The Inhibitive Effect On Human Colonic Cancer Cells By Targeting Recombinant Adenovirus

Colonic carcinoma is one of the most common malignant tumor with an extremely poor prognosis in the world.There were about 950,000 people invaded in the year 2000.And about 500,000 at last died from the disease.the attact rate upgrates year by year to the third number in all tumors with the change of living habits and social aging.Researches demonstrates that the condition of the disease in China with 53,000 death every year is getting close to developed countries.So,the study on colonic carcinoma is significent. Gene therapy is a new concept which can study the rules of diseases on the molecular level.Exogenous DNA orRNA was inserted to replace the unnormal genes.As a new technique developed with the molecular biology,gene therapy has become to a promising department .However,it is after all a young region .Many difficulties have no way to overcome yet.Among them,how to regulate exogenous genes expression only in tumor cells and regulate the amount of the expression are important questions today.scientists all over the world are diving for the focus to accelerate the process from laboratory to clinic. Adenovirus is a medium-sized, icosahedral virus that contained a double-stranded linear DNA genome. Adenovirus has many advantages include broad tissue tropism, stability, safety, high lever expression and replication-defective(E1A and E1B deletion) adenoviral vector can accommodate up to 7.5kb of foreign genes and can be amplified to high titers in 293 cells.We inserted the E2F-1 promoter into adenoviral vector to construct a targeting recombinant adenovirus. Only when the E2F-1 promoter combine with the dissociated E2F protein that lies in malignant cells but not nonproliferating cell lines,it can trigger the transcription of the DNA chains downstream.Due to the characteristic,the killing gene inserted downstream the E2F-1 promoter can be regulated to selectively express in tumor cells with no toxicity to normal cells.Moreover targeting recombinant adenovirus vector attenuates the immunogenicity because of the localized expression of variant genes. HIV-1 vpr is a 96-a.a. 14kDa protein associated with the HIV virus particle. HIV-1 vpr has been shown to affect tumor cells in ways similar to that of p53 and other tumor suppressor gene. Several laboratories have reported that vpr induces apoptosis following induction of G2/M cell cycle arrest in some tumor lines.In addition,someone reported that this two affect are independent one another.the vpr protein may not only be a single phasic to treat carcinomar but be an assistant remedy to advance the curative effect.The multiply ways for restraining tumor make the vpr gene a spectacularcurative factor for cancer.It is reported recently that vpr protein itself may be a selective treatment to malignant cells.If the conclusion is validity,the selective affect to tumor by our targeting recombinant adenovirus will be filtrated twice,which results in a stronger targeting biology phasic. Based on above consideration and theory, we useed Ad5 pAdEasy system to generate recombinant adenovirus by exploiting E.coli BJ5183 homologousrecombination machinery. We constructed a replication-defective adenoviral vector expressing vpr gene drived by E2F-1 promoter to achieve Colonic carcinoma -targeted gene therapy, named rvAdE2F-1/vpr. We confirmed that vpr gene and E2F-1 promoter gene have been integrated into adenoviral genome by PCR and other methods. In our study,we demonstrated the gene downstream E2F-1 promoter extremely expressed in colonic tumor cell line and slightly changed to protein in normal cells by check the amount of Green fluorescent protein(GFP).By the same way,the compare between the two results which rvAdE2F-1/GFP and rvAdCMV/GFP separately infected LS174T cell line approved that the effictiency of E2F-1 promoter was just lower than that of CMV promoter and that no statistic difference was detected. the effictiency of the two promoters arrayed by MTT sustained the conclusion too. The cell survival rate after transferction assayed by MTT method showed that the adenovirus containing vpr gene can inhabit about 70% LS174T cellular growth.Putting the sample LS174T cell transferred by rvAdE2F-1/GFP onto transmission electron microscope,we can detect the change of cellular ultramicrostructure of apoptosis. At last,Flow cytometry was used to assay cell-DNA distribution.The decrease of G1 phase cell and increased percentage of G2 phase cell were observed .Therefore,our recombinant adenovirus containing vpr gene can not only inhabit proliferation but induce apoptosis of human colonic carcinoma cell line LS174T. Form the above data and results, we concluded that rvAdE2F-1/vpr could...