| Background:Refers to the primary human liver cancer,modern clinical medicine including primary liver cancer and metastatic liver cancer,but actually known as hepatocellular carcinoma.In accordance with the type of cancer cells,liver cells can be divided into primary liver cancer,biliary tract cells and mixed type of liver cancer is hepatocellular carcinoma((Hepatocellular Carcinoma,HCC)are most common.Primary liver cancer is the most common malignant tumor and World Health Organization(WHO)released one of the ten malignant tumors,which ranked the fifth most common cancer in the world,and ranks third in the cancer-related cause of death.According the latest statistics on global HCC cases in the near future,new liver cancer cases worldwide each year about 60.7 million people.Particular in the most common primary liver cancer in Asia and Africa,in Europe and the United States also showed increasing trend in the population.And global disease incidence and mortality rates were on an upward trend,the clinical treatment and in early intervention strategy no delay.However,because the primary liver cancer and cancer is extremely aggressive or invasive,so most of the clinical effectiveness of first-line or second-line treatment is not ideal,because of liver cancer mortality of worldwide cases number of the disease remained in equilibrium.In addition,due to the features of primary liver cancer in the fast growth of cancer cells,cancer is highly malignant and often appear on drugs resistances,resulting in cancer liver metastases from an early stage,5-year survival rate is only about 5%.Therefore,from the basic medical sciences,clinical medicine,engineering,natural medicine,and integrative perspective,exploring and study on a kind of primary liver cancer treatment and early intervention strategies important in medical and clinical value.Gene therapy of cancer really is one of the hottest subjects in recent years,some studies have achieved breakthrough results,and cancer gene therapy techniques from experimental studies to clinical application,in which the suicide gene therapy of cancer is a major concern of cancer gene therapy.The basic mechanism of the therapy is to certain microorganisms(certain viruses and bacteria),genome prodrug-converting enzyme gene into the cancer cell,after the transfection of such genes encoding enzyme-specific features,original-toxic prodrug can be metabolized to toxic metabolites in the cancer cells,so as achieve the goal of destruction and kill cancer cells.Herpes simplex virus thymidine kinase(herpes simplex virus thymidine kinase,HSV-TK)and Escherichia coli cytosine deaminase gene(E.coli-cytosine deaminase,CD)gene is by the most important cancer double suicide gene,which for certain types of cancer cells,including human liver cancer cell destruction and killing of valid and important role.One of HSV-TK gene encoding specific thymidine kinase(TK),the high expression of TK by liver cancer and high activity of intracellular nucleoside analogs(NA)metabolism of liver cancer cells kill and exterminate triphosphate of the toxicity of compound,visible and effective anti-hepatocarcinoma effect.And the other a CD gene encoding specific cytosine deaminase and nontoxic in hepatoma cells of 5-fluorocytosine(5-FC)into destruction and killing hepatocellular 5-fluorouracil toxicity of metabolites(5-FU)to work on human hepatocarcinoma cells of high toxicity.In the double gene fusion consisting of double suicide gene on human hepatocellular carcinoma cells may play in the killing of a single greater biological effect of suicide gene.However currently used the cancer double suicide gene for clinical treatment of effect still not currently cancer treatment method reached of best treatment effect,and experiment effect and clinical effect still need to improve,especially cancer suicide gene therapy in the purpose gene transfection to cancer cells and transfection efficiency not high,and transfection carrier most for different of virus particles,plus its body vivo experiment and body drug mutual has obviously need the improvement,so the therapy still has further perfect of necessary and improve in future.Natural herbs contained in the natural medicine or functional components or bioactive substance because of its natural structure,with low side effects and high security has become a big advantage and characteristic of the field of cancer treatment.There are already large number of clinical and experimental studies shown,some compound,unilateral and monomer used alone,or in combination,or alternate application,or using the scientific method of integrated traditional and Western medicine to treat cancer better therapeutic effect can be achieved.3,5,4'-trihydroxystilbene resveratrol is a contains structure of non-flavonoid and more phenol compounds with the plant anti-toxins,Taxol its new of green natural anti cancer drug,its CIS form and Transform structure are anticancer effect,and in cancer occurred of starting.The development of stage of process in the anticancer biological activity are effect,they in cancer occurred of three of stage can play of regulation and inhibit and reversed role,that inhibit cancer to start,main rely to reduced has strong oxidation injury role of oxygen free base formed,and induced ? period drug generation enzyme increased,and anti-DIOXIN;inhibit COX,and inhibit hydrogen peroxide enzyme(CAT);When the inhibitory effects on the development of cancer,mainly relies on inhibiting the proliferation of cancer cells,induce cancer cell differentiation,speed inducing apoptosis in cancer cells and so on.Its anti-cancer effect of the mechanism may inhibit cytochrome enzymes,induction of detoxification enzymes,inhibit oxide synthase antagonism,inhibition of protein kinases,estrogen receptor,promotes tumor cell differentiation and apoptosis,including its joint biological effects.However,the mere effect of anticancer,and its mechanism is more complicated and may be the result of one or more effects,may also be cooperative or synergistic purposes other anti-cancer drug synergy,there may be other unknown pathway and its mechanisms.Particular in the effect of the mechanism may be RES in different types of cancer cell signaling,cell cycle and apoptosis mode,and there is a difference,so that its cancer-fighting properties have cancer species selectivity.Moreover,optimal anticancer effect of its time and dose-response is also different because of different cell types,that is,different types of cancer cell sensitivity to effects of pyrogallol is not the same or all different.Purpose:To more in-depth and system to explore and created more effect,and more features,and more with practical of clinical integrated Chinese and Western medicine combined anti-liver cancer therapy,this subject hope through established double suicide virus gene transfer system in people liver cancer cells HepG2 of experiment research,and observation people liver cancer HepG2 cell of experiment;while,this subject also hope exploration research natural resveratrol whether can tota collaborative double function fusion suicide gene CDglyTK,To improve and increase the anticancer effects of joint synergy between them,and this issue also hope true this study,exploring the natural effects of pyrogallol Masonic cooperative dual-function CDglyTK suicide Gene's best and dose-response,combining traditional Chinese and Western medicine in clinical application on human hepatocellular carcinoma in effective treatment and early intervention to provide new ideas and scientific basis.Method:1,RT virus Double suicide gene transfer system of established and stable infection liver cancer cells HepG2 of experiment research method:the part of subject general research method is:(1)pWZLneoCDglyTK plasmid transform,filter,and extraction,purification and identification;(2)carry double suicide gene RT virus inclusion,and identification and concentrated;(3)RT virus on liver cancer cells HepG2 cell staining:(4)Identification of infected human HepG2 cells,(5)and the comparative study on the biological characters of HepG2/CDTK cells and HepG2 cells.Specific method is:used genetic engineering technology,first using pWZLneoCDglyTK transcrip into to DH5 a competent bacteria,then from extraction of plasmid through lipidosome between guide will contains double suicide gene of RT virus carrier pWZLneoCDglyTK in to carry virus cell PA317 in,and after G418 filter with virus of PA317/CD+TK positive clone cell strains,for spread increased caltivate collection carry virus supernatant and for concentrated,Then Polybrene mediated transfection of human liver cancer cell line HepG2,G418 filtered and multiplication culture positive clones,to obtain the stable expression of double suicide gene HepG2/CD+TK cell strain and establish a stable of double suicide gene RT HepG2 cells,the last double suicide gene expression was detected by RT-PCR.2,Double function suicide gene CDglyTK on human liver cancer HepG2 cell line of lethal effect and resveratrol collaborative increased effect of experiment research of method:first used RT virus between double suicide gene,using 6418 filter produced virus of positive clone PA317/CD+TK cell strains,collect liquid supernatant with virus,concentrated and transfection HepG2 cell,again by 6418 filter,get the stable expression double suicide gene HepG2/CD+TK cell strains,Again with different concentration of resveratrol with human liver cancer HepG2 cell and human liver cancer HepG2 cell transfection double suicide gene of HepG2/CD+TK cell,at the end,distinguishing analysis used light microscope and electron microscope observation its cell morphology change;used apoptosis body cell morphology,and DNA Ladder,and TUNEL metod,and cell apoptosis related gene detection,and using cell cytometry observ its cell apoptosis;Use of MTT detection and observation cell clone formation,cell proliferation;cell cycle changes.Results:The results of this project are divided into two parts.The first part is:double suicide gene retroviral infection hepatocellular carcinoma HepG2 cells transfer system and its stability study results;the second part is:prodrugs with double suicide gene combined effect to human hepatoma HepG2 cells and transfection of HepG2/CD+TK cell-killing effect of double suicide genes.The first part of the experiment results1.double suicide gene retroviral transfer system and stable expression of results:(1)the plasmid transformation,expansion and identification of results:on pWZLneoCDglyTK plasmid DNA containing Amp resistance gene into positive strains on Amp-containing agar growth instead of transformed bacteria cannot grow without resistance to Amp,confirmed that outcome is the single bacterial clone of transformed colonies.(2)Minor plasmid identification results by using alkaline Lysis method:After extracting its DNA and purification,the plasmid pWZLneoCDglyTK joined the Loading Buffer and mix,then prepared 1%agarose gel,using UV transmission analyzers will presence of plasmid DNA.(3)Plasmid identification results:plasmid pWZLneoCDglyTK through BamH I single digestion can formed 1.19kb and 6.9kb of two electrophoresis;by EcoR I single digestion form 8.09kb of single electrophoresis;by BamH I and EcoR I double digestion can formed 1.19kb,and 1.31kb and 5.59kb of three electrophoresis,and this will confirmed plasmid with pWZLneoCDglyTK.using plasmid DNA as a template,then using CD and TK primers for PCR multiplication culture,can see the CD and TK multiplication electrophoretic band,and confirmed plasmid containing CD and TK genes into plasmid pWZLneoCDglyTK.And the build of double suicide gene DNA sequencing and sequences on genebank standards are comfirm and succes.Plasmid transcription cell strain with pWZLneoCDglyTK will multiplication culture,further large plasmid and electrophoresis analysis,positive results,prove the massive extraction of plasmid was successful.Finally,for extracting purity and concentration of pWZLneoCDglyTK plasmids were determined,results showed that the maximum absorption peak at 260nm maximum absorption at =0.525,280nm peak =0.29,DNA concentration(?g/?l)=1.81=5.25,DNA purity,consistent with the theoretical estimate 1.80.(4)Retroviral packaging,concentration and identification:retroviral vectors into after transcript,the PA317 cell microscopy,2nd day 80%of the cell will adherent cell growth,cells are fibroblasts,growth is good,strong proliferation,mitosis,cells,no granules in the cells,and passage once every 2-3 days.And this pWZLneoCDglyTK with liposome plasmids and plasmid PA317 pLXSN.GFP cellafter 48h visible cells in the control group with green fluorescence under a fluorescence microscope show that plasmid transfected,characteristically transfection efficiency was about 20-30%.(5)Transfection PA317 cell of PCR identification gene integration of results:on extraction non transfection cell PA317 and transfection cell PA317CD+TK of gene group DNA electrophoresis,visible with DNA electrophoresis band,proved gene group DNA extraction success;for template,respectively with CD,and TK gene,for CD,and TK gene PCR show multiplication increased of electrophoresis results,PA317/CDTK cell CD gene PCR product 730bp at has a special specific band,650bp TK gene PCR product has a specific band and PA317 CD,TK genes were negative,proof CD,TK genes on the genome that have been integrated into the PA317 cells.(6)Transfection PA317 cell of RT-PCR identification showing gene expression:on extraction transfection PA317 cell of total RNA for electrophoresis,results showed that does have RNA electrophoresis band with exists,proved RNA extraction success;will RNA RT transcription for PCR multiplication culture of electrophoresis results showed that,PA317/CDTK cell CD gene RT-PCR in 730bp at has a specific band,TK gene RT-PCR in 650bp has a specific band,PA317 CD,TK genes will negative,proof CD,TK gene has found expression in PA317 cells.(7)The results of electron microscopic observation of virus particles:the identification of the correct pWZLneoCDglyTK plasmid to inclosure cells PA317 in Liposomes,visible under electron microscope in the culture supernatant virus particles or gather the piles are ball-shaped,wavy edge,diameter approximately 100nm in size,the retroviral inclosure cells successful.2.Double suicide gene RT virus infection human liver cancer cells HepG2 of experiment results:(1)Resuscitation human liver cancer HepG2 cell of microscope check results:HepG2 cell resuscitation,second days showing adherent cell,replaced medium and continues to culture;cell showing epithelioid,growth well,see split phase,cell more transparent,under microscope showing little particles,may developed to organelle,cell passage on 2-3 days.(2)Experimental results of retroviral infection of HepG2 cells:transfected with retroviral cell culture supernatant and human hepatoma HepG2 polybrene 2-3D-mediated co-culture were observed under a fluorescence microscope,result transfer virus supernatant from human he-patoma HepG2 cells in the control group,numerous green fluorescence,infection till 60-70%(3)Filter results with Infection HepG2 cells:in polybrene induce,using PA317/CD+TK cell liquid supernatant in the RT virus transfection HepG2 cells microscope check found,cell within first day unchanged,with 500?g g/ml of G418 filter,2nd days only has minority cell shrink,and death,and formed cell debris;continues with 500 ?gg/ml of G418 pressure culture,survived cell continued proliferation,formed multiple cell clone,And cells are closely linked.(4)PCR identification of human hepatoma cell line HepG2 cells gene combination results:extraction not transfected HepG2 cells and transfection cell HepG2/CDTK DNA electrophoresis,DNA electrophoresis bands proved successful genomic DNA extraction.To its for template,respectively with CD,and TK gene of induce,for CD,and in TK gene PCR increased electrophoresis,results found HepG2/CDTK cell CD gene PCR product 730bp at has a specific band,TK gene PCR product 650bp at has a specific band with,and without transfection of blank HepG2 cell CD,and TK gene are negative,proved CD,and TK gene has integration to HepG2 cell of gene group.(5)Infection human liver cancer HepG2 cell of RT-PCR identification gene expression results:extraction transfection cell of RNA for electrophoresis,has RNA electrophoresis band exists,proved RNA extraction success;will RNA RT-PCR multiplication electrophoresis,results found,HepG2/CDTK cell CD gene RT-PCR product 730bp has a specific band,TK gene RT-PCR product 650bp has a specific band,and HepG2 cell CD,TK genes were negative,proof CD,TK gene has been expressed in HepG2/CDTK cells.3.Biological characteristics of human hepatoma HepG2/CDTK cells and HepG2 cells result:suicide gene transfected HepG2/CDTK cells and the control of HepG2 cells were passaged,cryopreservation,resuscitation after microscopic examination found in logarithmic when the General morphology of the two cells are basically the same epithelioid growth.Both basically same growth curve,and there is no significant difference(p>0.05).Using flow cytometry cell cycle detection for each of the two cells,found no significant difference in two cell cycle distribution.The second part of the experiment results1.Suicide genes prodrug(GCV and/or 5-FC)were to human hepatoma HepG2 and HepG2/CDTK cell 24h inverted microscope results:(1)Un-transfection suicide gene of human liver cancer HepG2 cell,application suicide gene prodrug GCV and/or 5-FC after 24h,human liver cancer HepG2 cell of General morphology as usual,cell still more transparent,rendering wall growth,and growth State good;and,the prodrug concentration Cytology form,and number and cell proliferation status are no obviously changes,with concentration and proliferation speed up or slows down of changes trend;And the two prodrug with GCV combined with 5-FC than either used alone,its target cells and had no significant difference,indicating that not using a suicide gene prodrug of human hepatoma HepG2 cells transfected with gene are not lethal effect.(2)Transfection suicide gene of HepG2/CD+TK cell,application suicide gene prodrug GCV and/or 5-FC after 24h,its and control group compared,with prodrug concentration of increased,HepG2/CD+TK cell within particles gradually increased,cell became increasingly shrink,wall growth performance weak,growth state variable poor,cell number variable less,but changes not obvious;and,with prodrug GCV and 5-FC joint application both or one separate application,Although their target cells growth condition worse,but after exposure to different concentrations of prodrugs in this experiment there were no cells apoptosis that using a suicide gene prodrugs of HepG2/CD+TK cells in a limited lethal effect..2.Resveratrol treat human liver cancer with HepG2 and HepG2/CD+TK cell after 24 husing inverted microscope observation results:respectively application different concentration of resveratrol on HepG2 and the HepG2/CDTK cell,with drug concentration increased,two species target cell are appeared cell growth state variable poor,and cell number reduced,and death cell increased,and dose correlation and concentration dependence,in resveratrol concentration for 0.20mmol/L,two species target cell are began appeared death;When it was 0.28mmol/L effect of increasing concentration of resveratrol to two target cells almost all died.However,the same effect of the concentration of respectively two target cells and the cell morphology and cell death does not see any different.3.Resveratrol combine suicide gene prodrug(GCV and/or 5-FC)were to human hepatoma HepG2 and HepG2/CD+TK cell result after 24h inverted microscope.(1)application of suicide gene prodrug(GCV and/or 5-FC)and the joint effect of resveratrol on human hepatoma HepG2 cells,with the purpose increase of drug concentrations,target cell growth variation,increase of cell number,cell death,and so on,target cell growth and effects of resveratrol was dose-and concentration-dependent manner.Also in the effects of RES at a concentration of 0.20mmol/L,target cells began to die,when the effects of concentration of RES 0.28mmol/L is reached,HepG2 cells almost all deaths and HepG2 cells alone effects no significant difference with RES.(2)Application of suicide gene prodrug(GCV and/or 5-FC)after exposure to and effects of RES combine with HepG2/CD+TK cells,with the purpose increase of drug concentration,target cells are apparent in cells worse and death,cell number,and so on,which is dose-and concentration-dependent manner.When RES respectively combine with GCV,and 5-FC,in RES concentration for 0.16mmol/L,its target cell that began appeared death;when RES concentration increased to 0.28mmol/L,its target cell almost all death;with RES combine with GCV,and 5-FC application,when RES concentration for 0.12mmol/L,its target cell on began appeared death,when concentration increased to 0.24mmol/L,its target cell almost all death.4.suicide genes prodrug combine(GCV/or 5-FC)were made for human hepatoma HepG2 and HepG2/CD+TK cell after 72h inverted microscope results(1)Un-transfection suicide gene of HepG2 cell,respectively application suicide gene with prodrug(GCV and/or 5-FC)after 72h,and after 24h of target cell form difference is unlikely,cell still more transparent,posted wall growth,growth State good,and target cell general form;cell number and cell proliferation are no changes,not with concentration and proliferation speed up or slows down of trend;prodrugs with GCV and 5-FC is either used alone when the cells no difference,but with prolonged effect,an increase in the cell density after 24h?(2)Un-transfection suicide gene of HepG2 cell,respectively application suicide gene with prodrug(GCV and/or 5-FC)after 72h,and after 24h of target cell form difference is unlikely,cell still more transparent,posted wall growth,growth State good,and target cell general form;cell number and cell proliferation are no obviously changes,not with concentration and proliferation speed up or slows down of trend;prodrugs with GCV and 5-FC is either used alone when the cells no difference,but with prolonged effect,an increase in the cell density after 24h.(3)Transfection suicide gene of HepG2/CD+TK cell,after application suicide gene prodrug GCV and/or 5-FC 72h,control group compared,with drug concentration increased,HepG2/CD+TK cell within of particles increased,and cell shrink situation after 24h of aggravated,cell posted wall performance and the growth State became more poor,cell number obviously reduced;and GCV and 5-FC combine application more than both one and separate application target cell of growth state variable poor,and Reduction in the number.When 5-FC 160 ?g,GCV 8 u g,cell death,and drug concentrations increased will increasing number of dead cells.5.RES respectively application to human liver cancer HepG2 and the HepG2/CD+TK cell after 72h inverted microscope observation of results:Using HepG2 and the HepG2/CD+TK cell respectively application different concentration of RES,and after24h compared,with drug concentration of increased,cell state more poor,and cell number more less,and death cell more,and has dose dependence,When RES concentration for 0.08mmol/L,this two species target cell are began appeared death;When concentration increased to 0.20mmol/L,and both target cells all died;but when the effects of three concentrations in the same two target cells,respectively,their target cell morphology and death had no significant difference.6.RES combine suicide gene with Prodrug(GCV and/or 5-FC)respectively for human liver cancer HepG2 and the HepG2/CD+TK cell after 72h inverted microscope observation:(1)with suicide and prodrug(GCV and/or 5-FC)and RES combine with HepG2 cell after 72h,and 24h compared,with purpose drug concentration increased,also appeared target cell growth State more poor,and cell number less,and more death cell,Target cell changes and effects of RES was dose-and concentration-dependent manner.Similarly,in effect when the concentration of RES 0.08mmol/L,HepG2 cells began to die,at a concentration increased to 0.20mmol/L,HepG2 cells all died,and HepG2 cells using effects no significant difference in RES.7.Application of suicide gene prodrug(GCV and/or 5-FC)and the combine effect of RES HepG2/CD+TK cell after 72h,and compared with 24h,with the purpose of drug concentration increased,HepG2/CD+TK cell morphology and growth is worse,more reduction in the number of cells,dead cells significantly increased,which is dose-and concentration-dependent manner.Effects of RES,respectively GCV,5-FC combine application,in the effects of three RES concentration of 0.04mmol/L,HepG2/CD+TK cell death begins when at a concentration increased to 0.16mmol/L,almost all of HepG2/CD+TK cell death;in effect of RES and GCV,5-FC combination at the same time,the effects of RES at a concentration of 0.04mmol/L,HepG2/CD+TK cell death begins,When at a concentration increased to 0.12mmol/L,almost all of HepG2/CD+TK cell death.8.Double suicide gene combine prodrug and effects of RES on hepatocellular carcinoma growth inhibition.(1)Suicide gene prodrugs inhibit the growth of HepG2 and HepG2/CD+TK:applied prodrug GCV or/and 5-FC to HepG2 cells,the cell proliferation was not affected,cell number no difference between the drug concentration.Application of suicide gene prodrug GCV or after exposure to 5-FC on HepG2/CD+TK cells,with the purpose of drug concentration increases,that cell proliferation is clearly inhibited cell growth.And,when combined with GCV and 5-FC,and with increasing drug concentration,cell growth inhibition is more obvious,and cell proliferation were significantly lower than those of the same concentration of GCV or 5-FC.(2)RES with HepG2 and HepG2/CD+TK of growth inhibit situation:application RES on HepG2 and HepG2/CD+TK cell,with purpose drug concentration of increased,the cell proliferation obviously by inhibit,and inhibit degree in two species cell between no significantly differences,and when RES dose increased to 0.20mmol/L,that into platform period,cell all was killed.(3)RES combine prodrug on HepG2 and the HepG2/CDTK of growth inhibit situation:application RES and Prodrug(GCV or 5-FC)on HepG2 cell,with purpose drug concentration of increased,the cell proliferation obviously by inhibit,but RES combine GCV and RES combine 5-FC,on the cell of proliferation no differences,and are and separate application RES group cell proliferation inhibit situation similar,when RES dose increased to 0.20mmol/L,the cell all killed.Application RES and prodrug(GCV or 5-FC)on HepG2/CD+TK cell,with drug concentration of increased,the cell proliferation obviously inhibit,RES combine GCV and RES combine 5-FC,on the cell proliferation no differences,but cell inhibit situation are obviously above separate application RES group,when RES dose increased to 0.16mmol/L,that into platform stage,cell full was killed.Applied effect of RERS and prodrugs(GCV and 5-FC)on the HepG2/CD+TK cells,with the purpose of drug concentrations increase,the cell proliferation was inhibited,cells were significantly higher than the other groups,when the effects of RES when the dose increased to 0.12mmol/L,will into platform stage cell were killed.9.Result of Wright's stain observation of apoptotic cells.After combine the prodrug GCV and/or 5-FC,HepG2 cell growth and proliferation well,no apoptosis,GCV,5-FC,GCV+5-FC,as well as the drug levels are no apoptosis.With after RES effects in low concentrations(0.04mmol/L),almost no apoptosis of HepG2 cells;and with the effects of increased concentration of RES,apoptosis increasingly obvious.After joining the effects of RES and prodrugs,alone with the effects of RES,low concentrations(0.04mmol/L),the basic no apoptosis of HepG2 cells;and with the effects of increased concentration RES,apoptosis gradually clear and the drug had no effect on apoptosis of HepG2 cells.After joining the prodrug GCV and/or 5-FC,at low concentrations(GCV lug/ml,and 5-FC 20ug/ml,and GCV lug/ml+5-FC 20ug/ml),almost no HepG2/CD+TK cells apoptosis as the prodrug concentration increases,apoptosis is becoming obvious;Apoptosis is a separate GCV+5-FC group GCV or 5-FC are more obvious.And HepG2 cells similar to combine after effects of RES in low concentrations(0.04mmol/L),almost no HepG2/CD+TK cells apoptosis with the effects of increased concentration of RES,apoptosis increasingly obvious.After joining the effects of RES and prodrugs,apoptosis in HepG2/CD+TK cells obvious,apoptosis occurs at low concentrations,and with increasing drug concentration,and increase the number of apoptotic cells,effects of RES,GCV,5-FC the combined use of the three separate applications,apoptosis is more obvious.10.DNA Ladder test on apoptosis:Combine different concentration of prodrug GCV(8?g g/ml and 4 ?g g/ml)and/or 5-FC(160 ?g g/ml and 80 ?g g/ml)after 72h,general method extraction 1%HepG2 cell DNA gene,Agarose gel Electrophoresis results showing DNA Ladder,After add low concentrations(0.04mmol/L)and in high concentrations(0.08mmol/L)were a glimmer of HepG2 cells DNA Ladder and with the increasing concentration of DNA Ladder is more obvious.After combine the effects of RES and prodrugs,as with separate combine effects of RES in low concentrations(0.04mmol/L)and in high concentrations(0.08mmol/L)are visible in HepG2 cells DNA Ladder and with the increasing concentration of DNA Ladder is more obvious.Add different concentrations of prodrugs GCV(8 ?g g/ml,4 ?g g/ml)and/or 5-FC(160 ? g/ml,80 ?g g/ml)after the 72h,the conventional method to extract the cell genome DNA,1%agarose gel electrophoresis of DNA Ladder;as the prodrug concentration increases HepG2/CDTK cell DNA Ladder is more and more obvious;GCV+5-FC group GCV alone or 5-FC are more obvious.And HepG2 cells similar combine and the after effects of RES in low concentrations(0.04mmol/L)and in high concentrations(0.08mmol/L)has a slight HepG2/CDTK cells are DNA Ladder and with the increasing concentration of DNA Ladder is more obvious.After joining the effects of RES and prodrugs,HepG2/CDTK cells DNA Ladder is a separate effect of RES or prodrugs more evident,apoptosis occurs at low concentrations,and concentrations with increase,increase the number of apoptotic cells,effects of RES,GCV,5-FC the combined use of the three separate applications,apoptosis is more obvious.11.Apoptosis result with using TUNNEL TestAdd different concentrations of prodrugs GCV(8ug/ml and 4ug/ml)and/or 5-FC(160ug/ml and 80ug/ml)after 72h,the cells have not been dyed Brown granules,TUNEL test results were negative.Join the after effects of RES,TUNEL detection showed that part of the cell nucleus is a light yellow,as weak,and with increased the concentration,nuclei yellow deepened.After joining the effects of RES and prodrugs,alone combine the effects of RES are no different,most of HepG2 cells weakly positive,and at the highest concentration,some of HepG2 cells are positive.Add different concentrations of prodrugs GCV(8?g g/ml,and 4ug/ml)and/or 5-FC(160? g/ml,and 80ug/ml),72h,TUNEL detection shows that each drug most HepG2/CD+TK cell is weakly positive at low concentrations,but in higher concentrations,a few cells were positive;when the combined use of two drugs,cells are mostly positive.And HepG2 cells similar combine after effects of RES,a part of the HepG2/CD+TK cell nucleus is a light yellow,which is weakly positive and yellow deepens of nucleus of high concentration effects of RES(Figure 29).After combine the effects of RES and prodrugs,HepG2/CD+TK cells were positive,and in high concentration cells were strongly positiveProdrugs GCV(8 ?g g/ml)or 5-FC(160 ?g g/ml)72h,HepG2 cells apoptosis rate were 5.43%and 5.49%,the same is applied and when combined,apoptosis rate of 11.09%,and combined prodrug after non-specific toxicity.Combine with RES(0.08mmol/L),a prodrug group,increased apoptosis of HepG2 cells,12.02%,illustrate effects of RES can cause apoptosis of HepG2 cells.After combine the effects of RES and prodrugs,HepG2 cells apoptosis rate higher than the separate effects of RES,apoptosis rate was 16.32%.Join prodrug GCV(8?g g/ml)or 5-FC(160 ?g g/ml)72h,compared with HepG2 cells,apoptosis rate increased significantly,11.11%and 14.37%,respectively;when the two are used in conjunction,apoptosis rate of 17.89%.combine the after effects of RES,HepG2/CDTK cells apoptosis of 15.81%,slightly higher than the HepG2.After joining the effects of RES and prodrugs,apoptosis HepG2/CD+TK cells were significantly higher,at 22.64%.1.2.Cell clone formed experimentCombine different concentration of prodrug GCV(8ug/ml and 4ug/ml)and/or 5-FC(160ug/ml and 80ug/ml)after 72h,the cell HepG2 clone formed... |
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