The Experimental Studies On Survivin Targeting For Therapy Of Human Gliomas

Part 1 Survivin expression and its relationship with proliferation, apoptosis and angiogenesis in human gliomasObject To explore the relationships of survivin expression with proliferation, apoptosis and angiogenesis of gliomas. Methods The expressions of survivin, Ki67 and Factor â…§-related antigen(Fâ…§Rag) were detected by immunohistochemistry and Apoptotic cells of tumors were screened by TdT-mediated dUTP nick end-labeling(TUNEL) in the paraffin-embeded sections of 73 gliomas and 10 cases normal brain tissue. Results No survivin expression was detected in the tissue samples from normal brain, while it was detected in 40 of 73 tissue samples from gliomas(54.8%). Expression of survivin was significantly associated with malignant grade of gliomas. Furthermore, survivin-positive gliomas had higher Ki67 labeling index(LI) and MVD than that of survivin-negative gliomas(P<0.05), and AI was inversely correlated with survivin expression. Conclusion Survivinoverexpressed in gliomas. Which may play an important role in malignant proliferation, antiapoptosis and angiogenesis of brain gliomas.Part 2 Effects of survivin antisense oligodeoxynucleotide on apoptosis of U251 cell and its mechanismsObjective To investigate the effects and the mechanisms of cell apoptosis on U251 cells after induction with antisense survivin-liposome complex, and to provide evidence in treatment for glioma . Methods We synthesized a 20mer ASODN target to the promotor region of survivin mRNA, The ASODN was transfected into U251 cell line at variety concentration using lipofectmine. Cell morphological change was observed with invert microscope. Apoptosis was observed by flow cytometry. Caspase-3 relative activity was analyzed by colorimetric assay.Results Abnormal morphological change of U251 cells was observed in ASODN transfected groups. Apoptosis of U251 cells was signigicantly increased compared with that in control and SODN group. There was no effect of survivin ASODN on the basic expression of caspase-3 in U251 cell line, but the activity of caspase-3 were increased in the cells treated by survivin ASODN in a dose-dependent manner.Conclusion The activation caspase-3 pathway play an important role in the apoptosis induced by survivin ASODN.Part 3 Survivin gene antisense oligodeoxynucleotide enhances the sensitivity of U251 cells to CisplatinObjective To explore the effect of antisense oligodeoxynucleotide (ASODN) of survivin gene on expression of survivin mRNA, protein and chemotherapy sensitivity of U251 cells. Methods MTT assay was applied to the determination of cell proliferation in U251 cells treated with different concentration survivin antisense compounds. By using RT-PCR and western-blot, the expression levels of survivin mRNA and protein were detected. Results Survivin ASODN inhibited the U251 cells proliferation, the inhibitory rate of different concentration ASODN were 22.0%, 41.5%, 57.3%, 62.7% respectively. Survivin ASODN down-regulated survivin expression(mRNA and protein) in a dose dependent manner. Its maximum effect was achieved at a concentration of 800 nmol/L. There was a significant increase of cell inhibitory rate after exposure to the combination of survivin ASODN and DDP(74.7%) as compared to DDP(33.6%) or survivin ASODN(38.4%) alone(both P<0.01). Conclusion Antisense survivin has significant inhibitory effect on growth of U251 cells through down-regulating survivin mRNA and protein and enhances the sensitivity of U251 cells to DDP. And antisense oligonudecleotides targeting survivin have a bright prospect for the therapy of glioma.