| Osteosarcoma is the most frequent highly malignant tumor of bone and exhibits a peak in manifestation during the second and third decade of life. It is difficult for clinical treatment and patient who accepted amputation alone often died of pulmonary metastasis within one year. As a result of the developments of surgical and imaging technology, the improvements of neoadjuvant chemotherapy and radiotherapy during last 20 years, an improvement in long-term survival rate from 10-20% to nearly 60-70% could be achieved. But many problems, such as metastasis, clinical recurrence remain to be unresolved.In the case of cancer, an abnormally increased cellular lifespan as a consequence of reduced apoptosis is ideal to favor the insurgence of genetic mutations, as well as to shield transformed cells from death induced by chemotherapy or radiotherapy, and to promote their survival at distant sites. Therefore, it is not surprising that manipulation of apoptosis has emerged as a new therapeutic strategy to help eliminate cancer cells. Survivin encode 142 amino acids, 16.5 kDa intracellular protein that belongs to the inhibitor of apoptosis (IAP) gene family. Its chromosomal location is 17q25. Survivin is abifunctional member of the inhibitor of apoptosis gene family that counteracts cell death and controls mitotic progression. Survivin possesses anti-apoptotic activity by binding and inhibiting the terminal effecter caspases 3 and 7. Accordingly, survivin prevents apoptosis induced by Fas (CD95), Bax, caspases and anticancer drugs. Survivin is expressed in the G2/M phase of the cell cycle and associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Survivin might participate in various aspects of mitosis, not only cytokinesis, and might play a dominant role in microtubule function. Reminiscent of oncofetal antigens, survivin is present during embryonic development, is undetectable in most healthy adult tissues and becomes prominently overexpressed in nearly all-human cancers. Predictive and prognostic detection of survivin has been implemented in some types of cancer. Two general considerations make survivin an attractive therapeutic target in cancer: it is selectively expressed in tumor cells and it is required for their viability.Objective: To investigate the expression of survivin in osteosarcoma and the specific inhibition of survivin gene expression by antisense RNA and small interfering RNA in MG63 cells.Methods: Part I :The clinic pathologic information of seventy-four cases of osteosarcoma was studied retrospectively. By using anti-survivin polyclonal antibody were investigated by immunohistochemical staining respectively. The above dates, together with the patients were analyzed statistically. Part II: Total RNA was extracted from osteosarcoma cell line MG63 cells using Trizol reagent. Coding sequence of survivin was amplified from MG63 mRNA by RT-PCR and then cloned into inducible eukaryotic vector pMDNA3. After the inducible survivin antisense vector was constructed, MG63cells were transfected with control pMDNA3 vector or pMDNA3-anti-survivin and selected by G418. Both of the control and survivin antisense transfectants were treated with ZnSO4. These cells cultured on cover slips were observed through immunohisto-chemistry staining, HE staining and electron microscopy. At the same time, the above cells were cultured and the numbers were counted every other day, thus growth curve was drawn. Apoptosis analysis was finished by Annexin V staining. Part HI: pSilencer 3.0-H1 neo, containing a neomycin gene had been used for the construction of specific RNAi plasmid for the survivin gene. MG63 cells were cultured and transfected with constructed RNAi vectors. The transfectants were selected with G418. Then, survivin mRNA and protein levels were checked with RT-PCR, immunofluorescence microscopy and western blotting. Assay of apoptosis was through Hoechst staining, flow cytometry and electron microscope observation. Cytotoxicity of ciplatin...
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