兰州大学机构库 >学院待认领
shRNA干扰沉默ENO1(enolase-α)基因对胃癌细胞增殖、侵袭和迁移的研究
Alternative TitleSilencing ENO1 by shRNA inhibits the proliferation, invasion and migration of Gastric Cancer cells
王玉凤
Thesis Advisor关泉林
2014-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword烯醇化酶-α shRNA 胃癌 增殖 侵袭和迁移
Abstract背景及目的:胃癌是世界范围内最常见的恶性肿瘤之一,是世界上死亡率位居第二位的恶性肿瘤。胃癌的发病具有广泛的地理分布特征,大约有2/3的胃癌病人位于发展中国家,其中中国的胃癌病人约占发展中国家胃癌病人数的42%,高危地区包括东亚、东欧和美国中部和南部的部分地区。在中国胃癌的发病率居各类肿瘤的首位。每年大约死于胃癌的人数有17万之多,基本上接近全部恶性肿瘤死亡人数的1/4。糖酵解酶ENO1在胃癌中呈高表达状态,并且与胃癌细胞的分化程度密切相关,ENO1在非小细胞肺癌中与肿瘤细胞的侵袭和转移存在密切相关。本篇研究通过将构建好的人烯醇化酶-α(enolase-α,ENO1)的慢病毒载体,转染人胃癌细胞株MKN45,探讨其体外增殖和侵袭迁移是否对人胃癌细胞产生影响。 方法:以蛋白质印迹法(Western Blot)筛选出胃癌细胞系中高表达ENO1的细胞株MKN-45。运用构建好的慢病毒质粒PLKO.1-ENO1shRNA转染胃癌MKN-45细胞株,并以空质粒PLKO.1-scramble shRNA作为阴性对照组。以荧光定量PCR检测mRNA水平,蛋白质印迹法检测蛋白质水平,验证干扰效果,以MTT法检测细胞的增殖,平板克隆实验检测成瘤能力,关于细胞的侵袭迁移能力,使用划痕实验、transwell实验检测。 结果:在高分化的胃癌细胞株MKN28上,ENO1表达相对较低,在低分化胃癌细胞株MKN45、SGC7901、BGC823中,ENO1表达相对较高。成功构建了稳定表达ENO1shRNA的人胃癌细胞稳定转染株MKN45-ENO1shRNA。与空载体PLKO.1-scramble shRNA组相比较,PLKO.1-ENO1shRNA组感染细胞后ENO1 mRNA水平和蛋白表达水平明显降低,差异有统计学意义( P < 0. 05 ) 。MTT及平板克隆实验结果表明ENO1shRNA的敲除,其细胞的增殖和成瘤能力,与空载体组和空白对照组相比,明显降低,差异有统计学意义(P < 0. 05)。化疗药物敏感性结果说明与空载体组和空白对照组相比较,ENO1shRNA敲除组的化疗药物敏感性显著增高,对细胞产生的抑制率也明显增加,差异有统计学意义(P < 0. 05)。侵袭和迁移实验结果表明,ENO1shRNA敲除组,细胞的侵袭和迁移能力明显降低,与两个对照组相比,差异有统计学意义(P < 0. 05)。ENO1基因沉默后,较大程度上抑制了胃癌细胞的增殖,对化疗药物的敏感性增加,并降低了体外胃癌细胞的侵袭迁移能力。 结论: ENO1与胃癌的侵袭和迁移相关。ENO1表达水平的下降,有效抑制胃癌细胞的增殖、侵袭和迁移能力,同时对化疗药物的敏感增加。 关键词: 烯醇化酶-α,shRNA,胃癌,增殖,侵袭和迁移
Other AbstractBackground and purpose: Gastric cancer is one of the most common malignant tumor in the worldwild, the mortality is the second of malignant tumor in the world. There are wide distribution characteristics with the incidence of gastric cancer, about two-thirds of the gastric cancer patients in developing countries, including China's stomach cancer patients (42%). There are some high-risk areas in worldwide, including Eastern Asia, Eastern Europe and some areas of Central and Southern of the United States. The incidence of gastric cancer ranks in the first of all kinds tumors in China. It is about 170,000 people died of stomach cancer each year, nearly a quarter of deaths of all malignant tumors. ENO1 is high expression in the stomach cancer, and is associated with the differentiation of gastric cancer cells. ENO1 is closely related to invasion and metastasis in non-small cell lung cancer. By this study, we will build a lentivirus vector of enzyme-alpha (enolase-α, ENO1), and transfect MKN45 cell lines of gastric cancer and explore the influence about proliferation, migration, invasion of human gastric cancer cells in vitro. Methods: The protein level of ENO1 in MKN28, MKN45, SGC7901, BGC823 was detected by Western Blot. The constructed PLKO.1-ENO1shRNA vector was transfected into 293T cells and used to infect gastric cancer cells MKN45 by using lentiviruses. The positive clones were selected with puromycin to establish stable transfection cell lines. Negative controls were generated by infection with viruses containing empty vector PLKO.1–scramble shRNA by the same protocol, and using wide type MKN45 cells as blank control. The silencing effect was confirmed by reverse transcription-PCR and Western blotting at mRNA and protein levels, respectively. Cell proliferation ability and chemosensitivity were tested by MTT assay. Cell migration and invasion ability were detected by using scratch experiment and transwell experiment. Results: ENO1 was expressed lower in high differentiated gastric cancer cell line MKN28, on the contrary, ENO1 was expressed relatively higher in poor differentiated gastric cancer cell lines MKN45, SGC7901 and BGC823. ENO1 ShRNA lentivirals vector was successfully transfected into MKN45 cells, the protein level and mRNA level of ENO1 after silencing ENO1 was assayed by western blot and RT-PCR. The results showed that ENO1 expression was significantly inhibited. MTT methods displayed that the growth of ENO1 was significantly inhibit...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223937
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
王玉凤. shRNA干扰沉默ENO1(enolase-α)基因对胃癌细胞增殖、侵袭和迁移的研究[D]. 兰州. 兰州大学,2014.
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