| BackgroundGastric carcinoma is one of the most common malignant tumors in the world. Since gastric carcinoma at advanced stages does not generally respond to current treatment, gene therapy represents an attractive alternative therapy. Among methods of gene therapy, suicide gene therapy is a promising strategy of gene therapy by its high effective and clinical useful potentiality.The specific expression of suicide genes in the tumor cells so as to selectively damage tumor cells could be realized by taking advantage of some tumor specific transcription modulating elements, such as promoter and enhancer. For example, AFP promoter was commonly applied in the suicide gene therapy for hepatic cancer and CMV promoter was introduced for breast cancer. As we known, in the process of the formation of tumors vascular, KDR is one of the receptors for vascular endothelial growth factor. VEGF stimulates endothelial cell proliferation and angiogenesis by KDR. Many research have demonstrated that KDR promoter could make genes of interest over-express exclusively in tumor cells and its neogenetic vascular endothelial cells.In vitro and in vivo studies revealed that TK-positive cells were toxic to the adjacent TK-negative cells when only a small percentage of tumor cells express TK, a phenomenon termed the bystander effect. Bystander effect has an important role in the treatmeat of suicide gene therapy, but it is not clear that the mechanism of bystander effect, so what the mechanism are known will contribute to increase the treatment of suicide gene on tumors.However, many problems such as low transduction efficiency, unsatisfactory antitumor , poor target work and unsafety still remain. In order to enhance antitumor effect and target, study was carried out to provide a further evidence to treat the stomach cancer by the way of double suicide gene system. ObjectivesStudy was carried out on the selectively killing effect and mechanism of the CDglyTK fusion gene driven by KDR promoter. Investigation was taken in the following steps (1) The recombinant adenovirus containing double suicide gene was amplified, detected by PCR technique and packaged by 293 cells, in order to get the recombinant adenovirus. (2) Study was made on the effect of AdKDR-CDglyTK system on cells in vitro . Firstly, influences of adenovirus-mediated CDglyTK gene on morphology and growth characteristics of cells. Secondly, adenovirus-mediated double suicide gene driven by KDR promoter selectively kills human gastric adneocarcinoma cell line SCG7901. Thirdly, Experiment study on the mechanism of bystander effect and the apoptosis of gastric cancer cells induced by double suicide gene system. (3) Study was made on effect of AdKDR-CDglyTK system on cells in vivo. Anti-tumor effect of the recombinant adenoviruses containing fusion suicide gene was obsvered in vivo. Experiment study on the apoptosis of gastric cancer in nude mice induced by double suicide gene system.Methods1.Amplification, purification and titre measurement of recombinant adenovirus vectorpAdEsayKDRP-CDglyTK was transgenic into 293 cells to generate recombinant adenoviruses AdKDRP-CDglyTK. The recombinant adenovirus was amplified in 293 cells. When flourished 293 cells approached 70～80% convergenation, they can used to be transgenic by adenovirus. Collect the cells when cells are observed reaching 90% lesions and freeze them in refrigerator. Freeze and melt the collected 293 cells repeatedly for four times. The adenovirus was purified by cesium chloride gradient ultracentifugation. The viral production was conveniently followed with the aid of green fluorescent protein. The recombinant adenoviruses were verified by PCR. 2.Study on effect of AdKDR-CDglyTK system on cells in vitro.The SCG7901, ECV304 and HepG2 cells were infected by the AdKDR-CDglyTK in which multiplicity of infection of Ads is 100 in virto. Firstly, The expression of CDglyTK fusion gene was confirned through the methods of RT-PCT and hybridization in situ. Furthermore, the changs on morphology of the transgenic cancer cells SCG7901, ECV304 and HepG2 cells are observed by light microscope, phase-microscope and transmission electron microscope. Moreover, the distribution of cell cycle was detected by flow cytometric assay and growth curve was detted by MTT test. Secondly, cell viability and bystander effect was measured using MTT test through the varies of treatment that includes different concentration and time of prodrugs. Thirdly, flow cytometry, transmission electron microscope and Hoechest33258 and PI fluorescein stain were used to observe change of cell cycle and apoptotic cells. At last, gap junction intercellular communication was determinated by fluorescence recovery after photobleaching on SCG7901, ECV304 , HepG2 and Hela cells of transgenic and untransgenic and use of medium with versulin. Moreover Transgenic and untransgenic cells were mixted with a proportion of 5% and 95% or 10% and 90% respectively. On the condidtion of mixture medium with versulin or without it. the cells survival rate were detected by MTT methods.3. In vivo toxicity studied with injections of the recombinant adenoviruses and prodrugsTo examine the therapeutic effect of the recombinant adenoviruses in vivo, two-to-four-week-old female Balb/c nu/nu ethymic mice were used as hosts for SCG7901 cells xenografts, and tumors were allowed to grow for two weeks before randomizing mice by size. All tumors were at least 0.5cm and mice were grouped according to treatment regimen (ie viruses+5-Fc+GCV, viruses only, 5-Fc+GCV only, blank). The growth curves of tumors were drawn and growth inhibition ratios of tumors were calculated, and the expression of fusion gene was detected by RT-PCR and hybridization in situ. Moreover, hematoxylin and eosin stain of tumor tissues were performed for histological examination and immunohistochemistry was used to detect to microvessel density of tumor tissues. In addition, systemic toxicity of recombinant adenoviruses and prodrugs were determined by examining histological change of heart, lung, liver, kidney and small intestine of nude mice after treatment. Lastly, transmission electron microscope and the TUNEL methods were used to observe change of apoptotic cells in vivo.Results1. Amplification and identification of Recombinant adenovirusAbout 24 hours after transfecting the recombinant adenoviral plasmid into 293 cells, the expression of green fluorescent protein could be seen and became intensified at 3-5 days. As lots of 293 cells turned round and plaques could form at 7-10 days, the 293 cells were gathered. Recombinant adenoviruses whose titer was as high as 2×1012pfu/ml have been achieved by repeatedly infecting 293 cells with recombinant adenoviruses supernatant, followed by CsCl banding procedure. The recombinant adenoviruses were further confirmed by PCR.2.Study on effect of AdKDR-CDglyTK system on cells in vitro2.1 SCG7901, ECV304 and HepG2 cells were infected with the recombinant adenovirusesThe infection rate of all cells were no difference, and it gradully increased with the increasing of the MOI of AdKDR-CDglyTK and AdCMV-CDglyTK. Almost all of them were infected as MOI of the recombinant adenoviruses was 200.2.2Influence of double suicide on morphology and growth characteristics of the cellsDouble suicide gene had no significant effect on the formation of SCG7901 ECV304 and HepG2 cells cultured in vitro drived by KDR promoter mediated by adenovirus. Moreover, the transgenic cells and untransgenic cells had the similar groth.2.3 Specific cytotoxicity of double suicide gene on SCG7901 and ECV304 cellsThree kinds of cells infected with Ad-KDR-CDglyTK and Ad-CMV-CDglyTKat MOI of 100 were maintained in culture medium of different concentration of GCV and/or 5-FC for 96 hours, it was revealed that the infected cells exhibited different sensibility to the two prodrugs: All the cells infected Ad-CMV-CDglyTK and the SCG7901 and ECV304 cells infected Ad-KDR-CDglyTK were highly sensitive to the prodrugs. Compared with them ,the HepG2 cells infected with Ad-KDR-CDglyTK appeared to be unsensitive to the two prodrugs. On the condition of prodrug with the same concentration, the cells survival rate of SCG7901 and ECV304 cells were dropped gradually as the culture time was prolonged. It showed that the survival rate were depended on the time in a shape. On the seventh day ,the survival rate of SCG7901 cells was (26.92±1.50) %,(31.66±1.25) % in the use of the GCV, 5-FC respectively, and in the combination of GCV and 5-FC the cells survival rate was (1.15+1.12) %, it showed that there were significance difference between the use of prodrugs. On the contrary , the survival rate of transgenic HepG2 cells had no relation with the culture time.2.4 The study of bystander effect of CDglyTK geneThe nontransgenic cells were cocultured with different ratios of the transgenic cells, bystander effect of CDglyTK gene on cells increased gradually with the increasing of rate of transgenic cells, as the ratio was 40%, survival rates of SCG7901, ECV304 and HepG2 cells were (12.47±1.92) %, (9.92±1.40) % and (97.46±1.98) % respectively, but killing effect by recombinant adenoviruses was not observed in HepG2 cells. In the use of GCV single ,killing cells effect were observered when the transgenic rate was 40%, and to 5-FC , bystander effect were seen when the rate was only 5%, but there were no difference on killing effect when the rate was 60%. In the combination of GCV and 5-FC, coeffect were existed in varies of mixture propotion. Furthermor the obviously coeffect was found when the transgenicl rate was 40%.2.5 The effect of prodrugs on cell cycle of transgenic SCG7901 and ECV304 cellsAt concentrations of GCV and 5-Fc were 100mg/L and 2000mg/L respectively by 72h after treatment, cell cycle of tranfected cells was monitored. It was revealed that the cell cycle of SCG7901 and ECV304 cells was arrested at S phase. By the methods of flow cytometry, apoptosis rate of SCG7901,ECV304 and HepG2 in control group were (2.01±0.32)%,(1.62±0.49)% and (1.56±0.16)%; the apoptosis rate in treatment group were (29.25±4.85)%,(27.37±4.16)%and(1.34±0.34)%, it showed that there were significance difference in apoptosis rate of each cell line between control group and treatment group.2.6 SCG7901 cells and ECV304 cells were observed by TEM after treatmentCell shrinkage, chromatin gathered along the nuclear membrane, cell budding. Chromatin condensation, fragmentation with intensive electron density and apoptotic body were observed by TEM in some cells.2.7 Hoechest33258 and PI fluorescein stainAccording to the fluorescence intensity of blue and red light, subdued bliue and red light was regarded as living cell; bright blue and weak was apoptotic cell; bright red and weak blue was dead cell. The apoptotic rate each group was (30.18±5.63) % A group; (26.39±4.40) % B group; (1.71±0.91) % C group; (1.34±0.72) % D group; (1.60±0.83) % E group. There were significant difference in the apoptotic rate betweeen each group.2.8 The functions of GJIC of cells was detected by FRAP techniquesThrough photobleaching techniques and laser scanning confocal microscope, the images of SCG7901, ECV304, HepG2 and Hela cells were observed on the initial dye distribution before bleaching and the fluorescence intensity of SCG7901, ECV304, HepG2 was gradually recovered at different times after bleaching, but the fluorescence intensity of Hela cells had no changes. The mean fluorescence recovery rates of SCG7901, ECV304, HepG2, and Hela cells had significant difference (F=5.97 P=0.006) .2.9 The functions of GJIC of versulin on cultured cells were observed by FRAP techniques.Through photobleaching techniques and laser scanning confocal microscope ,the images of SCG7901, ECV304, HepG2 and Hela cells cultured with versulin were obtained. images were observed on the initial dye distribution before bleaching and the fluorescence intensity of SCG7901, ECV304, HepG2 cultured with versulin was gradually recovered at different times after bleaching. In contrast to the cells cultured without versulin, the fluorescence recovery of the bleached cells cultured with versulin were obvious in the same time. But the fluorescence intensity of Hela cells cultured with versulin had no changes. The mean fluorescence recovery rates of SCG7901, ECV304, HepG2, and Hela cells cultured with versulin had significant difference (F=55.80 P=0.000) .2.10 The study of bystander effect of fusion gene and the effect of versulin on cultured cellsThe nontransgenic cells were cocultured with two ratios of the transgenic cells which rate was 5% and 10%, and bystander effect of fusion gene system on cells were observed. When the ratio was 5%, there was significant difference between cells survival of the groups related SCG7901cells (F=152.32 P=0.000) and the groups related ECV304cells (F=218.78 P=0.000) . When the ratio was 10%, there was significant difference between cells survival of the groups related SCG7901cells(F=407.83 P=0.000) and the groups related ECV304cells (F=523.67 P=0.000) .3. Study on effect of AdKDR-CDglyTK system on cells in vivo3.1 Anti-tumor effect of double suicide gene system in vivoHunan stomach tumors cells SCG7901 were injected into the corresponding syngeneic mice and allowed to established tumors of 50mm mean diameter. We injected the recombinant adenoviruses by introtumor, followed by GCV and 5-FC treatment, tumor weight and tumor growth inhibition rate were calculated. After prodrugs treatment ended, the weigt of A,B,C and D group were (501.94±4.50)mg,(25.04±3.09)mg, (503.79±6.27)mg, and (498.07±4.46)mg respectively, ratio of tumor growth inhibition were 79.01% in B group, it shoed that tumor weights had difference between groups (F=12727.42 P=0.000) .3.2 Histology of tumorThe nod of subcutaneously tumor seemed like circle, oval-shap and irregular, andit cannot be moved easily.Tumors with the recombinant adenoviruses were stained byimmunohistochemical method, the MDVvalue of A, B, C, D group respectively was(29.46±6.33) , (6..05±1.30) , (27.9±6.21) , (28.77±3.85) , there was significantdifference between the the MDVvalue of four groups (F=27.04 P=0.000) .3.3 Histology of heart, liver, lung, kidney and small intestineTo observe whether the recombinant adenoviruses and prodrugs contained systemic toxicity , liver, heart ,lung, kidney and small intestine of trentment group of nude mice were detected by H-E staining, it was revealed that the histology of heart, liver, lung, spleen, kidney and small intestine were't abnormal. 3.4 Cell Apoptosis rate was detected by TUNEL method in miceApoptosis cell was distributed in tumor under microscope , and apoptotic body were found. The apoptosis index of A, B, C, D group respectively was (1.94±0.53) %, (35.76±5.88) %, (2.02±0.60) %, (1.70±0.73)%. there was significant differenced between the apoptosis index of four groups (F=160.02 P=0.000) .In contrast to B group respectively, there was significant differenced in A, C and D group. Double suicide system driven by KDR may induce the cell apoptosis in mice.Conclusions1. Recombinant adenovirus was packed and amplified, and high purity and high transfection efficiency was made;2. The significant diffence have not been founded in morphology and characteristic of growth both the transgenic cells and untransgenic cells;3. KDR promoter may regulate the CDglyTK fusion gene system to selectively kill the KDR-expression SCG7901 and ECV304 cells. Moreover, a double approach is superior to a single suicide gene therapy.4. It is the first time to be observed that double suicide gene system may induce apoptosis of SCG7901 and ECV 304 cells and had bystander effect in vitro.5. Relation between intercellular communication and gap connection was observed by FRAP technique, which is firstly used by now.But Hela cell had no intercellular communication.Versulin may enhanced the effect of bystander killing cells.6. It is the first time to be observed that the mechanism of bystander effect of double suicide gene system may involve in gap connection and cell apoptosis in vitro.7. Stomtch tumor common mice model derived from human huamn cancer SCG7901 cells can be established stably, they can provide reliable materials to study treatment on human stomach cancer.8. Double suicide gene could be effectively expressed in the transgenic tumors in nude mice, and the suicide gene system displayed satisfactory antitumor efficacy in vivo, and involved tumor growth inhibition, reduction of microvessel density and tumor apopotisis.9. Double suicide gene system driven by KDR promoter may induce apoptosis of stomach cancer in nude mice.10. The histology of heart, liver, lung, spleen, kidney and small intestine in treatment group hadn't abnormal, it was revealed that the recombinant adenoviruses and prodrugs didn't exert systemic toxicity to nude mice. |
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