Antitumor Effects Of The Tumor Specific Adenovirus

People turn pale at the mention of the ill of cancer. The disease shows a high degree of malignancy. In the third survey of population, more than a quarter of the deaths are died of cancer in China. Cancer poses a grave threat to the human health. The common methods of treatment often can not achieve a satisfactory therapeutic effect. Currently, the treatment of cancer has entered the genome era. Researchers set out from the molecular mechanism of tumor, They make gene therapy emerged rapidly. Gene therapy has become a very promising method. It is considered the most likely cure for cancer treatment. At the same time, gene therapy can reduce the heavy financial burden of cancer patients, improve the quality of life and prolong survival in cancer patients. However, the specificity, safety, efficacy will still plague cancer gene therapy research. How to effectively break the key link and effectively integrate organic solidarity has become therapy problems to be solved in the field of cancer gene.In this study, we synthetic tumor-specific promoter PEG-3p. E1a gene of hAd5 placed downstream of PEG3p. We make use of CMV promoter to drive HN gene. We have constructed dual specific recombinant adenovirus with both tumorspecific restrain and tumor specific replication abilities. The purpose is to ensure the effectiveness of gene therapy, at the same time, enhance the anti-tumor specificity and improve the safety of gene therapy. In this study, RT-PCR and IFA were used to identify the recombinant adenovirus Ad-HP and the control groups of Ad-HN, Ad-EGFP and Ad-GP. The results show that the recombinant adenovirus are stable and can highly express in cells. The exosomatic anti-tumor effects of recombinant adenovirus on LLC cells were detected by MTT. The results show that When LLC cells were infected with recombinant adenovirus at an MOI of 100, the efficiency of cell-killing is maximal at 48 hr post .The suppression rate of Ad-HP on LLc cells is higher than Ad-HN, With longer infection times, the suppression rate of recombinant adenovirus infected tumor cells was inhibited. This may be related to the proliferation of adenovirus and the rate of proliferation in tumor cell. Control group of Ad-GP is similar to Ad-HN, which may be related to the ability of tumor-specific replication, Ad-EGFP is almost no effect on the LLC.By using observation of light microscope, AO/EB staining, DAPI staining, Annexin V staining and caspase activity detecting.We explore the ways of recombinant adenovirus Ad-HP in inhibiting tumor. The results showed that after infected by adenovirus Ad-HP and Ad-HN, the recombinant adenovirus induced the typical apoptotic characteristics. Such as nuclear coagulation, fragmentation, shrinkage, loss of membrane integrity, lipid membrane eversion,apoptosis-related Caspase activity increased, These results suggest that Ad-HP and Ad-HN cells play a role in inhibiting LLC by inducing apoptosis of LLC cellsIn this study, C57BL/6 mice model bearing LLC was constructed by transplanting LLC cells into the trachea of the mice he model animals were treated by Intravenous injection of the recombinant adenovirus, the results show that compared with saline and control groups, the growth of tumor is slow in Ad-HN, Ad-HP and Ad-GP. Compared with the Ad-HN, Ad-GP, Ad-EGFP and saline control, gross tumor volume of Ad-HP treated group is the smallest. That Ad-HP can significantly suppress the growth of tumor; The survival rates of each group (Ad-HP, Ad-HN, Ad-GP, Ad-EGFP, saline control group of mice) are 60%, 40%, 50%, 30 %, 30%, The result shows that Ad-HP can effectively improved the survival rate of mice.The results of the detection of cytokine of Th1/Th2 showed that the recombinant adenovirus Ad-HP and Ad-HN can up-regulated the level of interleukin -2,γ-interferon, interleukin -4 and interleukin -10. The level of IL-2 and IFN-γof mice of Ad-HP- and Ad-HN-treated groups was much higher than normal control; salin control and other groups. The level of related cytokine of mice of Ad-HP is higher than the Ad-HN treatment group. This result illustrated that Ad-HP lead the immune response to Th1 dominant, Meanwhile, because of the immunostimulation of adenovirus itself and tumor immunity, the levels of IL-4 and IL-10 of the mice of Ad-HP- and Ad-HN-treated groups were up-regulated.In summary, the dual specific anti-tumor recombinant adenovirus constructed by using RAPAd. I system basing on tumor specific promoter PEG3p and specific anti-tumor gene HN and Ad5 E1a in the study, which has both tumor specific restrain and tumor specific replication abilities, and the recombinant adenovirus can effectively inhibit the growth of LLC cells and prolong life span of animal model significantly and enhance the immune response. The researches can provide a useful data for the studies on the gene therapy...