| Malignant tumors are a king of diseases that severely threaten the life and health of human being.According to incomplete statistics,roughly 1.3 million people die of tumor each year in our country.On a worldwide scale,seven millions lives are taken away by tumors each year.Surgery,chemotherapy and radiotherapy could only cure 45%of all tumors;in another 55%of patients,tumors are incurable because the disease is in advanced stage or highly malignant.Prevention and treatment of tumor have become important missions for medical staff worldwide.Each and every country is investing huge quantity of human and material resource.Tumor vaccine,as an important biotherapy strategy against tumor,has become one hotspot in tumor research.By triggering cell immune response and humoral immune response,tumor vaccinations activate the natural anti-tumor responses of the human body and thereby achieve the goal of preventing and treating tumors.It meets the demand for targeting ability which is placed great emphasis in modern oncotherapy.Among numerous tumor vaccine strategies,genetic vaccine has unique advantages and attracts more attention.Genetic vaccine is a brand-new vaccine given rise by the development in the research field of genetic therapy.Honored as the third revolution of vaccine, genetic vaccine has become an important way to prevent and treat tumors.Malignant melanoma could occur in b th skin and mucosa.Malignant melanoma occurred in oral mucosa is the most malignant one among all oral malignant tumors,with frequent early and extensive metastasis.In 70%of cases,the disease spreads to regional lymph nodes,while metastasis in organs including lung,liver,bone and brain could also be seen.As much as 40%of cases show distant metastasis.Prognosis is extremely poor for malignant melanoma.Therefore,to perform investigation into the genetic vaccine against malignant melanoma is absolutely of major implication on our way to conquer tumors.By integrating the advantages of individual tumor vaccine designs at home and abroad,as well as combining genetic therapy and immune therapy,our study set to design a brand-new genetic vaccine in a broader sense,that is,to construct a dual-gene simultaneous-expression DNA vaccine based on mycobacterium tuberculosis heat shock protein 70(mtHSP70) gene and herpes simplex virus-thymidine kinase(HSV-TK) gene.Using a tumor targeting attenuated salmonella typhimurium as the vector,the vaccine was given by intratumor injection to treat solid melanoma.The scientific idea behind was to targetingly deliver the mtHSP70/HSV-TK genetic vaccine into tumor cells by attenuated salmonella typhimurium.Then A pro-drug of HSV-TK Gancivol(GCV) was given to initiate the cytocidal effect and bystander cytocidal effect of HSV-TK in tumor cells.Deceased tumor cells bound to mtHSP70 and created an mtHSP70-peptide complex.Such complex contained the fingerprints of all the tumor cell antigens,with antigenicity better than current genetic vaccines home and abroad which have used a common DNA vaccine design of targeting specific tumor antigen.Our vaccine might help solving the current issue of immune tolerance due to the low antigenicity of DNA vaccines,and achieve anti-tumor effect. â‘ To construct a eukaryotic plasmid PCMV-mtHSP70-IRES-TK that contains and expresses both mycobacterium tuberculosis heat shock protein 70(mtHSP70) gene and suicide gene HSV-tk and the control plasmids PCMV-TK-IRES-EGFP and PCMV-mtHSP70-IRES-EGFP for the experiments.â‘¡To study the in vitro expression of recombinant plasmids PCMV-mtHSP70-IRES-TK,pCMV-TK-IRES-EGFP,pCMV-mtHSP70-IRES-EGFP, PCMV-IRES-EGFP and the in vitro anti-tumor effect in mouse melanoma cell line B16.â‘¢To construct recombinant salmonella typhimurium SL7207/PCMV-mtHSP70-IRES-TK and its controls SL7207/pCMV-TK-IRES-EGFP,SL7207/pCMV-mtHSP70 -IRES-EGFP and SL7207/ pCMV-IRES-EGFP;to study the in-vitro expression of recombinant salmonella typhimurium and its invasiveness on B16 cellsâ‘£To determine the dosage and timeliness of recombinant salmonella typhimurium given by intratumor injection as well as the treatment scheme and to study the effect of intratumor injection of recombinant salmonella typhimurium SL7207/PCMV-mtHSP70-IRES-TK on mouse B16 tumor and its mechanisms.[Methods]:â‘ Login on genbank and search for mRNA sequences of HSV-TK and mtHSP70 genes.Specific primers for the amplification of full length cDNA of HSV-TK and mtHSP70 were designed by primer designing software DNA club.pcDNA3-TK plasmid and mtHSP70 plasmid DNA were used as template.PCR was performed with respective primers and primeSTAR HS DNA polymerase,cDNA segments of HSV-TK and mtHSP70 were obtained.With gel extraction reagent kit,target gene segments of 1192bp(TK) and 1898bp(mtHSP70) were collected.Collected DNA underwent addition of poly A tail and was then ligated onto the T vector pMD18-T. The vector was used to transform Escherichia coli(E.coli) TG1,which was then inoculated onto Amp resistant plate.A total of 200 positive clones were developed. Randomly selected eight clones of each group were inoculated into 2 mL EP tube containing 500μL of LB and were shaken.PCR was performed on the clones with specific primers of HSV-TK and mtHSP70 gene.The 1.2%agarose gel electrophoresis revealed PRC products of specific size in six out of eight clones of E. coli transformed by HSV-TK ligated product,suggesting that these positive clones contained TK gene recombinant plasmid.While 1898bp segments were obtained in six out of eight mtHSP70 transformed clones,which confirmed that they contained positive mtHSP70 recombinant plasmid.Upstream and downstream sequencing were performed on six positive pMD 18T-TK recombinant plasmids and six positive pMD 18T-mtHSP70 recombinant plasmids.By comparing the homology between the results of sequencing and the gene sequences,sequences of 2 recombinant plasmids of each type showed 100%homology with corresponding gene sequence,with completely correct sequences of introduced restriction enzyme sites.One of these plasmids of each type was selected for sub-cloning of HSV-tk and mtHSP70 genes. According to the restriction enzyme system,pCMV-TK-IRES-CD and pMD 18 T-TK plasmids were first digested by restriction endonuclease Notâ… .Electrophoresis revealed specific restriction enzyme digested map.Vector segments(around 6200 bp) and TK gene segments(1192 bp) were collected respectively.After CIP treatment,the vector segments underwent ligation reaction with the collected TK gene segments catalyzed by T4 DNA ligase.Ligation products were used to transform E.coli,which was then inoculated onto kana resistant plate.Positive clones were selected and shaken,and were extracted for small amount of plasmids which were then cleavage by Pstâ… enzyme.The results showed that 1424 bp and 423 bp cleavaged products were obtained when cleavaging recombinant plasmid of the number 3 clone, suggesting that TK was forwardly inserted on Notâ… site.A small amount of recombinant plasmids with forward insertion of TK(pCMV-TK-IRES-tk) were extracted;pCMV-TK-IRES-tk plasmid and pMD 18 T-mtHsp70 were treated with double restriction enzymes of EcoRâ… and Xhoâ… .Electrophoresis revealed specific restriction enzyme map.Collected 6200 bp and 1898 bp segments were used for ligation reactions.By replacing TK on the upstream of IRES with mtHSP70,a PCMV-mtHSP70-IRES-TK was constructed.Resulted positive clones were shaken and a small amount of plasmids were extracted.Restriction enzyme verification was performed on the plasmids with single restriction enzyme of Notâ… and double restriction enzymes of EcoRâ… and Xhoâ… ,respectively.Meanwhile,control plasmids PCMV-TK-IRES-EGFP and PCMV-mtHSP70-IRES-EGFP were also constructed by the methods as follow:pMD 18 T-TK and pCMV-IRES-EGFP plasmids were respectively cleavaged by restriction endonuclease Salâ… and BamHâ… .Cleavaged pMD 18 T-TK and pCMV-IRES-EGFP plasmids showed specific restriction enzyme maps in electrophoresis.TK gene segments of 1192 bp and vector segments of around 5300 bp were collected and underwent ligation reaction.TK gene was subcloned onto the upstream of IRES in pCMV-IRES-EGFP vector.PCR products of constructed plasmid PCMV-TK-IRES-EGFP showed specific bands of 1192 bp.A small amount of plasmids were extracted for restriction enzyme verification by Salâ… and BamHâ… . pMD 18 T-mtHSP70 and pCMV-IRES-EGFP were cleavaged by restriction endonuclease Xhoâ… and EcoRâ… .Electrophoresis on cleavaged pMD 18 T-mtHSP70 and pCMV-IRES-EGFP plasmids revealed specific restricion enzyme map.Collected 1898 bp mtHSP70 gene segments and 5300 bp vector segments were ligated. mtHSP70 gene was subcloned onto the upstream of IRES in pCMV-IRES-EGFP vector to constructe the plasmid PCMV-mtHSP70-IRES-EGFR Electrophoresis of the PCR products showed specific bands of 1898 bp.Restriction enzymes verification was performed with Xhoâ… and EcoRâ… .â‘¡When cells in passage culture achieved 50-70%confluency in the 24-well plates,transfection was performed according to the protocol of Lipofectamine 2000 transfection reagent kit.The cells were then incubated for another 24-48 hours in an incubator.Transfection efficiency was observed under fluorescent microscope.The cells underwent passage culture in 60 mm culture dishes.When the cells reached 50-70%confluency,transfection was performed according to the protocol of the Lipofectamine 2000 transfection reagent kit.RT-PCR and western blot assay were performed respectively to determine the mRNA and protein expression of mtHSP70/HSV-tk genes delivered by recombinant plasmids in B16 cells.A total of 2×106 cells were inoculated into 10 cm dish.When cells achieved 80%confluency, PCMV-mtHSP70-IRES-TK,PCMV-mtHSP70-IRES-EGFP,PCMV-TK-IRES-EGFP and PCMV-IRES-EGFP plasmids were transfected respectively using LIPOFECTAMINE 2000.At 12 hours after the transfection,cells were inoculated into 96-well plates.When cells were at logarithmic growth phase,Gancivol(GCV) was added.MTT assay was used to detect the tumor-cytotoxicity effect of TK gene on B16 cells in vitro.â‘¢Competent salmonella typhimurium SL7207 was prepared;plasmids PCMV-mtHSP70-IRES-TK,PCMV-IRES-EGFP,PCMV-mtHSP70-IRES-EGFP and PCMV-TK-IRES-EGFP were electro-transferred into salmonella typhimurium SL7207 respectively,so as to construct corresponding recombinant salmonella typhimurium.After restriction enzyme verification,4 verified recombinant salmonella typhimurium were respectively inoculated onto LB solid medium and were repeatedly passaged 20 times.Then plasmids were extracted and again underwent restriction enzyme verification for the stability of attenuated salmonella typhimurium SL7207 in carrying plasmid.Meanwhile,B16 cells were infected by each recombinant bacteria (ratio of cell to bacteria:1:50) and were incubated at 37℃for 30 minutes.The cells were then washed twice by serum-free medium containing gentamycin(50mg/L),so as to kill the bacteria outside the cells.Cells were then added with RPMI1640 medium containing fetal calf serum(100 mL/L) and were incubated for another four hours at 37℃.After that,tetracycline was added to the final concentration of 10 mg/L, in which cells were cultivated for another 36 hours.Expression of fluorescent protein was observed and photographed under fluorescent microscope.B16 cells infected by recombinant bacteria were collected for observation under electron microscope and for western blot assay to detect the expression of target genes.â‘£First of all,0.1 mL mouse melanoma B16 cells(cell concentration 107/mL) were subcutaneously inoculated into the back on the right side of C57BL/6J mice,so as to establish models of mouse melanoma.Pathological section examination confirmed that the tumor was melanoma.When tumor volume reached 100mm3, recombinant bacteria SL7207/PCMV-TK-IRES-EGFP was injected into the tumor at three gradient concentration levels of 107CFU/mL,109CFU/mL and 1011CFU/mL.At each concentration level,three mice were given the injection,with 100 uL of bacteria for each mouse.Abscess formation and rupture in the tumor lesion and life status of the mice were observed every day.Thereafter,animal model of mouse melanoma B16 was established in nine mice,so as to determine dosage of recombinant bacteria. An intratumor injection was given,with 100 uL of bacteria for each mouse.At day 2, day 4 and day 7 after the injection,three mice were executed respectively.Tumor tissue was obtained and prepared into frozen section.Fluorescent microscopic observation was performed to show the expression of the reporter gene EGFP.When dosage and timeliness of recombinant salmonella typhimurium given by intratumor injection and the treatment scheme has been established,cell concentration was 107/mL.In 60 inbred C57BL/6J mice(specific pathogen free animals certificate NO.0041126),subcutaneous injection of 100 uL B16 cell suspension was given in the back of the right side to establish B16 bearing animal model.Tumor diameter was measured with vernier caliper once every three days.Tumor volume was calculated with the formula described in related literature:V=length×width2×0.52.When tumor volume was around 100 mm3,the mice were randomly divided into five groups for the experiment,with 12 tumor bearing mice for each group.Experimental group 1:intratumor injection of recombinant salmonella typhimurium SL7207/PCMV-mtHSP70-IRES-TK(abbreviated as HT group).Control group 1:intratumor injection of recombinant salmonella typhimurium SL7207/PCMV-mtHSP70-IRES-EGFP(abbreviated as H group).Control group 2:intratumor injection of recombinant salmonella typhimurium SL7207/PCMV-TK-IRES-EGFP(abbreviated as T group).Control group 3:intratumor injection of recombinant salmonella typhimurium SL7207/PCMV-IRES-EGFP(abbreviated as Sa group).Control group 4:intratumor injection of PBS(abbreviated as PBS group).For experimental group and control group 1,2 and 3,100 uL of 109 CFU/mL recombinant salmonella typhimurium was given by intratumor injection via two injection sites.In control group 4,intratumor injection of 100 uL PBS was given via two injection sites.Only one dose of recombinant bacteria or PBS injection was given. At the second day,GCV 50mg/kg in 0.5 mL PBS was intraperitoneally injected,twice a day for five consecutive days.Each treatment course lasted for five days.In total, three successive treatment courses were given.Tumor diameter in each group was measured with vernier caliper once every three days.At the end of the third treatment course,tumor inhibition rate of each group was calculated based on measured tumor volume.From each group,five tumor bearing mice were randomly selected for peripheral blood sampling via orbital plexus.Blood sample 0.6 mL obtained from each mouse was put into anticoagulation tube for flow cytometric detection of CD3+\CD4+ and CD3+\CD8+ T lymphocyte and NK cell.Then the mice were executed by breaking the neck.Tumor tissue was obtained and weighed for the calculation of tumor inhibition rate.Afterward,each tumor tissue sample was divided into three portions;one was preserved at-80℃for measurement of IFN-γin the tumor tissue by Elisa method;the second portion of tumor tissue was fixed with 10% formalin,embedded with paraffin,and was then prepared into slices for detection of lymphocyte distribution inside the tumor by HE staining and immunofluorescence assay and detection of tumor cell apoptosis using Tunel method;the last portion of tissue was fixed with fixation buffer and was then observed under electron microscope for the changes in tumor cells.Organs including liver,kidney,lung, stomach and spleen were obtained;10%of tissue was obtained from each organ and fixed with formalin.After paraffin embedding and sectioning,the slices underwent HE staining for safety analysis of the vaccine.Remaining mice of each group were used for observation of survival duration.[Results]:â‘ Constructed plasmids pCMV-mtHSP70-IRES-TK underwent restriction enzyme verification by single restriction enzyme of Notâ… and double restriction enzymes of EcoRâ… and Xhoâ… ,respectively.Electrophoresis of cleavaged products showed specific restriction enzyme map,which confirmed that it was indeed recombinant plasmid pCMV-mtHSP70-IRES-TK;constructed plasmid pCMV-TK-IRES-EGFP was cleavaged by Salâ… and BamHâ… ,and specific restriction enzyme map was seen,confirming that it was indeed recombinant plasmid pCMV-TK-IRES-EGFE Constructed plasmid pCMV-mtHSP70-IRES-EGFP was cleavage by Xhoâ… and EcoRâ… and was verified to be mtHSP70 recombinant plasmid pCMV-mtHSP70-IRES-EGFP. â‘¡B16 cells were transfected by recombinant plasmids containing EGFP.At 48 hours later,fluorescence was significantly stronger;more than 70%of the cells expressed green fluorescent protein;fluorescence was evenly distributed around the whole cell.In B16 cells trans fected by recombinant plasmid PCMV-mtHSP70-IRES-TK,RT-PCR products contained 829 bp and 335 bp segments, which were consistent with primers of TK and mtHSP70 respectively in terms of size. In B16 cells transfected by recombinant plasmid PCMV-mtHSP70-IRES-EGFP, RT-PCR revealed 335 bp products,which was consistent with the primer of mtHSP70 in terms of size.In B16 cells transfected by recombinant plasmid PCMV-TK-IRES-EGFP,RT-PCR showed 829 bp products,which was consistent with the TK primer in terms of size.Results of western blot assay suggested expression of mtHSP70 and HSV-tk proteins in B16 cells transfected by pCMV-mtHSP70-IRES-TK plasmid;while B16 cells transfected by pCMV-mtHSP70-IRES-EGFP plasmid showed expression of mtHSP70 protein,and B16 cells transfected by pCMV-TK-IRES-EGFP plasmid showed expression of HSV-tk protein.Results of MTT assay indicated that the tumor-cytotoxicity effect of plasmid containing TK gene on B16 cells in vitro increased with the concentration of GCV.When the concentration of GCV was 50 ug/mL,tumor-cytotoxicity effects of recombinant plasmids PCMV-mtHSP70-IRES-TK and PCMV-TK-IRES-EGFP on B16 cells were 50.45%and 46.96%,respectively,with no statistically significant difference between them in vitro.â‘¢Plasmids were extracted from each recombinant salmonella typhimurium and underwent restriction enzyme verification based on corresponding restriction enzyme system.Electrophoresis of all cleavaged products showed specific restriction enzyme map,indicating successful construction of recombinant salmonella typhimurium. After being verified by restriction enzyme,four recombinant salmonella typhimurium were respectively inoculated onto LB solid medium and were repeatedly passaged 20 times.At this point,plasmids were extracted and underwent restriction enzyme verification by corresponding restriction enzyme system.The results were in line with what's expected,suggesting that the recombinant salmonella typhimurium were stably carrying the plasmids.After being infected by recombinant bacteria,B16 cells took on rounder shape as observed under light microscope.Fluorescent microscopic observation found strong expression of green fluorescent protein in cells infected with recombinant bacteria containing EGFP at 36 hours after infection.Electron microscopic observation showed multiple recombinant bacteria invaded into the cytoplasm of B16 tumor cells;B16 cells were swollen.Western blot assay revealed protein expression of relevant target genes in B16 cells after they were infected by corresponding recombinant bacteria.â‘£Mouse melanoma B16 cells 0.1 mL at the cell concentration of 107/mL was subcutaneously inoculated into the back on the right side of C57BL/6J mice;thereby a model of mouse melanoma was successfully established.At day 3 after the establishment of model,tiny tumor nodules could be palpated;at day 5,tumor was obviously developed;at day 7,tumor volume reached up to 100mm3;tumor formation rate was 100%.With HE(hematoxylin and eosin) staining,tumor tissue showed multi-lobulated appearance and extensive vascularization,with large amount of secreted melanin granules in the tissue.When given intratumor injection of 100 uL recombinant salmonella typhimurium at the concentration of 1011 CFU/mL,the mice were slightly listless and experienced mild diarrhea at the next day;at day 3,abscess developed in the tumor lesion;at day 5,necrosis occurred in the tumor.At two weeks afterward,solid tumor disappeared but all the mice survived.When given intratumor injection of 100 uL recombinant salmonella typhimurium at the concentration of 107 CFU/mL and 109 CFU/mL,the mice showed good life status;they did not experience diarrhea or local abscess formation or necrosis in the tumors lesion.To favor the analysis on the microenvironment of tumor after treatment,the dosage of 109 CFU/mL was therefore selected as the therapeutic dosage of recombinant salmonella typhimurium.At day 2,day 4 and day 7 after the intratumor injection of recombinant bacteria,three mice were executed.Tumor was obtained and prepared into frozen sections.Fluorescent microscopic observation was performed to show the expression of the reporter gene EGFP.The results suggested that at day 2 after the intratumor injection,expression of reporter gene EGFP could be seen in the tumor tissue.At day 4,expression of EGFP in the tumor tissue significantly increased.At day 7, expression of EGFP could still be found in tumor tissue,but expression level was lower,based on the experimental results mentioned above,we draw up a further treatment scheme of intratumor injection of recombinant bacteria for B16 bearing mice:model is established using 100 uL of 107/mL B16 cells;when tumor volume achieves 100 mm3,intratumor injection of 100 uL 109 CFU/mL recombinant salmonella typhimurium is given;at the second day after the injection of recombinant bacteria,a pro-drug of suicide gene HSV-tk,that is,GCV 50mg/Kg is intraperitoneally injected,twice a day for five successive days.Each treatment course lasts for five days.In total,three consecutive treatment courses are given.Next,we have injected the recombinant salmonella typhimurium SL7207/PCMV-mtHSP70-IRES-TK against mouse B16 tumor by intratumor,mouse melanoma B16 cell suspension 0.1 mL(cell concentration 107/mL) was subcutaneously inoculated into the back of the right side in C57BL/6J mice,so as to establish models of mouse melanoma.At day 3 after model establishment,tiny tumor nodules were palpated;at day 5,tumor was obviously developed;at day 7,tumor volume reached up to 100 mm3;tumor formation rate was 100%.For HT group,H group,T group and Sa group, 100 uL of 109 CFU/ml corresponding recombinant salmonella typhimurium was given respectively by intratumor injection via two injection sites;as for PBS group,100 uL PBS was given by intratumor injection via two injection sites.Only one dose of recombinant bacteria or PBS injection was given.At the second day,GCV 50mg/kg in 0.5 mL PBS was intraperitoneally injected,twice a day for five consecutive days. Each treatment course lasted for five days.Vernier caliper was used to measure tumor diameter once every three days.Consecutively three treatment courses were given.At the end of the third treatment course,statistical analysis was performed to reveal the differences in tumor volume and tumor mass.When treatment began,that is,at day 7 after model establishment,tumor volume was 101.54±4.52 mm3 in HT group, 101.54±4.36 mm3 in H group,100.37±3.74 mm3 in T group,100.18±6.41 mm3 in SA group and 97.27±10.95 in PBS group;variances were homogeneous,and analysis of variance suggested differences between groups were not statistically significant (P＞0.05).At the end of treatment,tumor volume was 1366.77±401.15mm3 in HT group,2351.34±819.075mm3 in H group,2511.09±809.105mm3 in T group, 2902.58±621.515mm3 in SA group and 6958.62±1200.40 in PBS group;variances were homogeneous,and analysis of variance suggested statistically significant differences between groups(P＜0.05).Furthermore,comparison between each two groups by LSD method revealed significant differences in tumor volume when comparing HT group to H group,T group,Sa group and PBS group,respectively (P＜0.05);the differences in tumor volume among H group,T group and Sa group were not significant(P＞0.05),while differences in tumor volume were significant when comparing these group with PBS group(P＜0.05);difference in tumor volume was not significant between T group and Sa group(P＞0.05),while differences in tumor volume between these two groups and PBS group were significant(P＜0.05); difference in tumor volume was significant between Sa group and PBS group (P＜0.05).In HT group,tumor grew at a slow pace;tumor growth was also retarded to a certain extent in H group,T group and Sa group,while tumor developed fast in PBS group.In relation to PBS group,tumor inhibition rate was calculated with the formula: (average tumor volume of PBS group-average tumor volume of experimental group)/average tumor volume of PBS group×100%.At the end of the third treatment course,tumor inhibition rate was 80.30%in HT group,66.20%in H group,63.91%in T group,and 58.29%in Sa group.When treatment ended,tumor mass was 1.9586±1.1095g in HT group,3.0433±1.6854g in H group,3.3943±1.5035g in T group,3.9327±0.6607g in Sa group and 8.9173±1.1803g in PBS group;analysis of variance suggested differences between groups were statistically significant (F=22.456,p=0.000＜0.05).Tumor inhibition rate of each group in relation to PBS group was calculated by:(average tumor mass in PBS group-average tumor mass in experimental group)/average tumor mass in PBS group×100%.Tumor inhibition rate was 78.04%in HT group,65.87%in H group,61.94%in T group,and 55.90%in Sa group.Survival duration in HT group was remarkably longer than those in varied control groups.Flow cytometric measurement was used to detect the distribution of CD3+\CD4+ and CD3+\CD8+T lymphocyte and NK cell.The results were as follow: in HT group,CD8:41.67±12.14,CD4:31.81±8.02,NK:20.41±15.63;in H group, CD8:26.19±2.29,CD4:34.19±4.23,NK:18.95±11.00;in T group,CD8:23.48±1.43, CD4:29.91±6.27,NK:15.65±9.15;in Sa group,CD8:23.75±1.25,CD4:27.12±5.29, NK:14.06±5.32;in PBS group,CD8:20.38±1.66,CD4:30.37±1.94,NK16.22±5.37. Variances in CD3+\CD8+ were heterogeneous between groups,and were thus analyzed by Welch test,which suggested significant differences between groups (P＜0.05),CD3+\CD8+ T cell in peripheral blood of HT group was remarkably higher than those of varied control groups;variances in CD3+\CD4+ and NK cell were homogeneous between groups,and analysis of variance suggested that differences between groups were insignificant(P＞0.05).Elisa assay was performed to detect IFN-γconcentration in tumor tissue of tumor bearing mice after treatment of recombinant vaccine.The results were as follow:150.4098±58.2892 in HT group; 67.6573±24.3157 in H group,57.7418±36.9552 in T group,42.5049±29.26963 in Sa group and 25.1185±11.6308 in PBS group.Variances in tissue IFN-γwere heterogeneous between groups,and were thus analyzed by Welch test,which suggested differences between groups were significant(P＜0.05,H=25.639,P=0.000); IFN-γof HT group was remarkably higher than those of varied control groups.In the tumor bearing mice of HT group treated with recombinant vaccine,HE stained tumor tissue slices showed gathering of numerous lymphocytes as well as large number of necrotic and apoptotic tumor cells;results of immunofluorescence assay showed remarkable CDS+ T lymphocytes;by Tunel method,large quantity of apoptotic tumor cells were detected in the tumor tissue from tumor bearing mice of HT group after treatment of recombinant vaccine.After the intratumor injection of recombinant salmonella typhimurium,electron microscope observation revealed numerous recombinant bacteria invaded into the tumor cells and rested in the cytoplasm of these cells;the bacteria proliferated by mitosis in the cytoplasm of tumor cells;cytolysis occurred in tumor cells.In both HT group and T group,tumor cells showed karyopyknosis,cellular apoptotic change such as nuclear margination,swollen endoplasmic reticulum,cytolysis and necrosis,and rupture of microvasculature;in both H group and Sa group,swollen endoplasmic reticulum,cytolysis and necrosis and apoptosis could be seen;while in PBS group,no obvious changes were seen in tumor cells under electron microscope;tumor cells were intact,with clear melanin granules.We performed safety assessment after treatment of recombinant vaccine. When given intratumor injection of 100 uL 109 CFU/mL recombinant salmonella typhimurium,the mice showed fine life status and did not experience diarrhea;no local abscess or necrosis occurred in the tumor lesion;no significant changes in body mass were observed during treatment.Tissues of liver,kidney,lung,stomach and spleen were obtained from the mice for HE staining.No significant pathological changes were seen in all these organs.[Conclusions]:â‘ As verified by restriction enzyme,our study has managed to construct recombinant plasmids pCMV-mtHSP70-IRES-tk,pCMV-TK-IRES-EGFP and pCMV-mtHSP70-IRES-EGFP.â‘¡Proper in-vitro expression of recombinant plasmids PCMV-mtHSP70-IRES-TK, pCMV-TK-IRES-EGFP,PCMV-IRES-EGFP and pCMV-mtHSP70-IRES-EGFP as well as plasmid containing TK gene showed anti-tumor effect on mouse melanoma cell line B16 in vitro.â‘¢Recombinant salmonella typhimurium were successfully constructed;attenuated salmonella typhimurium showed stability in carrying the plasmids and invasiveness against B 16 cells,and could express proteins encoded by target genes in B16 cells.â‘£Results of the animal experiment suggests intratumor injection of recombinant DNA vaccine containing and expressing both mtHSP70 and HSV-tk genes,as carried by attenuated salmonella typhimurium,has targeting ability against B16 tumor cell and induces strong cytotoxic T cell response,and could significantly inhibit tumor growth.Such strategy is safe and effective. |
PDF Full Text Request