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Objective To establish human pancreatic cancer xenografts in nude mice, and to investigate the antitumor efficacy of human endostatin expressed by replication-competent adenovirus AdTPHre-hE in vivo. Methods Pancreatic cancer cells AsPC-1 were injected subcutaneously in BALB/c nude mice to establish the xenografts. Tumor growth was observed and measured after AdTPHre-hE treatment. Expression of endostatin was detected by ELISA assay. The tumors were harvested for pathologic examination and immunohistochemical staining. Results Tumors grew more slowly in the AdTPHre-hE group and their sizes were markedly smaller than those of the Ad-hE group (P＜0.01)and control group(P＜0. 01). Endostatin levels were detected in the sera of nude mice in all treated groups, and endostatin expression in AdTPHre-hE group increased with time. The endostatin level in the AdTPHre-hE treated group was much higher(P＜0. 01)and increased faster than that in the Ad-hE treated group. Immunohistochemical staining for Hexon of adenovirus capsid showed more positive tumor cells in the tumor tissues treated with AdTPHre-hE. Immunohistochemical staining for FⅧ revealed a decreased microvessel density in the tumor tissues treated with AdTPHre-hE. Conclusion The replication-competent adenovirus efficiently expressed high-level endostatin and significantly inhibited tumor growth in vivo.
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Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.
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Objective To investigate the effect of a dual regulation of replicating adenovirus vector carrying human endostatin gene (AdTPHre-hEndo) on pancreatic cancer. Methods Human endostatin (hEndo) gene was cloned into the genome of replicating adenovirus specific for the tumor cells by virus recombination technology. The virus titer was 3.25 × 1010pfu/ml. A Balb/c nude mouse model carring sw1990 cells pancreatic cancer was established, the expression of human endostain and inhibition of tumor cells in vivo were detected. Results We successfully constructed AdTPHre-hEndo. The inhibition on pancreatic cancer cell line SW-1990 of AdTPHre-hEndo is better than Ad-hEndo (P ＜0. 01 ), and ONYX-015 ( P ＜ 0. 05 ). The endostatin expression of AdTPHre-hEndo group was significantly higher than Ad-hEndo group and the control group (P ＜ 0. 01 ). The intratumoral MVD also decreased significantly in the treated tumors(6. 8 ±2. 5 vs. 16. 0 ±4. 6、47. 2 ± 10. 0, P ＜0. 01 ). Conclusions The recombination adenovirus can express biologically active hEndo effectively, which results in inhibiting the growth of micro-blood vessels and proliferation slowly.
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Objective To construct the adenovirus vector containing mice CD40- IgG2aFc-IRS-GFP fusion gene and detect the expression of the fusion protein in transfeete 293cells.Methods The fragments of CD40, IgG2aFc were obtained and inserted into plasmid PDC316-IgG2aFc-GFP.After being verified by sequencing, Ad5-PDC316-CD40-IgG2aFc-GFP was contransfored with the bckbone vector into 293 cells.The virus titer was detected after replicating and purifing and the expression of the fusion protein was analyzed.Results A virus and plasmid were constructed successfully.The vitro infection showed that the virus can infect cells of which the fusion protein was confirmed by fluo-rescence.The expression of the fusion protein increased with the increased time and virus concentra-tion.The protein expression stopped increasing after the virus concentration came to a certain level.Conclusion CD40, IgG2aFc fragments are correctly ligated with plasmid PDC316-IgG2aFc-GFP, and the fusion protein can be expressed in 293cells.This might lay a foundation for further studies of the expresstion of virus, immune tolerance and mechanism of liver transplantation model in rats.