA method was established and applied to detect sera TN-C distribution. TN-C expression in OS tissues was explored with IHC. Transcription and expression of TN-C induced by EGF,IGF-1 and mechanical stress were identified by real-time RT-PCR and sandwich ELISA and its involved signaling were tested by specific inhibitors.Results: TN-C cDNA was cloned successfully and its recombinant proteins were expressed. Three strains of mAb specific for TN-C were prepared and all of them were applicable in IHC and western blot. Among them, 2 strains were used to establish sandwich ELISA method to detect TN-C. Sera TN-C concentrations of patients with OS were significantly higher than the normal controls. TN-C was positive in OS and most of it was among the tumor stroma. With different protein kinase inhibitors, it's found combined with cyclic strain, EGF and IGF-1 induced TN-C transcription through RhoA/ROCK signaling pathway. At the same time, EGF and IGF-1 also increased TN-C mRNA level by additional ERK 1/2 activation.Conclusion: 1. TN-C specific monoclonal antibodies were prepared and a sandwich ELISA method to detect TN-C was establiehed. 2. TN-C was positive within sera and tissue of patients with OS. 3. OS-related growth EGF,IGF-1 and mechanical stress synergistically up-regulated TN-C transcription and expression.