兰州大学机构库 >口腔医学院
Co-SLD对舌鳞癌细胞生物学行为的影响及其机制探讨
Alternative TitleEffect of Co-SLD on the biological behavior of tongue squamous cell carcinoma and its mechanism
李思睿
Thesis Advisor刘斌
2019-05-20
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Degree Discipline口腔临床医学
KeywordCo-SLD 舌鳞癌细胞 抗肿瘤活性 线粒体功能障碍 斑马鱼胚胎毒性
Abstract研究目的:研究了新型化合物含羰基钴的舒林酸衍生物对舌鳞癌细胞生物学行为的影响,并探讨其潜在机制。进一步为临床上口腔舌鳞癌的预防和治疗提供新思路和可能性。
实验方法:1)体内动物实验:配置不同浓度化合物Co-SLD溶液(0,DMSO,5,10,20umol/L)分别用于处理受精后4h(4hpf)的正常斑马鱼胚胎,培育至144hpf(每24h更换培养溶液),在不同时间点记录斑马鱼的孵化率、死亡率、畸形率、心率及幼鱼的游泳距离,使用吖啶橙(AO)染色法对48hpf的斑马幼鱼体内凋亡进行检测。
2)体外细胞实验:采用梯度浓度Co-SLD(0,DMSO,5,10,20umol/L)处理口腔舌鳞癌细胞CAL27,通过COX-2Elisa试剂盒检测Co-SLD作用CAL27细胞中COX-2的含量;采用生长曲线、克隆形成实验和Ki67的表达检测Co-SLD作用对CAL27细胞增殖影响;采用划痕实验检测Co-SLD对CAL27细胞迁移能力的影响;采用流式细胞术检测Co-SLD对CAL27细胞凋亡和周期的影响;并进行相关机制的探讨。
结果:1)Co-SLD安全性评估:斑马鱼胚胎早期发育期间,Co-SLD在低浓度(5和10μM)时没有明显的毒性,而浓度为20uM时对斑马鱼具有显著毒性。在24hpf测定的胚胎死亡率随着Co-SLD浓度的增加而上升,浓度在20μM时胚胎死亡率大约为9.8%;在48hpf测定胚胎孵化率,Co-SLD浓度为20uM时,孵化率降低至68.3%;72hpf畸形率的增加也证明了高浓度(20μM)对斑马鱼显著的毒性。高浓度(20μM)的Co-SLD还引发心肌细胞凋亡和心率降低,并抑制幼鱼的运动行为。
2) Co-SLD有效性评估:COX-2在CAL27细胞中高表达,Co-SLD作用24h后,浓度为10μM和20μM的Co-SLD作用于舌鳞癌细胞中COX-2表达明显下降,并抑制细胞增殖(与对照组相比减少39.8%),同时细胞的迁移能力也显著降低;细胞凋亡呈浓度依赖性,与对照组凋亡细胞0.63%相比,浓度为10μM或20μM时,凋亡细胞的比例高达6.96%或32.8%;细胞周期实验结果来看,阻滞主要发生在G1期,抑制DNA合成,导致细胞增殖受阻。随着化合物浓度增加,Co-SLD触发的线粒体功能障碍的特征表现为线粒体膜电位破坏,相比于对照组,10μM和20μM的Co-SLD处理组细胞的膜电位下降大约为26%和40%;同时高ROS的积累以及能量消耗,也呈浓度依赖性,并杀伤CAL27细胞。
结论:Co-SLD对舌鳞癌细胞存在很好的抗癌活性,且筛选出最佳浓度为10uM,不仅避免胚胎发育毒性,还通过破坏线粒体功能来增强对癌细胞的杀伤。综上,Co-SLD在口腔舌鳞癌的治疗中具有潜在的可行性。
 
Other AbstractObjective:The effects of a novel compound containing carbonyl cobalt sulindac derivatives on the biological behavior of tongue squamous cell carcinoma were studied and the underlying mechanism was explored.It provides new ideas possibilities for the prevention and treatment of oral tongue squamous cell carcinoma.
Methods: 1) In vivo animal experiment:Gradient concentration compound Co-SLD solution (0, DMSO, 5, 10, 20umol / L) was used to treat normal zebrafish embryos 4h (4hpf) after fertilization, and incubated to 144hpf (change culture solution every 24h) ), The hatching rate, mortality, deformity rate, heart rate and swimming distance of larvae were recorded at different time points. Apoptosis of 48 hpf zebrafish was detected by acridine orange (AO) staining.
2) In vitro cell experiments:The oral squamous cell carcinoma cell line CAL27 was treated with gradient concentration Co-SLD (0, DMSO, 5, 10, 20umol/L), and COX-2 content in CAL27 cells treated with Co-SLD was detected by COX-2Elisa kit. Growth curve was used. Cloning assay and Ki67 expression were tested for the effect of Co-SLD on the proliferation of CAL27 cells. The effect of Co-SLD on the migration of CAL27 cells was detected by scratch assay. Flow cytometry was used to detect the apoptosis of CAL27 cells by Co-SLD. The impact of the cycle; and the discussion of relevant mechanisms.
Results:1) Co-SLD Security Assessment:During the early development of zebrafish embryos, Co-SLD did not cause significant toxicity at low concentrations (5 and 10 μM), while zebrafish was significantly toxic at a concentration of 20 uM. The embryo mortality measured at 24hpf increased with the increase of Co-SLD concentration. The embryo mortality rate was about 9.8% at 20μM. The embryo hatching rate was measured at 48hpf. When the Co-SLD concentration was 20uM, the hatching rate decreased to 68.3. %; an increase in the 72hpf deformity rate also demonstrated a significant concentration of high concentration (20 μM) on zebrafish. High concentration (20 μM) of Co-SLD also induced cardiomyocyte apoptosis and heart rate reduction, and inhibited the behavior of zebrafish.
2)Co-SLD efficacy evaluation:2) Evaluation of Co-SLD effectiveness: COX-2 was highly expressed in CAL27 cells. After 24 h of Co-SLD treatment, the expression of COX-2 in tongue squamous carcinoma cells was significantly decreased and inhibited by Co-SLD at 10 μM and 20 μM. Cell proliferation (39.8% reduction compared with the control group), and the cell migration ability was also significantly reduced; apoptosis was concentration-dependent, compared with 0.63% of the control group, the concentration was 10 μM or 20 μM, apoptosis The proportion of cells is as high as 6.96% or 32.8%. As a result of cell cycle experiments, the blockage mainly occurs in the G1 phase, inhibiting DNA synthesis and causing cell proliferation to be blocked. As the concentration of the compound increased, the mitochondrial dysfunction triggered by Co-SLD was characterized by mitochondrial membrane potential destruction. Compared with the control group, the membrane potential of the 10 μM and 20 μM Co-SLD treatment groups decreased by about 26% and 40%. At the same time, the accumulation of high ROS and energy consumption were also concentration-dependent and killed CAL27 cells.
Conclusion:Co-SLD has good anticancer activity against oral squamous cell carcinoma, and the optimal concentration is 10uM, which not only avoids embryonic developmental toxicity, but also enhances the killing of cancer cells by destroying mitochondrial function. In conclusion, Co-SLD is potentially feasible in the treatment of oral tongue squamous cell carcinoma.
 
Pages57
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/341228
Collection口腔医学院
Affiliation口腔医学院
First Author AffilicationSchool of Stomatology
Recommended Citation
GB/T 7714
李思睿. Co-SLD对舌鳞癌细胞生物学行为的影响及其机制探讨[D]. 兰州. 兰州大学,2019.
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