發(fā)布時間：2018-06-12 11:29

  本文選題：KRAS + 腺相關(guān)病毒��； 參考：《華東師范大學》2017年碩士論文

【摘要】：癌癥是威脅人類健康的重大疾病。在人類所有癌癥中,癌基因RAS具有較高的突變率,參與多種高致死性腫瘤的發(fā)生和發(fā)展,如胰腺癌、肺癌和結(jié)腸癌等。因此,靶向RAS蛋白的藥物研發(fā)已經(jīng)成為癌癥治療的重要課題。同源細胞株及動物疾病模型為靶向藥物研發(fā)提供了有效手段,這些模型的構(gòu)建主要是通過基因編輯技術(shù)來實現(xiàn)的。目前應(yīng)用較為廣泛的基因編輯術(shù)主要有類轉(zhuǎn)錄激活因子效應(yīng)物核酸酶技術(shù)和CRISPR/Cas9技術(shù),其原理是通過同源重組和非同源末端連接的方式來實現(xiàn)基因敲除、敲入及過表達,從而改變生物體的遺傳信息。本研究利用腺相關(guān)病毒介導的基因編輯成功構(gòu)建了四種KARS不同突變位點的細胞模型(KRASG12D、KRASG12C、KRASG12S、KRASG12V)。在蛋白水平上對上述細胞株進行鑒定,結(jié)果顯示,四種KRAS突變型細胞中KRAS蛋白的表達量及KRAS蛋白的活化程度(KRAS-GTP)均表現(xiàn)出明顯的增加。KRAS突變會激活下游的信號通路,研究結(jié)果顯示,相對于野生型細胞,四種KRAS突變型細胞中磷酸化 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,表明細胞中突變的KRAS蛋白能介導細胞信號通路的活化。鑒于突變型細胞中KRAS信號通路已活化,本文對突變型細胞的生長情況進行研究。結(jié)果顯示,相比于野生型細胞,突變型細胞的生長速度、克隆形成速度、S期的細胞數(shù)目及S期相關(guān)蛋白CyclinE、CyclinA及其激酶磷酸化CDK2(p-CDK2-Thr160)的表達量均明顯的升高。這些結(jié)果提示,在突變型細胞中,KRAS點突變會促進下游信號通路的過度激活,使細胞快速增殖。最后,本文對KRAS突變型細胞株的藥物敏感性進行研究。本研究以RAS信號轉(zhuǎn)導通路為靶點選取了近20種臨床小分子抑制劑,通過分析藥物的半有效抑制濃度(IC50)來檢測細胞的藥物敏感性。結(jié)果顯示,KRAS突變型細胞株對三種MEK的抑制劑表現(xiàn)出明顯的耐受性,相對于野生型細胞約有10倍以上的差異,對Wee1的抑制劑和AKT的抑制劑表現(xiàn)出一定的敏感性。綜上所述,本研究成功構(gòu)建了四種KRAS不同突變位點的細胞株,該細胞株可為靶向KRAS突變腫瘤的藥物研發(fā)及靶點驗證提供良好的細胞模型。
[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.
【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R73-3

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy[J];遺傳學報;2012年05期

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本文編號：2009514