| Background&PurposeMetastatic hepatic carcinoma, also known as secondary hepatic carcinoma, is a advanced performance of malignant tumors. Malignant tumors in many organs prone to liver metastases, since the liver has blood supply dual blood supply of hepatic artery and portal vein. Liver metastasis of colon cancer is the most common metastatic liver cancer, which occurred on40～50%patients when they were diagnosed first or recurred, it most occur through the portal. Renal carcinoma, lung cancer, breast cancer, nasopharyngeal carcinoma, malignant melanoma may transferred into liver. The number of metastatic hepatic carcinoma patients is1.2times than the number of primary hepatic carcinoma patients in China, and it is20times in western countries, and it is rising. The therapeutic of metastatic hepatic carcinoma have an direct impact on the quality of life and survival time, however, the treatment of metastatic hepatic carcinoma is a difficult problem. Although at present various treatment methods more, but still can’t fundamentally change the prognosis of metastatic liver cancer.With the development of liver surgery techniques and perioperative management, partial hepatectomy has been considered as the only treatment that can prolong survival, whose3-year survival rate can reach50%. But only less25%patients are suitable for surgery,60%～70%patients who received resection of liver metastases may experienced recurrence. Traditional treatment methods (such as a simple intravenous chemotherapy) not be able to significantly prolong the survival of patients, who were diagnosed metastatic hepatic carcinoma. Though the research of combined with intravenous chemotherapy and targeted therapy have great achievements, treatment are limited by adverse reactions of drugs and resistance of tumor cells to chemotherapeutic drugs and the high costs of targeted drugs.Recent research data showed that IFN-β has a strong anti-tumor effect. The vitro study found that IFN-β has a strong effect on inhibiting n of tumor cell proliferation and inducing apoptosis of tumor cells. One study about inhibition of melanoma cell lines found that IFN-β has stronger role than IFN-α2in inducing tumor cell apoptosis, whose mechanism may be associated with TRAIL/Apo2L. The vivo study found that IFN-β can inhibit tumor angiogenesis. IFN-β may achieve its role by inhibiting genes encoding of VEGF and MMP9, directly inhibit tumor angiogenesis. Tumor angiogenesis plays a key role in the occurrence of metastatic liver cancer. That above-mentioned provide a basis for the use of IFN-β in the treatment of tumors. IFN-β has a short half-life in vivo and whose maximum allowable dose is lower than the effective dose, it is difficult be used directly in clinical practice. Some studies confirmed that IFN-β gene therapy using an adenovirus vector have good results in treatment of ovarian cancer, bladder cancer, glioma, lung cancer.Gene therapy is a novel treatment for the disease. Normal genes or genes which have therapeutic effect are imported into the target cells by vector, so the purpose of the treatment of diseases is implemented. In recent years, recombinant retroviral vector has been used as a more commonly used gene transfer tools in gene therapy and other research areas. In our study, we select IFN-β gene as the target gene, adenovirus as carrier. Murine model of colon cancer liver metastasis was built by the way of intraperitoneal injection.. We observed the efficacy of INF-β gene therapy on colon cancer liver metastases.Methods&ResultsThis experiment is mainly composed of three parts.1. Construction and identification of adenovirus vector that carried IFN-βIFN-β gene was inserted into adenovirus vector pIRES2-EGFP that containing the CMV promoter to build adenovirus shuttle vector pIRES2-EGFP-IFN-β. The target fragment IFNβ-IRES-EGFP was cloned into the intermediate vector pDONR221using BP recombination system of Invitrogen. After purified and amplified, the target fragment on Intermediate vector pDONR221-IFNβ-IRES-EGFP was cloned into adenovirus vector pAd/CMV/V5-DEST. Virus solution was purified, virus genome was identified by PCR. The adenovirus was named AdIFN-β-EGFP. The Titer of Virus AdIFN-p-EGFP was5.25×108TCID50/ml after293cell proliferation and cesium chloride density gradient centrifugation.2. Vitro effects of adenovirus that express IFN-β on murine colon carcinoma cells (C26)a. C26cells were infected by AdIFN-β-EGFP and AdEGFP, whose MOI were20,50,100,200.48hours later, the transfection efficiency was calculated under fluorescence microscopy. The result showed that the transfection efficiency increased with the increase of MOI. The transfection efficiency was respectively18%、45%、95%、99%with the increase of MOI. b. The express of IFN-β protein by C26cells, that were infected by adenovirus AdIFN-β-EGFP, was quantitative detected by ELISA. The express after2d、4d、6d and8d were915.18±407.40pg/ml, whose MOI was100.c. C26cells, which were respectively infected by AdIFN-β-EGFP and AdEGFP, were detected by MTT colorimetry, to detect its effect on cells growth. C26cells were respectively infected by AdIFN-β-EGFP and AdEGFP, whose MOI was100, and detected by MTT colorimetry respectively in1-5days. Cell growth curves were drew. After statistical analysis, we found that AdEGFP had no significant effect on proliferation of C26cells, AdIFN-β-EGFP could inhibit proliferation of C26cells. there was statistically significant(p<0.05).d. C26cells, which were respectively infected by AdIFN-P-EGFP and AdEGFP, were detected by flow cytometry to discover the effect on apoptosis of AdIFN-P-EGFP and AdEGFP. The result showed that the number of apoptosis after being infected by AdIFN-β-EGFP, there was no significant change after being infected by AdEGFP.e. C26cells, which were respectively infected by AdIFN-β-EGFP and AdEGFP, were detected by scratch test and transwell test to discover the effect on cell migration of AdIFN-β-EGFP and AdEGFP. The result showed that the ability of cell migration significantly reduced after being infected by AdIFN-β-EGFP, there was no significant change after being infected by AdEGFP.3. vivo study on adenovirus that express IFN-βand metastatic hepatic carcinomaa. Adenovirus was Injected intraperitoneally into murine, IFN-P vivo expression levels were detected by ELISA. The tails of murine were cut respectively after1、2、3、4、5、6、7、8、9、10d. Express of IFN-β in serum was detected. The result showed that IFNβcould be detected after1day, its express continued at a higher level and disappeared after10days. There was no significant increase in control group.b. Inhibition of IFN-beta gene therapy on colon cancer liver metastases. The establishment of colon cancer metastasis model in murine. The animals were divided into4groups. PBS、AdIFN-β-EGFP、AdEGFP were respectively injected intraperitoneally into murine of3groups after3d,9d. the murine of rest group were injected intraperitoneally with lml of0.8mg5-FU-0.9%Nacl. the livers were taken out after3days to observe the number and size of tumors. After biopsy staining, the number and size of intrahepatic tumors were detected under microscope. The result showed that the number and size of intrahepatic tumors in AdIFN-β-EGFP group and5-FU group was Less than PBS group and AdEGFP group. there was statistically significant. There was no significant difference between AdIFN-(3-EGFP group and5-FU group.c. The influence of IFN-(3gene therapy on Survival of Colon cancer liver metastasis mice. The survival mice in AdIFN-β-EGFP group and5-FU group was significantly prolonged, compared with the PBS group and AdEGFP group, there was statistically significant. There was no significant difference between AdIFN-β-EGFP group and5-FU group.ConclusionThe adenovirus AdIFN-P-EGFP with IFN-Pwas built successfully. The virus could efficiently infect cell strain C26of murine colon carcinoma. In vitro, it could inhibit the growth of C26cells, promote tumor cell apoptosis, reduce tumor cell migration. AdIFN-β-EGFP was injected intraperitoneally could express higher concentrations of IFN-β. Compared with control group, it could Inhibit the growth of colon cancer liver metastases mice and prolong the survival time of mice. Compared with5-FU group, there was no significant difference. Further study is needed in whether there is synergy between two treatments. |
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