The Effects Of Recombinant Adenovirus And Vaccinia Virus Carrying Lectin Genes On Liver Cancer Cells And The Underlying Mechanism

Section I The effects of recombinant adenovirus and Vaccinia virus carrying lectin geneson liver cancer cells and the underlying mechanismThe malignant tumor threat to human life and health seriously.Liver cancer is one of the common malignant tumors of human.With the progress of the development of molecular biology and gene technology,targeted gene-virus therapy has become new research hot spot of the treatment of liver cancer.Lectin is a kind of specific recognition of carbohydrate and protein or glycoprotein which combine with carbohydrate non-covalently and reversibly.Over the past few years,this research group applied the palm leaf pinellia lectin gene expressing in tumor cells,and made a thorough study of cytotoxicity in tumor cells.But there is no thorough research of killing mechanism of liver tumor cells which were infected carried different lectin recombinant adenovirus and vaccinia virus.This topic using Dicentrarchus labrax lectin(DIFBL),Strongylocentrotus purpuratus lectin(SPL),Haliotis discus discus lectin(HddSBL)and Pinellia pedatisecta agglutinin(PPA)construct adenovirus Ad-DIFBL 、 Ad-SPL 、 Ad-HddSBL and soluble tumor vaccinia virus Onco Pox-PPA、OncoPox-SPL,to infect liver tumor cells and explore the lectin toxic effects and mechanism of action of tumor cells.We used the same multiplicity of infectionl of Ad-DIFBL to infect SMMC-7721 which SLMAP and Striatin targets Knock Down.MTT results and fluorescent microscope showed there was significant difference between SMMC-7721 which SLMAP and Striatin targets Knock Down and the control group,found that killing effect of Ad-DIFBL to control tumor cells is much greater than the SMMC-7721 which SLMAP and Striatin Knock Down.Results show that SLMAP and Striatin these two genes may have a certain relationship with lectin toxicity,but the specific relationship between each other is not very clear,need further research.By Western blot,We discovered Histone H3 expression decreased using Ad-DIFBL,Ad-HddSBL to treat SMMC-7721;SMMC-7721 were infected by Ad-SPL,Ad-HddSBL,the expression of ISG15 decreased.SMMC-7721 were infected by Onco Pox-PPA,OncoPox-SPL compared with OncoPox-pCB,found that C-myc,SLMAP,Beclin1 expression level decreased obviously;the expression of β-catenin decreased a little;and produced β-tubulin’s dimer and trimer.And SMMC-7721 were infected by OncoPox-PPA、OncoPox-SPL compared with Onco Pox-pCB,took the samples to company for transcriptome data analysis,found that different genes had significant up and down regulation.Such as the SMMC-7721 were infected by Onco Pox-PPA compared with Onco Pox-pCB,the up-regulation gene Caspase8,FGFR1,MAVS,SIKE1,and the down-regulation gene TMEM173,STRIP1,IFIH1,CTTNBP2 NL,Striatin4,FGFR4.And the SMMC-7721 were infected by OncoPox-SPL compared with OncoPox-pCB,the up-regulation gene CTTNBP2 NL,Striatin4,FGFR2,FGFR3,SIKE1,and the down-regulation gene TMEM173,STRIP1,IFIH1,CTTNBP2 NL,Striatin4,FGFR4.To sum up,this topic used the same multiplicity of infectionl of AD-DIFBL to infect SMMC-7721 which SLMAP and Striatin targets Knock Down and control group,found that degree of the lesion of SMMC-7721 which SLMAP and Striatin targets Knock Down was lower than control group,show that there is some interaction between SLMAP,Striatin and lectin DIFBL,and the role of lectin DIFBL is still unknown,requires subsequent experiment verify.According to Western blot,immune coprecipitation,transcriptome data analysis results show lectin impact on a variety of genetic level.This study provides basis for the application of lectin gene in the treatment of liver cancer in the future.Section II The anti-tumor mechanism of marine lectins DIFBL and HddSBL fusedwith adenovirus receptorUsing two kinds of fusion protein sCAR-DIFBL,sCAR-HddSBL combined with lectin gene,we successfully induced the expression of the two fusion proteins by IPTG.Subsequently,we obtained high purity fusion protein by Ni ion affinity chomatography purification and inclusion body renaturation.The sCAR is the extra membrane fragments of adenovirus receptor.It can combine type 5 adenovirus.Using these fusion protein combined with Ad-EGFP to infect a variety cells,and observe different fusion protein on the efficiency of the Ad-EGFP infection in tumor cells by fluorescence microscope.Found that the two kinds of fusion protein can not onlyobviously increase the infection of Ad-EGFP in K562/ADR,but also in U87-MG in vitro.We also found that fusion protein can identify different tumor cells by FACS,the results show that the fusion protein lectin DIFBL and HddSBL can identify membrane glycoprotein of K562/ADR and U87-MG.Using Ad-DIFBL and Ad-PPA combined with fusion protein sCAR-DIFBL and sCAR-HddSBL effected jointly or seperatly infected U87-MG,MTT and Annexin V-FITC/PI double marking streaming results show that the fusion protein sCAR-DIFBL combined with Ad-DIFBL or Ad-PPA,can enhance the killing effect to the tumor cells;fusion protein sCAR-HddSBL also can enhancehe killing effect of Ad-DIFBL and Ad-PPA to the tumor cells,but inhibit the effect of adenovirus virus and protect the growth of tumor cells.According to the Western blot results,we found that protein expression of p-ERK rise,when U87-MG were effected by fusion protein sCAR-DIFBL,sCAR-HddSBL combined with Ad-DIFBL and Ad-PPA,illustrate lectin DIFBL and HddSBL has nothing with cytotoxic effect.And the expression of E2F-1 rise,when U87-MG were effected by fusion protein sCAR-HddSBL combined with Ad-DIFBL and Ad-PPA or only effected by fusion protein sCAR-HddSBL,indicate HddSBL may activate E2F-1,then protect tumor cells.