| Objectives: To prepare the high concentrated and purified adenovirus carrying the mouse endostatin gene (Ad-mEnd); to detect the transduction efficiency of Ad-mEnd on the effector cells and clarify mEnd transgene expression of the Ad-mEnd-transfered cells; the biological change of effector cells was observed.Methods and results:1. Ad-mEnd was amplified in 293 cells. After being purified, concentrated using cesium chloride gradient centrifugation method, the final titer of Ad-mEnd was 5.01 10 pfu/ml, assayed by TCID50 method. A similar strategy was used to amplify and detect the final titer of Ad-GFP, and it was 3.16 10 pfu/ml. When MOI 100, the transduction efficiency of Ad-GFP on G422 cells in vitro was above 90%.2. The G422 cells transfered by Ad-mEnd gene were seeded in culture flask for a few days, and harvested for further analyzing by Rt-PCR; a 540bp fragment was detected and the output was five times more than that of Actin. The measure squencing result conformed that the reaction product was endostatin gene. The 20KD endostatin protein was detected and identified from the supermatants of the transfered cells by Western blot.3. G422 glioblastoma cells were separated and cultivated to obtain the single cell suspension with digestive separaten. Several generatens of G422 cell were survival in DMEM culture medium without stickingculture-flask wall. Brain capillary endothelial cells were successfully extracted with differential centrifugation. When cultivated on DMEM culture medium for generatens in vitro, they were growing and forming road mental appearance after sticking culture-flask wall.4. The growth suppression effeciency of cells transferred by Ad-mEnd was assessed by microculture tetrazolium dye (MTT) assay. Study showed that the growth suppression effeciency of BMEC was 95%, ECV was 78%, G422 was none. In addition, the effect of Ad-mEnd gene transfer into target cells, involving BMEC, ECV and G422, was quantified by immunofluorescence flow cytometric analysis at different time. From the flow cytometric analysis, it was demonstrated that the apoptosis cells of endothelial cell lines showed a lot at 24h, with increasing from 48h to 60h. The apoptosis peak was seen from the cell cycle analysis graphs. The proportion of apoptopis cell could be seen significantly increased during G1 period. In contrast, the G422 apoptopis cells mass transfered by Ad-mEnd gene were not seen during the period of 12h to 60h and the cell cycles were normal too. The number of G422 apoptopis cells transfered by Ad-GFP or PBS was similar to the result of that by Ad-mEnd.Conclusions: Being purified and concentrated, the final titer of Ad-mEnd was 5.01 1010 pfu/ml. When MOI=100, the transduction efficiency of Ad-GFP on G422cells in vitro was more tnan 90%. The gene expression of mEnd carried by recombinant Ad-mEnd was demonstrated by Rt-PCR and Western blot. The recombinant Ad-mEnd inhibited the proliferation of endothelial cell and promoted the number of apoptosis cell. The inhibi ting effect on G422 cells was not seen. From above, we can further carry out the experiment in vivo.Part 2 Transduction efficiency assay of recombinantadenovirus in vivoObjective: to detect the transduction efficiency of Adenovirus vector inmice bearing-G422 glioblastoma, determine the best dosage and approach when gene therapy.Method: As construction and biological characteristics were similar to Ad-mEnd, Ad-GFP was taken as research subject. According to multiple factor and level analysis, involving the dosage, approach and time of injection, which were 60 levels in all. 195 Kunming mice bearing-tumour were treated with Ad-GFP. Having been treated with Ad-GFP correspondingly, each group mice were killed and separated the tumour tissue. Trypsinized, resuspended and other process, cell suspension was prepared. The cell suspension was coarsely screened by immunofluorescence microscope for identifying the positive cells, which was counted and recorded three times by flow cytometer analysis. The data was analyzed by SP...
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