Research Of Human Interleukin-21Gene Transfer Combined With γ-ray Radiation On Inhibiting Esophageal Cancer Cells

ObjectMalignant tumor is one of the most serious diseases threat to human health. Cancer treatment includes traditiongal surgical therapy, radiotherapy, chemotherapy and gene therapy,etc. Esophageal cancer is the nineth malignant tumor in the globe, it spreads in many areas, especially in the developing countries. China is the popular area of esophageal cancer. At present, radiotherapy is one of the most effective and safe treatments to esophageal cancer. Tumor gene therapy is to transfer the gene (DNA) to the tumor cells, express high concentrations of the product or restructuring related cells in vitro and then import expressed in somatic cells,to inhibite the malignant phenotype, selective kill the tumor cells without adverse significant effects on surrounding normal tissues, to realize the purpose of control the growth of tumor cells at the tumor site. Between radiotherapy and gene therapy exist the role of mutually reinforcing. Enhancement mechanism is increasing the transfection efficiency of genes in cells and (or) promote gene expression, also after gene transfection may increase tumor radiosensitivity.IL-21(interleukin21) is a member of cytokines,founded by Julla.etc [1] in2000, produced by activated CD4+T cells. IL-21and IL-2,IL-4and IL-15are highly homologous. Parrish-Novak.etc researcher cloned IL-21and and cDNA of its receptor (IL-21R).ORF of human cDNA IL-21codes162aa polypeptide precursor,its signal peptide31aa,mature protein131aa, calculate the molecular about15kD. This experiment aim is through transfering contains IL-21gene recombinant viral vectors (Ad-IL-21) into esophageal cancer EC109cells and culture in vitro. Observing IL-21gene anti-tumor effect,then combined with y ray,study both anti-tumor role,apply the rationale and experimental basis on the application of clininc esophageal cancer gene therapy.MethodsThis experiment on adenovirus Ad-IL-21gene is constructed and transfer esophageal cancer EC109cells in vitro, irradiate with6Gy137Cs γ ray after transfection,the empty virus titer is1.4×1012pfu/ml,adenovirus.IL-21titer is9×1011pfu/ml,MOI value is100.Experimental group design is divided into the control group, Ad-lacZ control goup, Ad-IL-21group, radiation alone group and Ad-IL-21combined with radiation group.Observe IL-21gene combined with radiation on the influence of cell growth, cell cycle and apoptosis by MTT and flow cytometry methods.Identify the expression situation on transfecting Ad-IL-21into esophageal cancer EC109cells through the PCR and Western Blotting methods.Results1. esophageal cancer EC109cells growth curve:the cell number of control group and Ad-lacZ group increased with prolonged culture period.Tumor cell growth in radiation alone group and Ad-IL-21combined with radiation group are inhibited apparently after tranfection2days,compared with Ad-IL-21group and Ad-lacZ group has different statistical significance(F=145.269P<0.05).Tumor cell growth in Ad-IL-21group is apparently inhibited after transfection3days compare with control group and Ad-lacZ group(F=7.181P<0.05).There is no different statistical significance between control group and Ad-lacZ group(P>0.05).Tumor cell growth in Ad-IL-21gene combined with radiation group are inhibited obviously.2.The condition of esophageal cancer EC109cells cycle and apoptosis:results of flow cytometry show,compare with Ad-lacZ group and control group,Ad-IL-21group, radiation alone group and Ad-IL-21gene combined with radiation group the number of cells at phase G1increase, apoptotic rates increase.Among them with Ad-IL-21gene combined with radiation group the proportion of cells at phase G1are the highest,achieve71.2%,apoptotic rates is13.88%.3.The changes on esophageal cancer EC109intracellular IL-21gene expression condition:after transfection, extract RNA from EC109cells, amplify expected IL-21genetic fragments(493bp) through RT-PCR, compared with GAPDH(300bp)..Analyzing the gray values by Quantity-one software, gene relative expression quantity in control group, Ad-IL-21group,Ad-IL-21gene combined with radiation group are respectively0.16,0.34,0.39. EC109cells IL-21gene expression in Ad-IL-21group is higher than control group, Ad-IL-21gene combined with radiation group is maximum,respectively is1.1times and2.44times than Ad-IL-21group and control group.4.The changes on esophageal cancer EC109intracellular IL-21gene protein expression:extract total protein from EC109cells after transfection Ad-IL-21, IL-21gene reveal a15KD protein band visualized by Western blotting,all groups use beta actine as control,appeared in42KD protein band.Analyzing the gray values by Quantity-one software:IL-21gene relative protein expression quantity in control group,Ad-IL-21group,radiation alone group,Ad-IL-21gene combined with radiation group are respectively0.36,0.75,0.39and0.79.Four groups express IL-21gene protein,in Ad-IL-21gene combined with radiation group IL-21gene protein expression quantity is highest.IL-21gene protein expression quantity in Ad-IL-21group is higher than in control group.Conclusions1.Recombinant adenovirus IL-21can inhibit the growth of esophageal cancer EC109cells,compare with Ad-lacZ,shows that tumor cells inhibition from IL-21gene,rather than adenovirus vector itself.2.Ad-IL-21group,radiation alone group and Ad-IL-21gene combined with radiation group all can inhibit EC109cell growth,Ad-IL-21gene acts in synergy with radiation,its inhibiting effect is strongest Ad-IL-21group,radiation alone group and Ad-IL-21gene combined with radiation group block esophageal cancer EC109cells at G1phase,increasing cells radiosensitivity,promoting cell apoptosis,apoptotic rates in Ad-IL-21gene combined with radiation group is the highest,effectively inhibiting the growth of tumor cells.3.Through RT-PCR and Western blotting detection show,IL-21gene expression and protein expression in control group,Ad-IL-21group,Ad-IL-21gene combined with radiation group are different,Recombinant adenovirus IL-21effectively transfer into cells,and stably express in the cells,IL-21gene expression and protein expression in Ad-IL-21gene combined with radiation group is highest,the results shows that IL-21gene can inhibit esophageal cancer EC109cells growth,and the inhibition influence of Ad-IL-21combined with radiation group is the most obvious,effectively inhibiting the growth of tumor cells,the experiment provides a new therapies for future malignant tumor therapy in clinical.