| Background:In China, the incidence of colon cancer is thrice more than it's twenty years ago as one in million,accompanying with it's rank changed from sixth to second in all cities' cancers.This incidence ascends rapidly at 4% annual growth rate,while no fundamental changes were seen in cure rate.It's urged to find new therapies since the traditional treatments had their limitations.Suicide gene therapy is an important strategy in cancer gene treatment,especially the selection and recombination of suicide gene,the optimization of vector system,the gene targeting transfer expression, and combined suicide gene therapy can increase the specific expression of suicide gene and enhance the effect to kill tumor cells.In the suicide gene therapy ,suicide gene is imported into the cancer cells and translated into enzymes which can convert the atoxic prodrug into cytotoxic metabolites,inducing the suicide of targeting cells and the death of cancer cells, meanwhile killing the non-transfected cancer cells through the bystander effect. The most investigated suicide gene system which approved into clinic trials by FDA in US are Herpes simplex virus type 1 thymidine kinase(HSV-TK)/ganciclovir(GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-FC),the anticancer effect of them have been approved in many tumors in vivo and vitro.The HSV-TK gene therapy has been used in prostate cancer clinical trials in Japan,Holland and Mexico.As shown in recent trials, in situ gene therapy was safe and effective in treating human prostate cancer, with good anticancer biologic activity.HSV-TK/GCV and CD/5-FC systems are most investigated in the suicide gene therapy. Some HSV-TK/GCV and CD/5-FC clinical treatment projects have been validated by FDA.Due to the different sensitivity of cancer cells to the suicide gene therapy,elevating drug resistance treated with only one system, complementarity in the mechanisms of CD/5-FC and HSV-TK/GCV, the combination of these two systems can not only increase the lethal effect of cancer cells, improving the treatment effect, but also enlarge the oncotherapy spectrum, reducing the drug resistance.It's well known that although the suicide gene therapy of cancer is popular, the low efficiency of purpose gene targeting and gene transfer are the major problems this method meets.The retroviral and adenoviral vectors were wildly used to transfect gene, compared with other viral vectors, the retroviral vector has low virus titer, only infects the division cells, while can contain the size of exogenous gene segment not more than 8kb.When the adenovirus infect cells, their DNA can not combine to chromatosomes but liberate in the nucleus, so it can not reach the long steady expression, and will cause the immune response when being used repeatedly, which limited the usage of these two viral vectors. The features of lentiviral vector such as infecting both division and non-division cells, holding larger segment size of exogenous gene , expressing long in vivo, lower immune response, better safety, producing high titer of virus in host cells at large range have been popular in the current gene therapy research. Meanwhile, this strategy will be more improved when the suicide gene can selectively affected and expressed in the cancer cells not normal cells. The cancer specific regulatory element such as erbB-2,α- hALA promoter were used to promote suicide gene therapy in breast cancer, AFP promoter was used in liver cancer, CEA promoter in intestine cancer and so on. Each specific promoter was used to treat specific type of targeting cancer cells in those treating protocols, while most tumor grows rapidly causing the nourishing vessel cannot supply all the tumor cells which prevents the effect of prodrugs. The suitable promoter can be selected to target the tumor vessel endothelial cells(VECs) and the tumor cells so that it can kill tumor cells and target destroy tumor nourishing vessels which leads to the tumor cells ischemic necrosis in the supplying area, highly amplifying the effect of the suicide gene. Amounts of studies indicated that KDR was expressed at low levels in normal human tissues, while highly expressed in VECs and most tumors cells. The expression of KDR was positively correlated with the VECs renewing rate and tumor malignancy which not only stimulated the division, proliferation of VECs, but also induced blood vessel hyperplasia, spured the growth and metastasis of the tumor cells. The expression of KDR in tumors is obviously higer than the normal endothelial cells, the genesis of vessels plays a key role in the growth and metastasis of tumor .So the application of KDR as a target is a novel theoretical support to diagnosis and treatment of tumor. KDR was reported highly expressed in gastric cancer ,colon cancer, rectal cancer, ovarian cancer, breast cancers and correlated with their biological behaviors, higher when the tumor grading ascends.Based on these studies, to infect ECV304 and LOVO colon cancer cells by lentiviral KDR promoted CD/TK fusion gene lentivirus and to observe the lethal effects in vitro of this therapy can not only overcome the shortage of using single suicide gene and the limited effect of some promoters which only available on specific tumor cells, but also exert the advantages of lentiviral vector, provide the basis for further study on this gene targeted therapy in colon cancer.Note: this study was supported by Natural science foundation of Guangdong province (013072). Objective:To investigate the selective killing effect of CD/TK double suicide genes drivbed by KDR promoter mediated by lentivirus on colon tumor cells. Firstly, the FUGW lentiviral vector was used to construct the recombinant lentivirus carrying double suicide gene medicated by KDR promoter which were used to infect ECV304 cells and LOVO cells. To determine the efficiency of infection, detect the expression of transgene, treat transgenic cells with prodrug (5-FC and /or GCV) and observe the sensitivity. At last, to build the nude mouse models with transplanted subcutaneous tumor on the base of LOVO cell line and to observe the tumor suppression of this therapy in vivo.Methods:1 .The construction of the recombinant lentivirus.The Ubiqutin promoter in the FUGW lentiviral vector was digested by enzyme, KDR promoter,CD gene sequence,TK gene sequence amplified by PCR were subcloned into the intermedial vector pcDNA3 to construct pcDNA3-KDRP-CD/TK, then the KDRP-CD/TK fragment was digested with restriction enzyme and inserted into the FGW, the lentiviral recombinant plasmid FGW- KDRP-CD/TK with CD/TK fusion gene regulated by the KDR promoter was constructed then.Then recombinant plasmid was packaged in the 293T cells, further amplified and purified, the package and cloning of recombinant virus were observed through the expression of green fluorescent protein(GFP) in 293T cells by fluorescent microscope, the recombinant lentivirus were identified by PCR.2. The lethal effect of KDRP-CD/TK fusion gene system mediated by lentivirus to LOVO and ECV304 cells in vitro.The recombinant lentiviral KDRP-CD/TK was used to infect LOVO, ECV304,LS174T(control) cells, the expression of objective gene was observed. The tumor cell survival rate, the bystander effect were detected under the treatment of prodrug 5-FC/GCV, the cell apoptosis and necrosis were observed through flow cytometry and transmission electron microscope respectively.3. The anti-tumor effect of FGW-KDRP-CD/TK fusion gene system mediated by lentivirus to the colon cancer LOVO cell athymic mouse model in vivo.The LOVO cell line in exponential phase of growth was vaccinated subcutaneously into the left axillary area of BALB/C female, 4 to 6 weeks old athymic mouse, to construct the colon cancer model. When the diameter of the tumor reached to 0.5cm, the tumor bearing nude mice were divided into group (ie viruses +5-FC+GCV,viruses only, 5-FC+GCV only,blank)at random. The growth of tumor was measured every five days, the weight of tumor and the tumor inhibitory rate were calculated, the tumor growth state was observed, the anti-tumor effect of the therapy was observed in vivo. After treatment, the CD/TK gene expression in tissue was detected, the routine pathological examination of tumor was made, and major organs such as heart, liver, spleen,lung,kidney were observed to find whether the pathological changes existed.Results:1. The identify of the recombinant lentiviral plasmids.1.1 The identify of the recombinant lentiviral plasmids KDRP-CD/TK.During the construction of recombinant lentiviral plasmid KDRP-CD/TK, the enzyme cutting and sequencing were made to identify. The CD ,TK fragment and KDR were united to T vector (pMD18-T) respectively and send to Shanghai Shenyou biologic technique LtD for sequencing, results were shown correct. The electrophoresis after enzyme restrictions of FGW-KDRP-CD/TK with Hind III and BamHI shew the product was 2.4kb (CDglyTK), which was correct.1.2 The package of recombinant lentiviral plasmid in 293T cells and identification of virus titer.The 10μg transfer vector, 7.5μg CMVAR8.2 packaging plasmid, 5μg VSV-G amicula plasmid were added in 130μL 2mol/L CaCL2, three plasmids were transiently cotransfected into 293T cells by calcium acid phosphate method, after 24h the GFP fluorescent expression was seen in fluorescent microscope, the supernatant was collected after 72h and filtered by 0.45μm filtrum, then centrifuged at 25000rpm for 90min, precipitate was solved in Hanks solution and stored at - 80℃for use. The gradient diluted virus solution was added into 293T cells, the expression of GFP was detected under fluorescent microscope after 72h, fluorescent cells was counted and the tite was calculated based on the virus dosage and dilution.2. the lethal effect of KDRP-CD/TK fusion gene system mediated by lentivirus to LOVO and ECV304 cells in vitro .2.1 The infection and identification of recombinant lentivirus in vitro.LOVO, ECV304, LS174T cells were added in the 200μl recombinant lentiviral suspension(100MOI) and cultured for 72h, the GFP gene expression was observed by fluorescent microscope, 80-90% cells had the green fluorescence, three types of cells shared similar infection rate. The expression of the objective gene was detected by RT-PCR in the LOVO,ECV304 cells except LS174T cells.2.2 The inhibition effect of recombinant lentivirus to cells in vitro.The 200μl recombinant lentiviral suspension(100MOI) was used to infect each cell lines, the different concentration of prodrugs were added and cultured for 72h, then the MTT was used to detect the inhibitory rate of cell growth.Results: the LOVO and ECV304 cells infected by lentiviral FGW-KDRP-CD/TK had higher sensitivity to the prodrug, the survival rate of the transgenic cells decreased when the concentration of prodrug increased. When the concentration of GCV and 5-FC were 100mg/L and 160mg/L respectively, the survival rate of LOVO cells was (5.40±1.85)%, the ECV304 was (5.26±1.82)%. While the LS174T cells infected by lentivirus were not sensitive to prodrug, the survival rate was (95.02±2.82)%, compared with other groups the significant difference existed (F=6567.806, P< 0.001) . The survival rate of FGW-KDRP-CD/TK infected LOVO,ECV304 and LS174T cells treated with 100mg/L GCV were (31.96±1.62) % ,(29.10±2.86) %,(96.57±1.89)% respectively; while treated with 160mg/L 5-FC the rates were (29.60±l .91)%,(25.23±2.05)%,(96.20±1.91)% respectively; and the combination of both drugs were (5.40±1.85)%,(5.26±1.82)%,(95.02±2.82)% respectively. This suggested that the combined use of two drugs had more lethal effect than either one in LOVO and ECV304 cells (F=803.123, P<0.001; F= 375.99, P<0.001; F=1.555, P=0.226) .2.3 Bystander effect.The obvious bystander effect could be observed in the different proportion mixed culture of the lentivirus infected cells and uninfected cells, the effect was obviously enhanced when the transgenic cells ratio increased. when this ratio was 40%, there were (32.40±1 .29)% LOVO cells and(29.95±1.96) % ECV304 cells existed respectively, while the survival ratio of LS174T cells was (97.74±1.74)%,the significant statistical difference was seen among them (F=5180.938,P<0.001) .2.4 The effect of recombinant lentivirus to the apoptosis in LOVO and ECV304 cells.The transgenic LOVO cells was dealt with prodrug for 24h( GCV: 1mg/L; 5-FC: 40mg/L) and then observed with flow cytometry: the apoptosis rate of LOVO cells treated group was 27.09±1.45%, significantly higher than the ratio of 2.96±0.34% in control group( P=0.007). The same procedure was done in the ECV304 transgenic cells , the apoptosis rate of ECV304 cells treated group was 10.85±1.50%, significantly higher than the ratio of 2.25±0.55% in control group( P=0.035).2.5 The transmission electron microscopy observation of prodrug dealt LOVO cells Cell body construction,chromatin fringe, irregular nucleus shape, nuclear fragmentation, several enhanced electron desity nuclear fragments in cytoplasm, apoptosis body, or the phagocytosis and degradation of entire apoptosis could be found in some cells under the transmission electron microscopy. Some cells may exhibit the necrosis signs like organelles dissolve.3. The anti-tumor effect of FGW-KDRP-CD/TK fusion gene system mediated by lentivirus to the colon cancer LOVO cell athymic mouse model in vivo .3.1 The growth of cancer in tumor bearing nude mice.The subcutaneous tumor node was found in the 10d after tumor cells injection.When the diameter of node reached 0.5cm, the nude mouse received treatment and the weight of tumor sample and tumor inhibited rate were recorded as follows: group I (21.34±5.72)mg,95.59%; group II :(466.61±67.89)mg, 3.58%; groupIII (451.91±48.29)mg, 6.61%; group IV: (483.91±51.60) mg;the significant difference was seen between four groups (F =165.805,P<0.001) ,weight in groups I decreased obviously,tumor weights hadn't difference between between II group,III group, IV group.3.2 Pathological changes and the detection of objective gene expression in tumor.Focal necrosis areas were seen infiltrated by amounts of inflammatory cells in the tumor tissue sections in group treated with prodrug and lentivirus. The expression of CD/TK fusion gene at mRNA levels was detected by RT-PCR in the recombinant lentivirus transgenic tumor bearing animal's tumor tissue.3.3 The histological observation in the nude mouse's major organs after treatment.In order to understand if the treatment system had toxic side effects to the nude mouse, the routine pathologic tests of heart, liver, spleen, lung,kidney were done in the treated group with no obvious abnormal findings.3.4 The visible colon cancer nude mice model. The visible subcutaneous transplanted colon cancer nude mice model was successfully constructed. The GFP green fluorescence could be observed under the LT-9MACIMSYSPULS general fluorescence imaging system. The features included:①Find the tumor as early as possible.②Observe the tumor growth and metastasis dynamically.Conclusions:1 .The KDRP promoted CD/TK suicide gene system of recombinant lentivirus was successfully constructed based on the lentiviral vector, the virus tite was 6×1010 pfu/ml2.The recombinant lentivirus could be highly transfected to LOVO cells, ECV304 cells and LS174T cells, the transfection efficiency was almost the same;3.The recombinant lentivirus could transfect the the KDRP promoted CD/TK fusion gene to LOVO cells and ECV304 cells and express effectively, with the satisfied targeting lethal effect which was concentration dependent. The combination of both prodrugs were more effective than either one;4.The mechanism of this therapy were direct tumoricidal activity and bystander effect to the targeting cells, causing the apoptosis and necrosis of target cells;5.The common and visible animal model of colon cancer transplanted nude mice could be constructed using LOVO cells, it's convenient to detect the growth, transfer;6.CD/TK fusion gene product could be expressed in the tumor tissues of recombinant lentivirus transgenic tumor bearing animals, made the satisfied anti-tumor effect in vivo, such as tumor growth inhibiting and tissue necrosis;7.After the KDRP promoted CD/TK fusion gene therapy, no obvious histological changes were seen in heart, liver, kidney, lung,spleen of the tumor bearing nude mice, suggested that this system had no toxic side effects to the tumor bearing nude mice.
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