| Hepatocellular carcinoma(HCC)is a common cancer with malignancy and high mortality,which seriously endangers human life and health.The traditional methods of treating liver cancer include surgery,radiotherapy and chemotherapy.The treatment effect is remarkable for early liver cancer patients,but weak for advanced liver cancer patients.Biotherapy of cancer can break through the limitations of traditional cancer therapy and treat cancer better.Gene therapy is a common biotherapy for cancer,but the deficiency of using gene therapy alone lies in the poor targeting of cancer genes to tumor cells and low transfection efficiency.Cancer targeting gene-viro-therapy combines gene therapy with oncolytic virotherapy to give full play to the respective advantages of the two anti-cancer strategies,with significant anti-tumor effect.The oncolytic adenovirus was modified as follows:the E1B gene of oncolytic adenovirus was deleted;wild type promoter of oncolytic adenovirus was replaced with survivin promoter.The mK5 anticancer gene and shPKM2 were inserted into the adenovirus vector to build the dual-gene oncolytic adenovirus OAd-mK5-shPKM2.PCR was used to detect contamination of wild type adenovirus.The expression of E1A,mK5 and PKM2 was detected by Western blot assay.CCK8 cell proliferation assay was used to detect the lethality of oncolytic adenovirus to liver cancer cells.The apoptosis induced by oncolytic adenovirus was detected by using Hoechst 33342 and Annexin V-FITC/PI assays.The antitumor effect of oncolytic adenovirus in vivo is detected by using Huh-7 liver cancer xenograft model in BALB/c nude mice.PCR experiments have proven that the oncolytic adenovirus used in the experiment was free of wild adenovirus contamination.Western blot assay showed that the expression of E1A,mK5 and PKM2 is in normal.CCK8 cell proliferation assays showed that the antitumor efficacy of OAd-mK5-shPKM2 is stronger than OAd-mK5 or OAd-shPKM2.Hoechst 33342 and Annexin V-FITC/PI assays indicated that OAd-mK5-shPKM2 can induce more cancer cells apoptosis than OAd-mK5 or OAd-shPKM2.In vivo,OAd-mK5-shPKM2 had an enhanced antitumor effect than OAd-mK5 or OAd-shPKM2.Therefore,OAd-mK5-shPKM2 has the remarkable anticancer effect and may be a novel potent therapy for hepatocellular carcinoma. |
