Research On The Mechanism Of Antitumor By Intracellular Expression Of Lectin Pinella

Currently malignant tumors have taken over the cardiovascular and cerebrovascular diseases and become the first-leading lethiferous disease. With the morbidity and mortality of tumor patients have increased dramatically each year,topical tradition medicine has been unable to achieve the purpose of treatment of cancer.The development of traditional therapy of tumor is very difficult,so people must find out more effective and safe treatment of concepts and methods to achieve the purpose of curing underlying causes. As Prof. Liu propose a novel strategy for cancer treatment named"Targeting Gene-Virotherapy of Cancer", we can use viral vectors that carring one or more cancer therapeutic gene in order to target tumor cells,then kill them, which has brought new light and hope to cancer treatment.Besides, the virus carrying therapy gene which promote by the specific promoter has more advantage on killing effect of tumor cells.Therefore, when constructing viral vector, choosing a good therapeutic genes is particularly important.Pinellia Pedatisecta Agglutinin, PPA is a very basic mannose-binding lectin that accumulates in the tuber of P. pedatisecta,which could specifically recognize and bind mannose. PPA has become a very useful tool in biomedical and glycoconjugate research, and it also has been extensively used to study the invasion, metastasis and prognosis of tumors.Current researches on the PPA are as anti-tumor drugs for external use,the pharmacological mechanism of PPA have no further study.We transfect PPA gene into tumor cells, make it express and then study the lethal mechanism as follows.First of all,we constructed the Eukaryotic expression vector pCDNA3/flag and pCDNA3/flag-PPA.Transfected tumor cells using pCDNA3/flag-PPA,detected by Western bolt and shows the expression of PPA;and then detected how PPA induced cell death in the mechanism of cell signaling pathway ;at the same time, using pulldown and Mass spectrometry to find and identify proteins that interact with PPA,in order to study whether PPA is interaction with other proteins to induce tumor cell death,and the target of PPA;Finally, we also constructed a replication-defective adenovirus which PPA gene is under control of the survivin promoter.In vitro experiment of MTT confirmed that our purpose virus can specifically kill tumor cells,meantime has a significant of antitumor effect;CPE experiment shows that the recombinant virus has lethality of cancer cells and security of normal cells,which lay the foundation of PPA for the feasibility of therapy gene.Meanwhile, for studying the function of PPA protein domains,we use the constructed Eukaryotic expression vector that carrying part of PPA gene to transfected cells,the results showed that EGFP-PPAn proteins distributed at the beginning in the form of scattered cells,and then gathered in an area cells,and overlapped with the nuclear dye staining Hoechst33342.The results showed that fusion protein distributed in the nucleus area.After 48 hours,compare with complete nuclear,it appears depolymerization and diffusion of the state.The results suggest that protein can enter the nucleus,binding with DNA and lead nucleus to further disintegration.Our research on PPA provides a basis for the future of further transformation and applying in cancer gene therapy .