| Canine mammary tumors is a neoplastic diseases in clinical veterinary. Canine breast cancer has many likenesses with human breast cancer, as an excellent model for human breast cancer. Recent years, although the study of canine mammary tumor cells has obtained the corresponding achievements, but the cell cycle synchronization and the cycle regulation mechanism remains to be studied. Serum starvation and thymidine(TdR) double blocking methods are effective ways to make the cells synchronized at a particular stage, but the methods used in canine mammary tumor cells have not yet been reported. Paclitaxel as the20th century, the natural anti-tumor drugs, now widely used in human ovarian cancer, breast cancer, non-small cell lung cancer, and clinical treatment of melanoma, which is more prominent effect caused widespread attention. But the effects of paclitaxel on canine mammary tumor cell cycle and tumor-related factors need to be further confirmed. The experiment conducted in-depth study on this series of questions.This study used breast tumors cells in vitro as the materials, by the concentration of0%,0.1%,0.2%,0.5%,1%,0%FBS of DMEM for24h,32h,42h,54h respectively. Morphological changes observed using an optical microscope, by propidium iodide(PI) staining and flow cytometry to detect G0/G1phase synchronization effect under different conditions. Use2.5mmol/L TdR detect the synchronization effect on canine mammary tumor cell cycle. Using a concentration of0.1μmol/L and0.05μmol/L paclitaxel for24h,48h and72h respectively to detect their effects on cell cycle. Using the LightCycler2.0real-time quantitative PCR instrument and SYBR dyemethod method to test p27mRNA、p53mRNA、bcl-2mRNA, the LightCycler Software4.1is used to analysis the experiment data to evaluate the impact of paclitaxel on tumor-associated factors.The result showed that low serum concentrations made the cells lack nutrition, with the serum concentration slowly increased cell growth appeared to improve, but when the time reaches54h, most of the cells has shown a broken state, no complete cell morphology. After the cells by TdR double blocking, black particles in the cytoplasm increased significantly, and some vacuoles, and cell refractive poor. Paclitaxel on canine breast tumor cell proliferation inhibition, increased with the increase of drug concentration and time, and produce obvious apoptosis in canine breast tumor cells. When the concentration of serum was0.5%, the incubation time for42h, G0/G1phase synchronization reached most efficient87.47%. TdR double-blocking method can well synchronized cells in the G1period, S-phase cells reached93.74%when released4h. After releasing8h, the vast number of cells across S period, G2/M phase ratio was67.74%. Paclitaxel caused G2/M phase arrest and0.1μmol/L paclitaxel is more significant than0.05μmol/L. P27gene variation was gradually increased and the difference was significant(P<0.01), at the same time. p53gene also showed a gradually increasing trend with time-dose effect. As paclitaxel concentration increased, the role of time in the extended gradually, gradually reduced the bcl-2gene, the same concentrations of paclitaxel, different roles within the set time differences were very significant (P<0.01).Conclusions of all studies:Serum starvation method synchronized the vast majority of cells in G0/G1phase,0.5%FBS of DMEM hunger42h reached the best synchronization effect, serum concentration plays an important role in cell growth, the lower the concentration, cell growth situation worse. TdR double-blocking method can make a good canine mammary tumor cells synchronized in G, period. Release culture4h, cell synchronization is very good at the effect of the S phase; releasing cultured8h, cells can be synchronized to better G2/M phase and G2/M phase cells most vulnerable to falling death. Paclitaxel of G2/M phase retardation on canine breast tumor cell is apparent, can induce tumor suppression gene p27mRNA, p53mRNA expression rising and decrease the expression of oncogen-bcl-2mRNA. |
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