| Objeative To construct and transfect HIF-1α(hypoxia-induced factors, HIF-1) gene recombinant antisense plasmid pEGFP-HIF-1αand make use to detect the fuction of the HIF-1αprotein. Methods Recombant antisense plasmid pEGFP- HIF-1αwas cloned into an eukaryotic expression vector,and transfected into Hela cells by lipofectamine mediated gene transfer. The efficiency of tranfection was determined by microscopy and the block of HIF-1αwere detected by Western blot. Results The recombinant antisense plasmid pEGFP- HIF-1αwas correctly cloned showed in the electro of the endoenzyme cleavage production.The expression of HIF-1αgene was detected after transfection distinctly. Conclusions It was suggested that antisense pEGFP- HIF-1αeukryotic expression vector was effective cloned and transfected,which could be used to further study of fuction of HIF-1αprotein. Objective To investigate the cytotoxicity and proapoptotic activity of topotecan(TPT) on cisplatin-resistant human ovarian cancer cell lines COC1/DDP and its mocular mechanisms. Methods The cytotoxicity of TPT on COC1/DDP was monitered using MTT assay and soft agaragar assay. TUNEL and flow cytometry were employed to observe the apoptosis of COC1/DDP induced by topotecan. Western blot were used to determine the expression of bcl-2, bax and caspase-3, in addition to the activity of caspase-3. Results TPT had strong cytotoxicity on COC1/DDP cell in vitro, and the cytotoxicity was dose-and time-dependent. TPT-treated cells demonstrated apoptosis property while the apoptotic rate was increased from 8.54% to 23.16%(P﹤0.05). Although the expression of bcl-2 was not affected, the expression of bax and caspase-3 were upregulated and the caspase-3 activity was promoted in the course of COC1/DDP apoptosis when treated with topotecan. Conclusion The cytotoxicity and proapoptosis activity of TPT, which is significantly observed in cisplatin- resistant human ovarian cancer, may be related to the increased bax expression and the promoted caspase-3 activity. |
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