Research Of Adenovirus-mediated IL-21 Gene Vector With γ-ray Radiation On Inhibiting Cervical Carcinoma Hela Cells

ObjectMalignant tumor threats seriously to human health and life,at present cancer treatment is the key point of medical research,including traditional radiotherapy and chemotherapy,surgical therapy and gene therapy,etc.the best therapeutics in middle-advanced cerical cancer is Radiotherapy,and assured curative effects.But problem on normal tissue which is near the tumor radiation injury and some tumor Radiation resistant etc,affecting the curative effect and application of Radiotherapy. Therefore,how to decrease or eliminate its adverse reactions,enhance the radiation effect has been an important subject of radiation oncology research.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 is in recent years a kind of cytokines.Parrish-Novak.etc researcher cloned interleukin (IL-21) 21 and cDNA of its receptor (1L-21R).ORF of human cDNA IL-21 codes 162aa polypeptide precursor.its signal peptide 31aa,mature protein 131aa calculate the molecular about 15kD.IL-21 has four spiral beam structure domain,IL-21and IL-2,IL-4 and 1L-15 are highly homologous.the position in two pairs Cys residues of IL-21 and IL-15 are the same,the position in one paire Cys residues is the same as IL-2, IL-4 and gm-csf,the other pair Cys residues is peculiar to IL-21 and IL-15. human IL-21 (hIL-21) gene is located in 4q26-q27,about IL-2 gene 180 KB,distance is futher than IL-15 genes.This experiment aim is through transfering contains IL-21 gene recombinant viral vectors (Ad-IL-21) into cervical cancer Hela cells and culture in vitro.Observing IL-21 gene anti-tumor effect,then combined with y ray,study both anti-tumor role,apply the experimental basis on the application of clininc cervical cancer gene therapy combined with radiotherapy.MethodsThis experiment on adenovirus Ad-IL-21 gene is constructed and transfer cerical cancer Hela cells in vitro,irradiate with 6Gy 7Csγray after transfection,the empty virus titer is 1.4×1012 pfu/ml,adenovirus IL-21 titer is 9×1011 pfu/ml,MOI value is 100.Experimental group design is divided into the control group, Ad-lacZ control goup, Ad-IL-21 group, radiation alone group and Ad-IL-21 combined with radiation group.observe IL-21 gene combined with radiation on the influence of cell growth and cell cycle,apoptosis by MTT and flow cytometry methods.identify the expression situation on transfecting Ad-IL-21 into cerical cancer Hela cells through the PCR and Western Blotting methods.Results1.Cervical cancer Hela cells growth curve:the cell number n control group and Ad-lacZ group increased with prolonged culture period,tumor cells are inhibited apparently after tranfection 96 h,compared with control group and Ad - lacZ group has different statistical significance (F=7.181 P<0.05),there is no significant difference between control group and Ad-lacZ group (P>0.05).Hela cells in radiation alone group and Ad-IL-21 gene combined with radiation group appear apparent inhibition after transfection 48h,Ad-IL-21 gene combined with radiation group and radiation alone group compare with Ad-lacZ group and Ad-IL-21 gene group have different statistical significance (F=249.88 P<0.05),especially Ad-IL-21 gene combined with radiation group compares with radiation alone group and Ad-IL-21 gene group after transfection 96h,cell growth are inhibited obviously (F=42.981 P< 0.05).2.The condition of cervical cancer Hela cells cycle and apoptosis:results of flow cytometry show,compare with Ad-lacZ group and control group,Ad-IL-21 group, radiation alone group and Ad-IL-21 gene combined with radiation group the number of cells at phase G1 increase,the number of cells at phase S decrease,increase apoptotic rates.Among them with Ad-IL-21 gene combined with radiation group the proportion of cells at phase G1 are the highest,achieve 88.9%,apoptotic rates is 15.3%.increasel5.8% than Ad-lacZ group,the number of cells at phase S are the lowest,1%, apoptosis rate increases 11.1%.3.The changes on cervical cancer Hela intracellular IL-21 gene expression condition,after transfection I extract RNA from Hela cells, amplify expected IL-21 genetic fragments(493bp) through RT-PCR.Analyzing the gray values by Quantity-one software, gene relative expression quantity in control group, Ad-IL-21 group,Ad-IL-21 gene combined with radiation group are respectively 0.17,0.35, 0.38.Hela cells IL-21 gene expression in Ad-IL-21 group is higher than control group, Ad-IL-21 gene combined with radiation group is maximum,respectively is 1.1 times and 2.24 times than Ad-IL-21 group and control group.4.The changes on cervical cancer Hela intracellular IL-21 gene protein expression:I extract total protein from Hela cells after transfection Ad-IL-21, IL-21 gene reveal a 15K.D protein band visualized by Western blotting,all groups use beta actine as control,appeared in 42K.D protein band.Analyzing the gray values by Quantity-one software:IL-21 gene relative protein expression quantity in control group.Ad-IL-21 group.radiation alone group,Ad-IL-21 gene combined with radiation group are respectively 0.42,0.92.0.61 and 0.99.Four groups express IL-21 gene protein,in Ad-IL-21 gene combined with radiation group IL-21 gene protein expression quantity is highest,obviously higher than control group and radiation alone group.Conclusions1.Recombinant adenovirus IL-21 can inhibit the growth of cervical cancer Hela cells,compare with Ad-lacZ,shows that tumor cells inhibition from IL-21 gene,rather than adenovirus vector itself.2.Ad-IL-21 group,radiation alone group and Ad-IL-21 gene combined with radiation group all can inhibit Hela cell growth,Ad-IL-21 gene acts in synergy with radiation,its inhibiting effect is strongest,tumor cells suppression in Ad-IL-21 group is equivalent to radiation group.Ad-IL-21 group,radiation alone group and Ad-IL-21 gene combined with radiation group block cervical cancer Hela cells at G1 phase,increasing cells radiosensitivity,promoting cell apoptosis,apoptotic rates in Ad-IL-21 gene combined with radiation group is the highest,effectively inhibiting the growth of tumor cells.3.Through RT-PCR and Western blotting detection show,IL-21 gene expression and protein expression in control group,Ad-IL-21 group,Ad-IL-21 gene combined with radiation group are different,Recombinant adenovirus IL-21 effectively transfer into cells,and stably express in the cells,IL-21 gene expression and protein expression in Ad-IL-21 gene combined with radiation group is highest,the results shows that IL-21 gene can inhibit cervical cancer Hela cells growth,and the inhibition influence of Ad-IL-21 combined 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.