本文選題：宮頸癌 + 跨損傷DNA合成�。� 參考：《復(fù)旦大學(xué)》2014年碩士論文

【摘要】：目的REV3L是DNA聚合酶ζ(Polζ)的催化亞基,在跨損傷DNA合成通路(TLS)中起關(guān)鍵作用。本課題旨在研究REV3L基因?qū)m頸癌細(xì)胞化療敏感性的影響,探討REV3L基因在宮頸癌中作為分子靶點(diǎn)的價值。方法采用免疫組化法檢測123例宮頸癌組織及17例正常宮頸組織中Polζ蛋白的表達(dá)水平。構(gòu)建REV3L基因過表達(dá)載體pcDNA-REV3L后導(dǎo)入REV3L低表達(dá)的宮頸癌細(xì)胞系,構(gòu)建REV3L基因干擾載體shRNA-REV3L并轉(zhuǎn)染REV3L高表達(dá)宮頸癌細(xì)胞系后,通過CCK-8法檢測對細(xì)胞增殖、流式細(xì)胞儀檢測細(xì)胞周期變化、平板克隆形成實(shí)驗(yàn)檢測克隆形成率的變化,進(jìn)一步通過CCK-8法檢測REV3L基因?qū)︺K類藥物耐受性的影響,通過流式細(xì)胞術(shù)檢測藥物作用后細(xì)胞凋亡情況,并通過蛋白免疫印跡法檢測凋亡相關(guān)蛋白Bcl-2、Bcl-xl及Bax等的變化情況。通過免疫熒光法檢測順鉑處理后REV3L基因?qū)-H2AX焦點(diǎn)形成情況的影響,蛋白免疫印跡法檢測Y-H2AX等蛋白的變化。結(jié)果宮頸癌組織中Polζ蛋白表達(dá)水平較正常宮頸組織高(P0.05)。REV3L基因沉默后宮頸癌細(xì)胞株增殖減慢,REV3L基因過表達(dá)后宮頸癌細(xì)胞株增殖加快：沉默REV3L基因通過G1/S期阻滯抑制細(xì)胞周期進(jìn)展,而REV3L基因過表達(dá)后可以越過G1/S期細(xì)胞周期檢查點(diǎn)促進(jìn)細(xì)胞周期進(jìn)展；REV3L基因沉默后細(xì)胞平板克隆形成率減少,而過表達(dá)后細(xì)胞克隆形成率增加。下調(diào)宮頸癌細(xì)胞系REV3L基因表達(dá)后CCK-8檢測示宮頸癌細(xì)胞對順鉑的敏感性增加；流式細(xì)胞儀檢測順鉑作用后早期凋亡率增加；Western blot檢測促凋亡蛋白升高,而抗凋亡蛋白降低；免疫熒光檢測細(xì)胞內(nèi)Y-H2AX焦點(diǎn)形成增多,Western blot檢測Y-H2AX蛋白表達(dá)升高。REV3L基因過表達(dá)后CCK-8示細(xì)胞對順鉑的抵抗性增加；流式細(xì)胞儀檢測順鉑作用后早期凋亡率降低；Western blot檢測促凋亡蛋白降低,而抗凋亡蛋白升高；免疫熒光檢測細(xì)胞內(nèi)Y-H2AX焦點(diǎn)形成減少,Western blot檢測Y-H2AX蛋白表達(dá)降低。結(jié)論抑制REV3L基因在宮頸癌細(xì)胞中的表達(dá),可提高細(xì)胞對順鉑的敏感性,而過表達(dá)REV3L基因增加其對順鉑的抵抗性。REV3L可能作為宮頸癌化療增敏的分子靶點(diǎn)。
[Abstract]:Objective REV3L is a catalytic subunit of DNA polymerase 味 Pol 味, which plays a key role in transcriptional DNA synthesis pathway. The purpose of this study was to investigate the effect of REV3L gene on the chemosensitivity of cervical cancer cells and to explore the value of REV3L gene as a molecular target in cervical cancer. Methods Immunohistochemical method was used to detect the expression of Pol 味 protein in 123 cases of cervical carcinoma and 17 cases of normal cervical tissues. REV3L gene overexpression vector (pcDNA-REV3L) was constructed and introduced into cervical cancer cell line with low REV3L expression. The REV3L gene interference vector shRNA-REV3L was constructed and transfected into REV3L overexpression cervical cancer cell line. The proliferation of cervical carcinoma cell line was detected by CCK-8 assay and cell cycle change was detected by flow cytometry. The effect of REV3L gene on the tolerance of platinum drugs was detected by CCK-8 assay and apoptosis was detected by flow cytometry. The changes of Bcl-xl and Bax were detected by Western blot. The effect of REV3L gene on the formation of focal point of Y-H2AX was detected by immunofluorescence, and the changes of Y-H2AX and other proteins were detected by Western blot. Results the expression level of Pol 味 protein in cervical cancer tissue was higher than that in normal cervix tissue. The proliferation of cervical cancer cell line slowed down after the silencing of P0.05N. REV3L gene and the proliferation of cervical cancer cell line increased after overexpression of REV3L gene. The silencing of REV3L gene inhibited cell cycle progression through G 1 / S phase arrest. After overexpression of REV3L gene, the cell cycle progression could be promoted by crossing the checkpoint of G 1 / S phase cell cycle. After the silencing of REV3L gene, the cell flat clone formation rate was decreased, but the cell clone formation rate increased after overexpression. After down-regulation of REV3L gene expression in cervical cancer cell line, CCK-8 assay showed that the sensitivity of cervical cancer cell line to cisplatin was increased, the early apoptosis rate was increased by flow cytometry, and the pro-apoptotic protein was increased by Western blot, but the anti-apoptotic protein was decreased by flow cytometry. The increase of Y-H2AX focus formation in cells by immunofluorescence assay and the increase of Y-H2AX protein expression by Western blot. After overexpression of REV3L gene, CCK-8 showed increased resistance to cisplatin. Flow cytometry was used to detect the early apoptosis rate of cisplatin. Western blot was used to detect the decrease of pro-apoptotic protein while anti-apoptotic protein was increased. Immunofluorescence detection of Y-H2AX focus formation decreased and Western blot detected the decrease of Y-H2AX protein expression. Conclusion inhibiting the expression of REV3L gene in cervical cancer cells can increase the sensitivity of the cells to cisplatin, while overexpression of REV3L gene can increase its resistance to cisplatin. REV3L may be a molecular target for chemosensitization of cervical cancer.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 朱峰,楊軍,徐方,余應(yīng)年;人REV3基因啟動子區(qū)的克隆和生物信息學(xué)分析及其對MNNG的反應(yīng)[J];浙江大學(xué)學(xué)報(醫(yī)學(xué)版);2003年05期

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