| Chemokine with low molecular weight can induce the traffic function of white blood cells. It was reported that IP-10, a member of a- chemoattractant family, is an inhibitor of tumor angiogenesis, and can inhibit neovascular differentiating into capillary vessles. Many researchers have demonstrated that IP-10 could inhibit tumor angiogenesis, and chemotherapy combined with antiangiogenic therapy had enhanced effects. But the combination of IP-10 gene with gemcitabine in anti-tumor has not been reported. This study was designed to explore the antitumor effects and mechanisms of IP-10 gene combined with gemcitabine in bearing-tumor mice. First, pBLAST-EP-10 was transferred into E.coli JM109, and then the monoclon coliform was selected and cultured. IP-10 plasmid was prepared, purified and identified by restricted enzyme. IP-10 plasmid was transfected into COS cells assisted by lipofectamine. Conditioned medium were collected and confirmed by Western Blot analysis and ELISA to determine the content of IP-10 protein. Human umbilicus vein endothelia cells (HUVECs) were cultured in 96 wells plate. Adding the culture media with IP-10 or combined with gemcitabine, the growth of HUVECs was observed. The results showed that COS cellstransfected with IP-10.plasmid could express protein with activity in vitro, and IP-10 protein combined with gemcitabine could inhibit the prolferation of HUVECs.To investigate the expression of IP-10 plasmid in mice, different doses of IP-10 plasmid were injected into mice, and serum at different times were collected and determined by ELISA. The results demonstrated that IP-10 was expressed at high level for a long time after injection of plasmid. And the peak of IP-10 level occurred at 100 μg per injection. Administration with 200 μg IP-10 plasmid resulted in similar level protein in serum compared with 100 μg per injection.The antitumor efficacy of IP-10 plasmid in vivo was dose-dependent. To observe the antitumor effects and investigate the mechanism of IP-10 plasmid combined with gemcitabine, H22 hepatocarcinoma models were established in BALB/c mice, and Lewis lung cancer models were established in C57BL/6 mice. Then the survival rate of mice, tumor volume and side effects of treatment were observed. Compared with the controls, tumor volume of mice in the combination treatment group had significant regression, even disappeared, and the survival time was prolonged. Angiogenesis within tumor tissues was estimated counting the number of microvessels on the section staining with an antibody reactive to CD31. The combined treatment group resulted in apparent inhibition of angiogenesis in tumors compared with control groups. Apoptosis of tumor cells was observed by TUNEL. Combined treatment induced increased apoptosis of tumor cells. Alginate encapsulation assay also showed that combined treatment inhibited angiogenesis apparently, compared with control groups. No obvious side effects on mice were found during the treatment andpathologic changes in normal tissues (e.g.heart, spleen, kidney) were not observed by hematoxylin and eosin (H&E) staining.In conclusion, gene therapy with DNA encoding pBLAST IP-10 combined with gemcitabine had improved efficacy in antitumor, compared with gene therapy or gemcitabine administered alone. This study suggested that antiangiogenic gene therapy with IP-10 DNA combined with chemical agent gemcitabine may be a new approach in cancer therapy. Our findings may provide a strategy for cancer therapy through inhibition of angiogenesis and inducing apoptosis of tumor cells and may be of important in the exploration of the potential clinic application of gene therapy combined with chemotherapy. |
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