蜂毒素对人膀胱癌T24细胞P16基因表达及甲基化的影响-外科学专业论文.docxVIP

三峡大学硕士学位论文 三 峡 大 学 硕 士 学 位 论 文 PAGE IV PAGE IV Abstract Objective: To investigate the effect of melittin on the P16 expression and methylation in human bladder carcinoma T24 cells and its probable mechanism involved. Methods: Human bladder carcinoma T24 cells were cultured in vitro, and treated with different concentration of melittin (0，1，2，4，8，16 and32 μg/mL). The proliferation inhibition was detected by MTT assay. Invented microscope was observe variation of cell morphology. FCM was tested the cell cycle. RT-PCR was used to detect to variation of P16 mRNA. Western blotting was used to detect the expression of P16 protein. MSP method analyses the CpG island methylation status of P16 gerne of T24 cells. Results: Melittin obviously inhibited proliferation of human bladder carcinoma T24 cells in a dose-dependent manner, but the effect of inhibition was not obvious enhancement with the extention of time. Using invented microscope observed decreasing density of T24 cell, a part of cells suspending in culture medium and cell apoptosis morphology changing in a dose-dependent manner. T24 cells treated by melittin arrested significantly S- phase cell cycle. The expression of P16 mRNA gradually increased with increasing concentration of melittin. In protein level the expression of P16 gradually increased in a dose-dependent manner. MSP method detected tumor suppressor gene P16 in the human bladder carcinoma T24 cells show a part methylation status, Melittin have demethylation effect in a dose-dependent manner. Conclusion: Melittin can inhibit the proliferation of human bladder carcinoma T24 cells in a dose-dependent manner. P16 gene 5’ promoter region existed a part CpG island methylation status in human bladder carcinoma T24 cells, P16 gene methylation in human bladder carcinoma development process maybe a more important molecular mechanisms. Melittin can become a de-methylation drug which can make silence or induce the P16 re-expression. Key words: Melittin Bladder ca