兰州大学机构库 >学院待认领
塞来昔布通过PI3K/Akt通路调控胃癌细胞凋亡的分子机制
Alternative TitleThe molecular mechanism of celecoxib regulating apoptosis in gastric cancer cells via PI3K/Akt pathway
李春梅
Thesis Advisor周永宁
2011-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword塞来昔布 胃癌细胞 凋亡 自噬 PI3K/Akt通路
Abstract目的:探讨选择性COX-2抑制剂塞来昔布通过PI3K/Akt通路调控人胃癌细胞株SGC-7901凋亡的分子机制。 方法:不同浓度、不同时间塞来昔布干预人胃癌细胞株SGC-7901后,采用二苯基溴化四氮唑蓝(MTT)法检测细胞增殖活性,吖啶橙荧光染色观察细胞形态学改变,DNA原位末端标记法(TUNEL)检测细胞凋亡,透射电子显微镜观察细胞超微结构的改变,流式细胞术检测细胞凋亡率,实时荧光定量RT-PCR法和Western Blot法检测天冬氨酸特异性半胱氨酸蛋白酶-8(caspase-8)、天冬氨酸特异性半胱氨酸蛋白酶-9(caspase-9)、蛋白激酶B(Akt)的表达。 结果:塞来昔布对人胃癌细胞株SGC-7901有明显的增殖抑制作用,呈时间和剂量依赖性。125μmol/L塞来昔布处理SGC-7901细胞72h时,药物对细胞生长的抑制率最高达85.6%。吖啶橙荧光染色观察到凋亡小体,TUNEL法检测到凋亡细胞。透射电镜下观察到典型的凋亡改变:细胞核膜皱缩、染色质高度凝聚并边缘化产生凋亡小体。值得注意的是,我们还在胞浆的多个位置观察到自噬空泡和自噬体,自噬体内的细胞器清晰可见;流式细胞术检测到细胞凋亡率从2.24%上调到35.67%;塞来昔布干预后,实验组Akt mRNA表达水平的改变无统计学意义,P-Akt蛋白的表达下调并呈时间和剂量依赖性,差异有统计学意义(P<0.05);实验组凋亡起始蛋白caspase-8 mRNA水平上调,呈时间和剂量依赖性,差异有统计学意义(P<0.05);实验组凋亡起始蛋白caspase-9 mRNA表达水平较对照组均上调,但125μmol/L塞来昔布作用24h组mRNA表达量与对照组相比差异无统计学意义,48h和72h组与对照相比差异有统计学意义(P<0.05)。塞来昔布作用72h后,75μmol/L组caspase-9 mRNA表达量与对照组相比差异无统计学意义,100μmol/L和125μmol/L组与对照相比差异有统计学意义(P<0.05);实验组procaspase-8和procaspase-9蛋白被活化,表达量下调,呈时间和剂量依赖性,差异有统计学意义(P<0.05)。 结论:1.塞来昔布可能通过PI3K/Akt通路诱导细胞凋亡和细胞自噬两种程序性死亡方式导致细胞死亡。2.塞来昔布对人胃癌细胞株SGC-7901有显著增殖抑制作用。3.塞来昔布诱导胃癌细胞凋亡的分子机制为线粒体途径和死亡受体途径。
Other AbstractObjective:To investigate the mechanism of celecoxib regulates apoptosis in human gastric cancer cell line SGC-7901 via PI3K/Akt signaling pathway. Methods:The effect of celecoxib on the growth of human gastric cancer cell line SGC-7901 were studied by MTT assay. Acridine orange staining was used to abserve the morphological changes of cell. Transmission electron microscopy(TEM) was used to abserve the structure changes of cell. Apoptosis was assessed by terminal deoxynucleotidyl transferse-mediated dUTP nick and labeling(TUNEL). The apoptotic rate was observed by flow cytometry(FCM). Real time quantitative RT-PCR and western blot were used to analyse the changes of caspase-8、 caspase-9、 Akt mRNA and protein levels in SGC-7901 cell after treatmented with celecoxib. Results:The proliferation of SGC-7901 cells were deareased in celecoxib-treated group, This changes were in a time- and dose-dependent manner. Apoptotic bodies were observed by acridine orange staining. A lot of apoptotic tumer cells were measured by TUNEL. Typical apoptotic changes such as nuclear membrane shrinkage、chromatin condensation、 chromatin marginalized and apoptotic bodies were observed by TEM. Interestingly, intense autophagic vacuolization and autophagosome was detected in distinct cytoplasmic areas of the affected cell. The SGC-7901 cell treated with celecoxib for 72h, FCM showed the apoptotic rate increased from 2.24% to 35.67%. The expression of Akt mRNA have no significant changes, p-Akt protein was decreased in treated cells. This changes were in a time- and dose-dependent manner(P<0.05). The expression of caspase-8 mRNA was increased sharply in SGC-7901 cell treated with celecoxib. This changes were in a time- and dose-dependent manner(P<0.05). The expression of caspase-9 mRNA was increased in SGC-7901 cell treated with celecoxib,but the changes of caspase-9 mRNA treated with 125μmol/L celecoxib 24 hours compared with control group have no difference. There is difference between the expression of caspase-9 mRNA treated with 125μmol/L celecoxib 48 hours and 72 hours compared with control group(P<0.05). The changes of caspase-9 mRNA treated with 75μmol/L celecoxib 72 hours compared with control group have no difference. There is difference between the expression of caspase-9 mRNA treated with 100μmol/L and 125μmol/L celecoxib 72 hours compared with control group(P<0.05). This changes were in a time- and dose-dependent manner. The expression of procaspase-8、proc...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222781
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
李春梅. 塞来昔布通过PI3K/Akt通路调控胃癌细胞凋亡的分子机制[D]. 兰州. 兰州大学,2011.
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