Experimental Study For Therapy Of Human Breast Carcinoma By Tumor-targeted Induction Of Apoptosis Combined With Anti-angiogenesis Strategy

Objective:To construct the canstatin gene secretory eukaryotic expression vector under the hTERT promoter by recombinant DNA technology. Then to observe its tumor-specific expression in MCF-7/ADR cell by RT-PCR and Western blotting analysis and to detect its specific apoptosis-inducing effects in ECV204 cells by MTT,morphologic changes, PI stained flow cytometry (FCM) and AnnexinV-FITC/PI stained FCM. To observe the effects of canstatin gene combined with TRAIL gene on implanted human breast cancer of nude mice.Methods:1. Construction of the secretory eukaryotic expression vector phTERT-canstatinSpecific forward and reverse primers for the canstatin gene were designed and both of them contained restrictive enzyme site (NheI and HindIII, respectively) at the 5' end. Then the canstatin gene(684bp) with signal sequence of human IgG γ chain was specifically amplified using pETC as a template by PCR, the product was then extracted and orientationly ligated into phTERTlucl digested with the same restrictive enzymes, ligation product was transformed into E. coli. expression host DH5α, to screen and identify the positive recombinants, the positive clones were digested by restrictive enzymes, PCR, automatic DNA sequencing after plasmid isolation.2. Expression of the secretory plasmid phtert-canstatin in MCF-7/ADR cells...