| Objective1.To investigate the expression,selective killing efficacy and bystander effect of ecoli-cytosine deaminase/herpes simplex virus-thymidine kinase(CDglyTK)gene delivered by lentiviral vector in rat breast cancer SHZ-88 cells.2.To observe the expression of CDglyTK fusion gene in the flaps following the recombined vector infecting rat free superficial inferior epigastric artery(SIEA)flaps by intra-artery perfusion.3.To explore the anti-tumor effect of the CD/TK double suicide gene and prodrug(5-fluorocytosine plus ganciclovir,5-FC+GCV)system on the rat transplanted breast cancer in vivo.4.To detect whether genetically modified free flaps could minimize systemic toxicity in SD rats or not.Methods1.The amplified recombined expression plasmid/control plasmid,packaging plasmid and envelope plasmid were transferred into HEK 293T cells.The recombinant lentivirus was harvested by collecting the supernatant of virus producing cell culture and concentrated by ultracentrifugation.For the determination of virus titer,HEK 293T cells were seeded in 96-well plate and infected with serial 10-fold dilutions of the recombinant lentivirus.The number of green fluorescence protein(GFP)-positive cells per well was counted under a fluorescence microscope and the virus titer was determined by the following formula:virus titer = the number of GFP-positive cells/the infective dose of virus.2.SHZ-88 cells were infected with the recombinant lentivirus at multiplicities of infection(MOI)ranging from 0 to 200,and the GFP-positive cell percentage was counted under fluorescence microscopy.Then,the Cell Counting Kit-8(CCK-8)solution was used to measure the effect of recombinant lentivirus on SHZ-88 cell growth.Reverse transcriptase polymerase chain reaction(RT-PCR)and western blot were performed to evaluate the expression of CDglyTK fusion gene in SHZ-88 cells transfected with recombinant lentivirus.The transfected SHZ-88 cells were treated with various concentrations of 5-FC and GCV separately or in combination,cellular survival rates were detected by the CCK-8 assay,cellular apoptosis and the percentages of the cells in the G0/G1,S or G2/M phases of cell cycle were performed by the MuseTM Cell Analyzer.SHZ-88 cells were cultured in the supernatant from the infected cells which were treated with certain concentrations of 5-FC and GCV,afterwards,the CCK-8 assay was used to measure the cellular survival rates of the SHZ-88 cells.3.We dissected SIEA flaps of SD rats to research the blood supply system of the flaps and confirmed the blood supply area of superficial inferior epigastric artery in the SIEA flap by perfusing methylene blue through femoral artery.SHZ-88 cells were injected subcutaneously into the SIEA flap,transplanted tumor growth and survival of the flap was observed every three days.4.We designed and raised SIEA flaps of SD rats.The recombinant virus or phosphate-buffered saline(PBS)was perfused through afferent artery.The virus was allowed to incubate within the free flaps for 1 h at 37?,then re-anastomosed the native vessels and replanted the flaps in its original position.RT-PCR and western blot were used to evaluate the expression of CDglyTK fusion gene in the gene modified flap,the underlying flap bed and the internal organs including heart,lung,liver,spleen,kidney,small intestine at 7 days after operation.The SIEA flaps were infected with LV-CDglyTK,LV-GFP and PBS respectively and then replanted in the original location as described above.SHZ-88 cells were injected subcutaneously into the flaps.The intraperitoneal injection of 5-FC + GCV was administered daily from the first postoperative day.The dimensions of the tumors were detected every third day,tumor volume was calculated and the growth curves of tumors were drawn.Serum was collected and analyzed for aminotransferase(ALT)and aspartate aminotransferase(AST)every week.The tumors and internal organs including heart,lung,liver,spleen,kidney and small intestine were harvested on days 42.The changes of tumor weight and tumor-inhibition rate were evaluated.The hematoxylin and eosin(H&E)staining of the tumors and the major organs was performed for histological examination.Results1.The lentivector packing and titration:(1)48 hours after the three plasmids were transferred into HEK 293T cells,more than 95%of the cells had green fluorescence,which indicated that the recombinant lentivirus was constructed successfully.(2)As for the determination of virus titer,when the amount of the recombined lentivirus was 10-5 ?l,two of the cells infected with LV-CDglyTK had green fluorescence and only one of the cells transfected with LV-GFP expressed green fluorescence protein.The virus titers of LV-CDglyTK and LV-GFP were 2E+8 TU/ml and 1E+8 TU/ml respectively,which was calculated by the formula as described above.2.The direct cytotoxic effect and bystander effect of the CD/TK double suicide gene and prodrug system on SHZ-88 cells:(1)The transduction efficiency of LV-CDglyTK and LV-GFP was quite similar in SHZ-88 cells.The percentages of GFP-positive cells increased and the green fluorescence enhanced gradually with the increase of the MOI of recombinant lentivirus.When the MOI was 100,95%of the cells had green fluorescence.When the MOI was 200,the GFP-positive cell percentage was nearly 100%.According the results of CCK-8 kit detected,it did not effect on the transfected cell growth when the MOI of recombined virus was less than 100.However,when the MOI was over 200,it had cellular toxicity and significantly inhibited the infected cell growth.(2)The expression of CD/TK fusion gene could be detected in the SHZ-88 cells transfected with LV-CD/TK by RT-PCR and western blot analysis,but not identified in the cells of the empty lentivirus infected group and control group.(3)The cells in the objective gene infected group were highly sensitive to prodrugs(5-FC+GCV)and the cell survival rate decreased significantly in the presence of the prodrugs in a dose-dependent manner(P<0.001),but the cell growth of the negative control group and the blank control group was not markedly inhibited.In addition,the inhibited effect of the combined use of 5-FC+GCV was much stronger than either one used alone to the cells infected with LV-CDglyTK(P<0.01).Compared with the negative control group and the blank control group,the cell apoptosis rate and the percentage of cells in the GO phase in the experiment group following treatment with 5-FC+GCV were significantly increased(P<0.01).(4)The supernatant from the experiment group which was treated with 5-FC and GCV could obviously decrease the growth of normal SHZ-88 cells(P<0.001),but the supernatant from the negative control group and the blank control group did not affect the normal cell growth.3.The blood supply system of SIEA flap and transplanted tumorigenesis in SD rats:(1)We found that the rat SIEA flaps had stable blood supply system following dissecting SIEA flaps of SD rats.The femoral artery constantly gave off a branch called superficial inferior epigastric artery about 1.4cm away from the groin,which was the main blood supply to the SIEA flap.The distribution of methylene blue in the SIEA flap was more than over 2.5cm×2.5cm after perfusing it through the femoral artery.(2)The transplanted tumor grew stability following injecting SHZ-88 cells into the SIEA flap subcutaneously with good reproducibility.4.The anti-tumor effect of lentivirus-mediated CDglyTK gene transfecting rat SIEA flaps on the rat models of breast cancer by intra-artery perfusion:(1)At 7 days after operation,the expression of CD/TK fusion gene could be detected in the SIEA flap(including cephalic flap tissues,pedicle and caudal flap tissues)of the experiment group by RT-PCR and western blot assay,but not identified in the flaps of the negative control group and the blank control group.In addition,no evident expression of CD/TK gene was detected in the underlying flap bed and the internal organs of the experiment group.(2)Following the recombined virus or PBS perfusing free SIEA flaps as described above,SHZ-88 cells were injected subcutaneously into the flaps and the intraperitoneal injection of prodrugs was performed daily till 30 days.Final tumor volume(mm3)and final tumor weight(mg)in the experiment group(104.00±7.53,122.25±6.93)were obviously smaller than those in the negative control group(690.10±41.95,693.56±26.91)and the blank control group(706.57±21.22,720.17±23.15)(P<0.01).Meanwhile,the tumor inhibition rate in the experiment group(83.02%)was significantly higher than those in the negative control group(3.70%)and the blank control group(P<0.01).The results of HE stained sections showed obvious necrosis in the transplanted tumor of the experiment group while no abnormal histology observed in the two control groups.(3)The levels of ALT and AST in the serum of the three groups did not have wide fluctuation within 42 days after operation.The infiltration of inflammatory cells and the metastasis of rat breast cancer cells were not found in the internal organs of the experiment group.Conclusions1.The recombinant lentivirus LV-CDglyTK and LV-GFP could be constructed successfully with a three-plasmid lentivirus packaging system.2.The expression of CD/TK fusion gene could be detected in the SHZ-88 cells transfected with LV-CD/TK.The double suicide CD/TK and prodrug system had selective killing efficacy on SHZ-88 rat breast cancer cells,meanwhile,it could increase the apoptosis of the infected cells and arrest the cell cycle at the G1 phase.In addition,the inhibited effect of the combined use of two prodrugs was much stronger than either one used alone to the transfected cells.The double suicide CD/TK and prodrug system also showed significant bystander effect.3.The rat SIEA flap had stable blood supply system and the blood supply area of superficial inferior epigastric artery in the SIEA flap was over 2.5cm×2.5cm.The transplanted tumor grew stability following injecting SHZ-88 cells into the SIEA flap subcutaneously with good reproducibility.4.Following LV-CDglyTK transfecting a rat SIEA flap by intra-artery perfusion,the expression of CD/TK fusion gene was found within the flap,but not detected in the underlying flap bed and the internal organs of the SD rat.The double suicide CD/TK and prodrug system showed obviously anti-tumor effect on the rat transplanted breast cancer in vivo.The genetically modified free SIEA flaps could minimize systemic toxicity in SD rats. |
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