Study On Adenovirus-mediated Mda-7/IL-24 Gene Selective Arrest The Growth Of The Hepatocellular Carcinoma Cell Lines

HCC is one of the world's most common malignancy. World about 1.25 million new cases annually, which were about 50% concentrated in China. The earliest occurrence of liver metastasis was through the liver blood, and also more common. Forming a small cell tumor thrombus in the portal vein could be violated. The shedding tumor thrombus formation could be planted in the liver and other place, which to be multiple metastatic lesions . Surgical resection of liver cancer was just removed surrounding tissue, however athe small tumor thrombus and satellite lesions surgery is powerless. For chemotherapy, as the majority of HCC cells was tolerance to chemotherapy drugs. Increased the dose of chemotherapy drugs in patients would cause systemic toxicity and caused multiple organ failure. The same as the HCC cells was not sensitive to radio-therapy, radiotherapy in the treatment of liver cancer had great limitations. Research show that if the HCC cells were combining treated with radiotherapy and chemotherapy its invasion had increased. Moreover the liver was also an important metabolism and immune organ in human. If it damaged by surgery and drugs and radiation, liver metabolism and immune function decreased significantly, this was also an important reason the treatment of liver cancer patients would be rapidly deteriorating condition after a major reason. Therefore, gene therapy for HCC was a hot topic in recent years. Gene therapy showed the strong performance of specific targeting and destruction. The development of molecular biology and immunology of liver provided some new theoretical basis and technological methods to cancer gene therapy research. If a tumor suppressor gene can not only promote cell growth inhibition and apoptosis but also could active the bypass way which would lead a strong anti-tumor effect (which offset the effect of low transduction), Other synergies amplification treatment (radiation, chemotherapy and / or antibody therapy). Immune amplification process (for further strong increase on the tumor cells injury); and the role of a anti-angiogenesis (inhibition of tumor blood supply and transfer). So not only can find and discover selective killing of tumor cells without affecting normal cells. also can inhibit and kill all the cells in the treatment of liver cancer research program has been a hot topic.MDA-7/IL-24 was such a gene. Pual.B.Fisher professor at Columbia University in the United States in 1993 to use subtraction from melanoma detailed compatriots on the human interferon-beta and inquired (mezerin PKC activprogram has been a hot research topic. This gene is MDA-7/IL-24 gene. Pual.B.Fisher professor at Columbia University, using gene technology in 1993 from Asia subtraction melanoma cells by the human interferon-beta and inquired (mezerin. PKC activator), which was induced . MDA-7 been named, but according to their biological properties and IL-24 chromosome location has been named. Initial studies have found that the gene expression and expression of melanoma cells with malignant melanoma cells were negatively correlated, The gene was transfected into malignant melanoma cells and found it has a strong growth block promote differentiation and apoptosis, but more high-grade tumors better. even be reversed malignant melanoma . Subsequent research found that the gene not only can cause melanoma cell growth inhibition. but also can promote cancer cell growth inhibition and apoptosis in various other, including melanoma, lung cancer, breast cancer, pancreatic cancer , glioma, such as ovarian cancer and prostate cancer. Comparing with the p53 tumor suppressor gene, MDA-7/IL-24 inhibit the growth of cancer cells and those of other conditions unrelated to the tumor suppressor gene (p53 3, Rb, or p16ink4) and therefore can be more effectively applied in cancer therapy. Moreover, the MDA-7/IL-24 gene can induce apoptosis in tumor cells. However, the differentiation of normal cells but not cytotoxicity, thereby suggesting that the gene has a strong tumor-specific options. As an ideal tumor suppressor gene, and hepatoma cells at home and abroad for its biological function less. The subject topics to be from adenovirus vectors encoding MDA-7/IL-24 eukaryotic expression vector, MDA-7/IL-24 study of apoptosis and proliferation of hepatoma cell line to block, Ectopic expression of MDA-7/IL-24 gene study in hepatoma cell lines to chemotherapeutic drugs doxorubicin and cisplatin. 5-FU drug resistance movement and migration of the change and the change. It shows that MDA-7/IL-24 role in liver cancer cell lines specific cell pathway. After the gene therapy for liver cancer to provide an effective molecular target Part one Construction of adenovirus vector carrying MAD/IL-24 gene and its expressing in normal live cell lines and hepatocellular carcinoma cell lineObjective: to construct adenovirus vector expressing MAD/IL-24 geneMethods: Ad-EasyTM system was used to construct MAD-7/IL-24 by recombination in E.conli. The virus was packaged in EK293 cells and subsequently identified valid.Results: The construction of adenovirus vector expressing MAD/IL-24 gene was the Ad.mda-7.Conclusion: the Ad.mda-7 vector can took the gene into the gene into the normal live cell line and HCC cell lines Part two Ad.mda-7 selective kill the HCC cell lines in vitroObjective: to observe the change between the liver cell line lo2 and the HCC cell lines HepG2,SMMC7721,Hep3B after infect Ad.mda-7 and Ad.VEC. Study the change, we can take the theory in the gene therapy.Methods: Ad.mda-7 infect the liver cell line lo2 and HCC cell lines HepG2, SMMC7721, Hep3B. MTT and the PI staining in FCM were to describe the cells growth changing and the cell cycle. Using Adriamycin, CDDP, 5-FU in alliance with Ad.mda-7 treated the liver cell line lo2 and HCC cell lines HepG2, SMMC7721 cell lines. Using the MTT and the PI staining in FCM were to describe the change in the cell lines which were resistant to the chemotherapeutics.Result:1. MAD-7/IL-24 gene was not expressed in the normal liver cell and the HCC cell lines. Ad.mda-7 infected the those cell line could take the MAD-7/IL-24 gene into those cells. MAD-7/IL-24 gene and the protein can be detected after infecting. Ad.mda-7 can induce the apoptosis in the HCC cell lines and had no cell toxicity in the normal liver cell line. The ability of MAD-7/IL-24 gene induced the cell apoptosis had the direct proportion to the dose of the Ad.mda-7. Ad.mda-7 can arrested the HCC cell lines in the G2/M cell cycle.2. Ad.mda-7 can enhanced the chemotherapeutics sensitiveness to the HCC cell HepG2.Conclusion: Ad.mda-7 could selective kill the HCC cell line and had not use to normal liver cell. The Ad.mda-7 enhance the enhanced the chemotherapeutics sensitiveness to the HCC cell and cut down the invasion and the metastasis ability in the Hepatocellular carcinoma cell line. Part three The mechanism of the Ad.mda-7 induce apoptotic signalingObjective: To observe the change of BCL-2 family, BAX, GADD family and stat-3 protein after Ad.mda-7 infect the normal liver cell line and the HCC cell lines by turns。Methods: Ad.mda-7 and AD.VEC were infected liver cells and liver cells, in 12 hours 24h,36h,48h cytosolic proteins were extracted. BCL-2/BCL-XL using western blot analysis. BAX and stat-3 and p-stat-3 stat-3 and protein at each time point changes. Transwell experiments demonstrated that the combined effects of tyrosine kinase inhibitor AG490. Note role in the inhibition of phosphorylation stat-3, Ad.mda-7 induced apoptosis of hepatoma cell line change.Results : In 24 hours, hepatoma cell line cytosolic proteins P-stat3 stat-3 peak Apoptosis is not unanimous. However, the use of AG490 stat-3 inhibit protein phosphorylation. Ad.mda-7 can effectively inhibit apoptosis induced hepatocellular carcinoma cell lines. MDA-7/IL-24 gene to the liver cells and change migration and the impact of exercise capacity.Conclusion:After Ad.mda-7 infected HCC cells, Ad.mda-7 can increase the activity of cytosolic protein stat-3. Inhibited the P-stat-3 of protein can markedly lower the Ad.mda-7 cell cytotoxic in vitro.