發(fā)布時(shí)間：2019-03-25 19:27

【摘要】：背景:CRISPR(Clustered Regularly Interspersed Short Palindromic Repeats)即規(guī)律間隔成簇短回文重復(fù)序列,它能抵御入侵的病毒和外源DNA,通過(guò)sg RNA引導(dǎo)Cas9核酸內(nèi)切酶切割其定位的雙鏈DNA,達(dá)到敲除的效果;ABCG2基因高表達(dá)與腫瘤多藥耐藥性密切相關(guān),在癌細(xì)胞中過(guò)表達(dá)ABCG2將引起多耐藥性導(dǎo)致治療失敗,然而卻少有報(bào)道它在結(jié)直腸癌中除了多耐藥性外和其他惡性生物行為的關(guān)系,本研究通過(guò)應(yīng)用CRISPR-Cas9敲除ABCG2基因觀察其對(duì)結(jié)直腸癌的多藥耐藥性和其他惡性生物行為的影響。方法:1、構(gòu)建Lenti CRISPR v2載體;2、構(gòu)建ABCG2敲除細(xì)胞株;3、MTT法檢測(cè)穩(wěn)定敲除細(xì)胞株對(duì)藥物敏感性的改變;4、藥物積累實(shí)驗(yàn)評(píng)估穩(wěn)定敲除細(xì)胞株外排功能性的改變;5、通過(guò)MTT法、流式細(xì)胞術(shù)、劃痕實(shí)驗(yàn)、Transwell侵襲實(shí)驗(yàn)、軟瓊脂糖克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞生物行為的改變。結(jié)果:1、Lenti CRISPR v2載體構(gòu)建成功;2、ABCG2穩(wěn)定敲除細(xì)胞株構(gòu)建成功;3、ABCG2基因敲除細(xì)胞株的藥物敏感性提高;4、敲除ABCG2使結(jié)直腸癌細(xì)胞胞內(nèi)藥物積累增加;5、敲除ABCG2對(duì)結(jié)直腸癌細(xì)胞生長(zhǎng)的影響不大,顯著使細(xì)胞周期分布,遷移、侵襲克隆形成能力都減弱。結(jié)論:應(yīng)用CRISPR-Cas9技術(shù)敲除ABCG2能抑制結(jié)直腸癌的惡性生物學(xué)行為。
[Abstract]:Background: CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) is a regularly spaced cluster of short palindromic repeats, which can resist invading viruses and exogenous DNA, to induce Cas9 endonucleases to cleave its localized double-stranded DNA, through sg RNA to achieve knockout effect. Overexpression of ABCG2 gene is closely related to multidrug resistance in cancer cells. Overexpression of ABCG2 in cancer cells may lead to treatment failure. However, there are few reports about its relationship with other malignant biological behaviors in colorectal cancer besides multidrug resistance. In this study, we observed the effect of CRISPR-Cas9 knockout ABCG2 gene on multidrug resistance and other malignant biological behaviors of colorectal cancer. Methods: 1, construction of Lenti CRISPR v2 vector; 2, construction of ABCG2 knockout cell line; 3, detection of drug sensitivity of stable knock-out cell line; 4, drug accumulation test to evaluate the functional changes of the exocytosis of stable knock-out cell line; 5. MTT assay, flow cytometry, scratch test, Transwell invasion test and soft agarose clone formation test were used to detect the changes of cell biological behavior. Results: 1, Lenti CRISPR v2 vector was successfully constructed; 2, ABCG2 stable knock-out cell line was successfully constructed; 3, ABCG2 gene knockout cell line was more sensitive to drugs; (4) knockout ABCG2 increased intracellular drug accumulation in colorectal cancer cells; 5. Knockout of ABCG2 had little effect on the growth of colorectal cancer cells, and significantly weakened the cell cycle distribution, migration and invasive clone formation ability. Conclusion: CRISPR-Cas9 knockout of ABCG2 can inhibit the malignant biological behavior of colorectal cancer.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34

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本文編號(hào)：2447247