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  • br Tumor xenograft experiments br The xenograft models were

    2020-08-12


    2.12. Tumor xenograft experiments
    The xenograft models were established via subcutaneous injection of PC-3-luc A83-01 (1 £ 106/100 ml) into the right legs of mice. When the tumors had formed clearly (usually 7 days), the mice were divided randomly into 5 groups (n = 50). Group 1 was injected with 1 £ 108 plaque forming units (PFU) of Ad-VT in 100 ml of PBS. Group 2 was injected with 1 £ 108 PFU of Ad-T in 100 ml of PBS. Group 3 was injected with 1 £ 108 PFU of Ad-vp3 in 100 ml of PBS. Group 4 was injected with 1 £ 108 PFU of Ad-mock in 100 ml of PBS. Group 5 was injected with 100 ml of PBS. The xenograft models were infected with recombinant ade-novirus via intratumoral injection. After successfully estab-lishing xenograft models of nude mice, injections were given twice a week for 3 consecutive weeks. Starting from 0 week, the tumor site of the nude mice was photographed once a week using in vivo living imaging equipment (Merc, Berlin, German), and photographed continuously for 5 weeks. Then, the measurement time (week) was taken as the abscissa and the average bioluminescence value of the tumor (mean pho-tons/s) was taken as the ordinate to plot the average biolumi-nescence curve of the tumor. The length and width of the xenograft tumors were measured weekly using Vernier cali-pers from 0 week, and were continuously measured for 5 weeks. The tumor volume was calculated using the following formula: 0.52 £ (smallest diameter) 2 £ (largest diameter). The percent tumor inhibition was calculated using the for-mula: (1 - treatment group tumor weight/control tumor weight) £ 100% [30,32,35]. After successfully establishing xenograft models of nude mice, the survival of nude mice was recorded every day, and from 6 weeks was recorded continuously. The survival curve of the nude mice was plot-ted with survival time (day) as the abscissa and survival rate as the ordinate.
    2.13. Statistical analysis
    Statistical analysis was conducted using data from at least 3 independent experiments. SPSS or SigmaStat 3.5 (Systat Software) was used for the analysis. P < 0.05 was
    considered to indicate statistical significance. Data are pre-sented as the mean § standard deviation (SD).
    3. Results
    3.1. Construction and identification of PC-3-luc
    The cells were seeded into 96-well plates at 5 £ 103 cells per well, and after 48 hours, luciferase activities of different clones were determined. The two clones with the highest RLU were Clone 17 and Clone 21 (Fig. 2A); these two clones were retained for luciferase stability assay.
    Clone 17 and Clone 21 were amplified and passaged, and the RLU was tested every fifth generation. Clone 17 had the highest RLU when it was passed to the 40th genera-tion, and the RLU measured by this clone in the 40th pas-sage was not significantly different from the initial value (P > 0.05) (Fig. 2B), which indicated that Clone 17 could stably express luciferase after 40 passages.
    Clone 17 was sequentially diluted and seeded into a 96-well plate at a ratio of 1:2. After the addition of the fluorescein sub-strate, the bioluminescence intensity and cell number of PC-3-luc presented a certain linear relationship (R2 = 0.9982), and the number of cells that could be detected was less than 100. The luminescence intensity increased with the increase in cell number of cells, further proving that Clone 17 could stably express luciferase (Fig. 2C andD).
    3.2. Characterization of PC-3-luc
    PC-3 cells and PC-3-luc cells were seeded in 96-well plates, and the OD values of PC-3-luc cells and PC-3 cells were detected by adding WST-1 solution at different time points. As shown in Fig. 2E, the growth trend of PC-3 cells and PC-3-luc cells was similar, and the growth curve was basically consistent, indicating that the construction process had no significant effect on the growth characteristics of the cells (P > 0.05) (Fig. 2E).
    The collected PC-3-luc cells and PC-3 cells were per-meabilized, stained with PI/RNase, and then placed in a flow tube and the cell cycle was detected by flow cytome-try. The results showed that both cells were mainly in G1 phase, and the proportion of G2 phase and S phase was also basically the same, with no significant differences in the cell cycle between the 2 cell types (P > 0.05) (Fig. 2F and G). Furthermore, we used western blot to detect cell cycle regulators of PC-3 and PC-3-luc cells. The results showed that there was no significant difference in the expression levels of Cyclin D1 and CDK2 in PC-3 and PC-3-luc cells (P > 0.05) (Fig. 2H).
    3.3. Inhibitory effect of recombinant adenoviruses on the proliferation of human prostate cancer cells