隐丹参酮诱导胃癌细胞凋亡机制的研究

发布时间：2018-04-28 07:24

本文选题：隐丹参酮 + 胃癌　；参考：《黑龙江八一农垦大学》2017年硕士论文

【摘要】：目的:胃癌是常见的消化系统恶性肿瘤之一。在我国,因胃癌死亡的人数远高于世界平均水平,严重威胁人们的健康及生命。因此,寻找一种高效、安全、价格低廉的抗癌药物势在必行。隐丹参酮是由唇形科植物丹参的干燥根和根茎中提取出来化合物单体,具有抗菌、抗炎及抗癌等多种药理作用,但其具体的分子药理机制尚不清楚。隐丹参酮具有抗肿瘤活性,并已经在肺癌及前列腺癌细胞中得到证实。本研究利用异种移植瘤裸鼠模型和胃癌细胞系,从动物、细胞及分子水平上,阐明了隐丹参酮对胃癌细胞的杀伤作用、阻滞细胞周期作用、诱导凋亡作用及相关信号转导途径,为临床治疗胃癌提供一定的理论依据。方法:本论文利用噻唑蓝比色法检测细胞存活率;利用Annexin V/PI双染法、流式细胞术及蛋白质免疫印迹法检测细胞凋亡情况;利用流式细胞术和蛋白质免疫印迹法检测细胞周期阻滞情况;利用蛋白质免疫印迹法检测细胞内MAPK、AKT及STAT3信号通路相关蛋白的表达情况;利用流式细胞术和蛋白质免疫印迹法检测隐丹参酮诱导细胞凋亡过程中对活性氧水平的影响;利用异种移植瘤裸鼠模型检测肿瘤生长情况及药物毒性作用;利用免疫组织化学染色法检测移植瘤裸鼠的肿瘤组织中MAPK和STAT3信号通路相关蛋白的表达情况。结果:体外实验结果显示:隐丹参酮对12种胃癌细胞均具有良好的杀伤作用;隐丹参酮能够通过抑制Bcl-2和pro-caspase-3蛋白的表达;促进Bad和cleaved-caspase-3蛋白的表达,诱导胃癌AGS细胞发生凋亡;隐丹参酮通过抑制cyclin B1和CDK 1/2蛋白的表达,诱导胃癌MKN-28细胞G2/M期周期阻滞;隐丹参酮能够促进p-JNK和p-p38蛋白的表达,抑制p-ERK、p-AKT及p-STAT3的表达;隐丹参酮能够加剧细胞内活性氧的累积进而诱导胃癌细胞的凋亡。体内实验结果显示:隐丹参酮能够抑制移植瘤裸鼠皮下肿瘤的生长,同时移植瘤裸鼠体重无变化,对血常规指标无影响,对主要脏器无毒副作用;隐丹参酮能够促进移植瘤裸鼠肿瘤组织中p-JNK、p-p38及cleaved-caspase-3蛋白的表达,抑制p-ERK蛋白和p-STAT3蛋白的表达。结论:隐丹参酮通过细胞内活性氧介导的MAPK、AKT及STAT3信号途径,诱导胃癌细胞发生凋亡及周期阻滞,同时抑制体内肿瘤的生长。
[Abstract]:Objective: gastric cancer is one of the most common malignant tumors of digestive system. In China, the number of deaths due to gastric cancer is much higher than the world average, which seriously threatens people's health and life. Therefore, it is imperative to find an effective, safe and inexpensive anticancer drug. Cryptotanshinone is a compound monomer extracted from dried root and rhizome of Salvia miltiorrhiza, which has many pharmacological effects, such as antibacterial, anti-inflammatory and anti-cancer, but its specific molecular pharmacological mechanism is not clear. Cryptotanshinone has anti-tumor activity and has been confirmed in lung cancer and prostate cancer cells. In this study, we used xenotransplantation tumor nude mice and gastric cancer cell lines to elucidate the killing effect of cryptotanshinone on gastric cancer cells, blocking cell cycle, inducing apoptosis and related signal transduction pathways at the animal, cellular and molecular levels. To provide a certain theoretical basis for the clinical treatment of gastric cancer. Methods: cell viability was detected by thiazolyl blue colorimetry, apoptosis was detected by Annexin V/PI double staining, flow cytometry and Western blotting. The cell cycle arrest was detected by flow cytometry and Western blotting, and the expression of MAPK K T and STAT3 signal pathway related proteins were detected by Western blotting. Flow cytometry and Western blotting were used to detect the effect of cryptotanshinone on the level of reactive oxygen species (Ros) in the process of apoptosis induced by Cryptotanshinone, and the tumor growth and drug toxicity were detected by xenotransplantation tumor model in nude mice. Immunohistochemical staining was used to detect the expression of MAPK and STAT3 signal pathway related proteins in nude mice. Results: Cryptotanshinone had a good killing effect on 12 kinds of gastric cancer cells in vitro, cryptotanshinone could inhibit the expression of Bcl-2 and pro-caspase-3 protein, promote the expression of Bad and cleaved-caspase-3 protein, induce the apoptosis of gastric cancer AGS cells. Cryptotanshinone induced G _ 2 / M phase arrest in gastric cancer MKN-28 cells by inhibiting the expression of cyclin B1 and CDK 1 / 2 protein, cryptotanshinone promoted the expression of p-JNK and p-p38 protein, and inhibited the expression of p-ERK _ (1) -AKT and p-STAT3. Cryptotanshinone can increase the accumulation of reactive oxygen species and induce apoptosis of gastric cancer cells. The results in vivo showed that Cryptotanshinone could inhibit the growth of subcutaneous tumor in nude mice, while the weight of xenografted nude mice had no change, had no effect on blood routine indexes, and had no toxic and side effects on the main organs. Cryptotanshinone could promote the expression of p-JNK-p38 and cleaved-caspase-3 protein and inhibit the expression of p-ERK protein and p-STAT3 protein in nude mice. Conclusion: Cryptotanshinone induces apoptosis and cell cycle arrest in gastric cancer cells through intracellular reactive oxygen species mediated MAPK- AKT and STAT3 signaling pathway, while inhibiting tumor growth in vivo.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

【参考文献】