| Objective To construct a novel human homologous gene E2F-1 retroviral expression vector, explore the role of E2F-1 gene over-expression in gastric cancer cell proliferation.Methods E2F-1 was cloned through gene recombination technique, and inserted into the retroviral expression vector pLXSN.The recombinant retroviral expression vector pLXSN-E2F-1 was identified by restriction endonuclease digestion and RT-PCR, then the recombinant vector and the empty vector were infected into the packaging cell line PA317.Transfect the recombinant retroviral expression vector pLXSN-E2F-1 into the MGC-803 cell line. Western bolt and RT-PCR were used to detect the expression of the mRNA and protein of E2F-1 gene. Colony forming assay and MTT assay were used to detect the proliferation.Results (1) PCR, restriction endonuclease digestion revealed that the E2F-1 gene was cloned into the retroviral expression vector pLXSN successfully. (2) The recombinant vector pLXSN-E2F-1 was identified by RT-PCR. (3) By infecting NIH3T3 cell line, we determined the virus titer was 2.4×105CFU/ml.(4) compared to the MGC-803 group and MGC-803/PLX group, the expression of E2F-1 in MGC-803/PLX-E2F-1 group was significantly up-regulated than the other two groups (P<0.05). (5) In the MGC-803/PLX-E2F-1 group, the colony forming efficiency was 48%, which was significantly lower than the MGC-803 group and MGC-803/PLX group(P<0.01). The result of MTT assay revealed that the growth rate of MGC-803/PLX-E2F-1 group was significantly slower than the MGC-803 group and MGC-803/PLX group(P<0.01).The prolifertation rate of MGC-803/PLX-E2F-1 group was 26.61%.Conclusion Successfully construct the retrovirus packaging cell line PA317/E2F-1. The recombinant retroviral expression vector pLXSN-E2F-1 was successfully infected MGC-803 cells. The over-expression of E2F-1 gene can inhibit the proliferation of the MGC-803 cells. Objective To study the effects of pre-built recombinant retroviral vector expressing the E2F transcription factor 1 (E2F-1) gene (pLXSN-E2F-1) on the expression of E2F-1 gene and the growth of human gastric subcutaneous tumor of nude mice.Methods BALB/C nude mice were inoculated subcutaneously with MGC-803 cells to establish the nude mouse gastric cell carcinoma model; and the models were randomly divided into pLXSN-E2F-1 group, pLXSN group and NS group. The corresponding agents were injected (0.2 ml per time) for a consecutive of 7 times on every two days, and measured the tumor volumes at the same time. Nude mice were killed after 15 days, and analyzed them by statistics; After HE staining, histopathologieal characteristics of subcutaneous tumor were observed by microscope; Immunohistochemistry was used to detect the expression of E2F-1 in the subcutaneous tumor; The expression of E2F-1 mRNA and protein was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting methods respectively; The apoptosis of tumor xerographs was measured by in situ end labeling technique (TUNEL).Results The mean rate of tumor growth in the E2F-1 treatment group was much slower than that in the pLXSN group and NS group (P<0.05); The tissue got from the nude mice had been diagnosed thatwas malignant tumor under microscope after HE staining. Immunohistochemistry results showed that there were many brown granules in pLXSN-E2F-1 group, while were almost not found in the other two groups; Compared with pLXSN group and NS group, the expression level of E2F-1 mRNA and protein were increased (P<0.05) in the pLXSN-E2F-1 group; The apoptotic rate in the pLXSN-E2F-1 treatment group was (15.4±4.1)%, significantly higher than that in the other two groups ((8.3±2.2)% for pLXSN and (8.1±2.3)% for NS(P<0.05)).Conclusion Intra-tumor injection of retrovirus pLXSN-E2F-1 has significantly inhibitory effect on the growth of human gastric subcutaneous tumor of Nude Mice. |
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