蟾酥对Hep-2细胞系的体外生长及生存素Caspase-9蛋白表达的影响

Effect of Toad Vemon on Hep-2 Cells In Vitro Growth and the Expression of Survivin and Caspase-9

目的:肿瘤是威胁人类生命安全的疾病之一。寻求高效的抗肿瘤药物一直是研究的热点。研究表明,蟾酥具有抗肿瘤等多种活性。本研究通过对体外培养的Hep-2细胞系进行药物干预,研究蟾酥对肿瘤细胞生长的抑制作用及其对Survivin蛋白和Caspase-9蛋白表达的影响,探讨蟾酥抗肿瘤的分子机制,为药物的临床应用提供新的理论基础和实验依据。方法:Hep-2细胞的传代培养。MTT法分别检测不同时间(6、12、24小时)、药物浓度(18、9、4.5μg/ml)的生长抑制率。Western blot检测4.5μg/ml浓度药物作用不同时间(0、6、12、24、48小时)Survivin和Caspase-9蛋白表达的影响。结果:蟾酥对Hep-2细胞的生长具有抑制作用。其抑制程度与蟾酥水溶物浓度高低和作用时间成正比,组间有显著差异(P<0.05)。Western blot结果显示:与对照组相比,受到蟾酥水溶物作用的Hep-2细胞系的Survivin与Caspase-9蛋白的表达呈现下降趋势,具有时间依赖性(P<0.05),统计学上具有显著差异。结论:Western Blot结果显示由于蟾酥水溶物的影响,Hep-2细胞的Survivin蛋白的表达减少、Caspase-9蛋白的表达减少。MTT检测结果显示随着药物浓度的升高、时间的延长,蟾酥水溶物对Hep-2细胞的生长抑制效果增强。

Objective： Cancer was one of the diseases that were serious threaded to the safety of the human life. To seek highly effective anticancer drugs and treatment methods had been the focus of the modern scientific research. The toad was one of the ancient animals,and toad vemon was processed from the secretion of toad’s ear and skin gland. It was a traditional precious Chinese medicine. Modern medical research showed that the toad vemon had anti- tumor,analgesic,anti- epileptic and other activities. Survivin and caspase molecules,especially survivin,due to its particular mechanism and its specific tissue distribution,had become a new target of cancer diagnosis and treatment. Academic studies showed that the distribution of survivin protein was different from other inhibitor of apoptosis protein family members,most of normal human organizations were undetectable for the expression of survivin. But it had a high expression in most tumor tissues. Studies had indicated that survivin mainly played its anti- apoptotic role via the direct inhibition of the activity of caspase- 3 and caspase- 7. Some scholars speculated that the anti- apoptotic effect of survivin was activated by inhibiting Caspase- 9 apoptotic pathway. In recent years,a lot of extensive research had been done on the anti- tumor effect of toad venom and its cellular molecular mechanisms. But the specific mechanism was still unclear,and therefore to carry out its mechanisms had a great significance. In this study,Hep- 2 cells in vitro were drug- intervented,in order to study the inhibition of the water solution of toad venom on tumor cells and the protein expression of survivin and caspase- 9,and explore the molecular mechanisms of toad venom anti- tumor drugs in clinical application to provide a new theoretical foundation and experimental basis. Methods： 1. Hep- 2 cells cultured. Culturing Hep- 2cells in T- 25 flasks with DMEM. 2. Using MTT method to detect the inhibition of growth rate of Hep- 2 cells at different times（ 0,6,12 hours） and different drug concentrations（ 18,9,4. 5μg/ml）. 3. Western blot analysis the protein expression of survivin and caspase- 9 at the drug concentration of 4. 5μg / ml at different times（ 0,6,12,24,48 hours）. Results： 1. The impact of the toad vemon on Hep- 2 cell growth. MTT analysis showed that the water solution of toad venom had a inhibition on Hep- 2 cells growth. The extent of inhibition was proportional to the concentration and time of the water solution of toad venom the difference between each group was statistically significant（ P 〈 0. 05）. 2. Effect of toad vemon on the survivin and caspase- 9 protein expression of Hep- 2 cell. Western blot analysis showed that compared with the controlled group,by the water solution of toad venom extract,the role of Hep- 2 cell’s survivin and caspase- 9 protein expression displayed a downward trend,with a time- dependent manner. The difference between each group was statistically significant（ P 〈 0. 05）. Conclusion： 1.Western blot results show that the water solution of toad venom reduce the expression of survivin and caspase- 9 in Hep- 2 cell line.2. MTT test results show that with increase of the drug concentration and time,the cell growth inhibition effect enhancing.