KRAS突变型细胞株的构建与鉴定

发布时间：2018-06-12 11:29

本文选题：KRAS + 腺相关病毒　；参考：《华东师范大学》2017年硕士论文

【摘要】：癌症是威胁人类健康的重大疾病。在人类所有癌症中,癌基因RAS具有较高的突变率,参与多种高致死性肿瘤的发生和发展,如胰腺癌、肺癌和结肠癌等。因此,靶向RAS蛋白的药物研发已经成为癌症治疗的重要课题。同源细胞株及动物疾病模型为靶向药物研发提供了有效手段,这些模型的构建主要是通过基因编辑技术来实现的。目前应用较为广泛的基因编辑术主要有类转录激活因子效应物核酸酶技术和CRISPR/Cas9技术,其原理是通过同源重组和非同源末端连接的方式来实现基因敲除、敲入及过表达,从而改变生物体的遗传信息。本研究利用腺相关病毒介导的基因编辑成功构建了四种KARS不同突变位点的细胞模型(KRASG12D、KRASG12C、KRASG12S、KRASG12V)。在蛋白水平上对上述细胞株进行鉴定,结果显示,四种KRAS突变型细胞中KRAS蛋白的表达量及KRAS蛋白的活化程度(KRAS-GTP)均表现出明显的增加。KRAS突变会激活下游的信号通路,研究结果显示,相对于野生型细胞,四种KRAS突变型细胞中磷酸化 cRaf(p-cRaf-Ser338)、磷酸化 ERK1/2(p-ERK1/2-Thr202/Tyr204)、磷酸化 AKT(p-AKT-ser473)以及磷酸化MEK(p-MEK-Ser217/221)蛋白水平均升高,表明细胞中突变的KRAS蛋白能介导细胞信号通路的活化。鉴于突变型细胞中KRAS信号通路已活化,本文对突变型细胞的生长情况进行研究。结果显示,相比于野生型细胞,突变型细胞的生长速度、克隆形成速度、S期的细胞数目及S期相关蛋白CyclinE、CyclinA及其激酶磷酸化CDK2(p-CDK2-Thr160)的表达量均明显的升高。这些结果提示,在突变型细胞中,KRAS点突变会促进下游信号通路的过度激活,使细胞快速增殖。最后,本文对KRAS突变型细胞株的药物敏感性进行研究。本研究以RAS信号转导通路为靶点选取了近20种临床小分子抑制剂,通过分析药物的半有效抑制浓度(IC50)来检测细胞的药物敏感性。结果显示,KRAS突变型细胞株对三种MEK的抑制剂表现出明显的耐受性,相对于野生型细胞约有10倍以上的差异,对Wee1的抑制剂和AKT的抑制剂表现出一定的敏感性。综上所述,本研究成功构建了四种KRAS不同突变位点的细胞株,该细胞株可为靶向KRAS突变肿瘤的药物研发及靶点验证提供良好的细胞模型。
[Abstract]:Cancer is a major disease threatening human health. Among all human cancers, the oncogene Ras has a high mutation rate and is involved in the occurrence and development of many highly fatal tumors, such as pancreatic cancer, lung cancer and colon cancer. Therefore, the research and development of Ras protein targeting drugs has become an important subject of cancer treatment. Homologous cell lines and animal disease models provide an effective means for the development of targeted drugs. These models are constructed mainly by gene editing techniques. At present, gene editing techniques have been widely used in transcription activator effector nucleases and CRISPR-Cas9 techniques, which are based on homologous recombination and non-homologous terminal junctions to realize gene knockout, knockin and overexpression. Thus altering the genetic information of the organism. In this study, four cell models of KRASG12DN KRASG12C KRASG12SN KRASG12SN KRASG12VN were successfully constructed using adeno-associated virus mediated gene editing. The results showed that the expression of KRAS protein and the activation degree of KRAS protein in the four KRAs mutant cells were significantly increased with the increase of KRAS-GTP-), and the downstream signaling pathway was activated by the mutation of .KRAS. The results showed that phosphorylated cRafp-cRaf-Ser338C, phosphorylated ERK1 / 2 + -ERK1 / 2-Thr202 Tyr204N, phosphorylated AKTP-AKT-ser473 and phosphorylated MEKp-MEK-Ser217221) were increased in four KRAs mutant cells compared with wild-type cells. These results suggest that the mutant KRAS protein can mediate the activation of cell signaling pathway. In view of the activation of KRAS signaling pathway in mutant cells, the growth of mutant cells was studied in this paper. The results showed that compared with wild-type cells, the growth rate of mutant cells, the number of cells in S phase of clone formation rate and the expression of cyclin E cyclin A and its kinase phosphorylated CDK2 p-CDK2-Thr160) were significantly increased. These results suggest that the KRAs point mutation in mutant cells can promote the excessive activation of downstream signaling pathway and make the cells proliferate rapidly. Finally, the drug sensitivity of KRAS mutant cell line was studied. In this study, Ras signal transduction pathway was used as the target to detect the drug sensitivity of cells by analyzing the semi-effective inhibitory concentration (IC50) of 20 clinical small molecular inhibitors. The results showed that KRAS mutant cell lines showed obvious tolerance to three MEK inhibitors, which were more than 10 times different from those of wild type cells, and showed a certain sensitivity to the inhibitors of Wee1 and AKT. To sum up, we successfully constructed four KRAS cell lines with different mutation sites, which can provide a good cell model for drug development and target verification of KRAS mutant tumors.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3

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