本文選題：宮頸癌 + HPVE7��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：背景:宮頸癌是全球最常見的婦科腫瘤之一,人乳頭瘤病毒(Human papillomavirus,HPV)的持續(xù)感染是其首要啟動因素。HPV是一種雙鏈環(huán)狀DNA病毒,其編碼的早期蛋白E6、E7是導(dǎo)致宮頸細(xì)胞惡性轉(zhuǎn)化的關(guān)鍵蛋白。然而,僅有HPV的感染不能導(dǎo)致細(xì)胞的惡性轉(zhuǎn)化,它僅作為宮頸致癌過程的第一次打擊。HPV感染宮頸上皮后所誘導(dǎo)的宿主細(xì)胞內(nèi)一系列的分子信號通路的改變,最終導(dǎo)致細(xì)胞發(fā)生了惡性轉(zhuǎn)化。然而,目前HPV致宮頸癌發(fā)生發(fā)展的機(jī)制仍有待進(jìn)一步闡明。凝縮蛋白Ⅰ復(fù)合體的非染色體結(jié)構(gòu)維持蛋白的亞單位H(Non-SMC CondensinⅠ Complex SubunitH,NCAPH)基因,主要參與間期核染色質(zhì)轉(zhuǎn)變成高度螺旋的核染色體,以往有研究顯示:NCAPH與非小細(xì)胞肺癌的不良預(yù)后有關(guān),這提示NCAPH可能與腫瘤的發(fā)生發(fā)展有關(guān),但機(jī)制不明。目的:本課題擬通過體外實(shí)驗(yàn)初步闡明NCAPH在宮頸癌中的表達(dá)情況、對宮頸癌細(xì)胞生長的影響及其可能機(jī)制,初步揭示HPVE7與NCAPH之間的調(diào)控關(guān)系。方法:1)收集10例正常宮頸及22例宮頸癌的新鮮組織標(biāo)本,提取組織內(nèi)的總RNA并逆轉(zhuǎn)錄為cDNA,同法提取三種宮頸癌細(xì)胞系中的總RNA并逆轉(zhuǎn)錄為cDNA,實(shí)時熒光定量PCR法檢測NCAPH的表達(dá),對比其在正常宮頸及宮頸癌中的表達(dá)差異。收集65例正常宮頸組織及149例宮頸癌的石蠟標(biāo)本,免疫組織化學(xué)方法檢測其中NCAPH蛋白的表達(dá),對比正常宮頸及宮頸癌中NCAPH蛋白的表達(dá)差異;2)培養(yǎng)宮頸癌細(xì)胞系HeLa和SiHa,瞬時轉(zhuǎn)染靶向NCAPH siRNA,利用CCK-8試劑盒、平板克隆形成實(shí)驗(yàn)檢測NCAPH對細(xì)胞增殖的影響;利用transwell遷移與侵襲實(shí)驗(yàn)檢測NCAPH對細(xì)胞遷移與侵襲能力的影響;3)培養(yǎng)HeLa和SiHa,轉(zhuǎn)染NCAPH siRNA,利用Western Blot和實(shí)時熒光定量PCR法檢測相關(guān)蛋白的表達(dá)變化;4)利用JASPAR及PROMO軟件預(yù)測NCAPH啟動子區(qū)的轉(zhuǎn)錄因子,挑選評分最高的轉(zhuǎn)錄因子E2F1;在HeLa和SiHa中干擾和過表達(dá)轉(zhuǎn)錄因子E2F1,檢測NCAPH的mRNA和蛋白表達(dá)變化;5)將NCAPH基因啟動子區(qū)序列連接到pGL3-Basic載體上,構(gòu)建轉(zhuǎn)錄因子E2F1的過表達(dá)載體,然后共轉(zhuǎn)染293T細(xì)胞,利用雙熒光素酶報告檢測系統(tǒng)驗(yàn)證轉(zhuǎn)錄因子E2F1是否可以結(jié)合NCAPH啟動子區(qū),激活NCAPH基因的轉(zhuǎn)錄;6)培養(yǎng)HPV16E7高表達(dá)的細(xì)胞系RPE1 E7及其對照細(xì)胞系RPE1,檢測E7過表達(dá)對NCAPH及轉(zhuǎn)錄因子E2F1的表達(dá)的影響;在HeLa和SiHa中干擾E7,檢測NCAPH及轉(zhuǎn)錄因子E2F1的表達(dá)變化;在RPE1E7細(xì)胞中干擾轉(zhuǎn)錄因子E2F1的表達(dá),檢測敲低E2F1能否影響E7對NCAPH表達(dá)的影響。結(jié)果:1)NCAPH在3種宮頸癌細(xì)胞系中均有表達(dá);與正常宮頸組織相比,NCAPH在宮頸癌組織中的表達(dá)水平顯著升高;2)CCK-8、平板克隆形成實(shí)驗(yàn)結(jié)果顯示:干擾宮頸癌細(xì)胞中NCAPH的表達(dá)后,細(xì)胞增殖能力明顯下降,克隆形成能力顯著降低;3)干擾宮頸癌細(xì)胞中NCAPH的表達(dá)后,細(xì)胞的遷移和侵襲能力均受到明顯抑制,上皮相關(guān)的蛋白ZO-1表達(dá)水平顯著升高,而間葉相關(guān)的蛋白表達(dá)水平Vimentin、Snail顯著下降;4)干擾宮頸癌細(xì)胞中NCAPH的表達(dá)后,細(xì)胞內(nèi)的總AKT沒有明顯變化,而絲氨酸473位點(diǎn)磷酸化的AKT蛋白的表達(dá)顯著降低;5)轉(zhuǎn)錄因子E2F1可以結(jié)合在NCAPH的啟動子區(qū)并上調(diào)NCAPH的轉(zhuǎn)錄,HPV的關(guān)鍵致癌蛋白E7可以通過調(diào)控E2F1上調(diào)其下游的NCAPH的表達(dá)。結(jié)論:NCAPH可能作為一種新的癌基因參與宮頸癌的發(fā)生發(fā)展。它可以顯著促進(jìn)宮頸癌細(xì)胞的增殖,并通過促進(jìn)EMT增強(qiáng)宮頸癌細(xì)胞的遷移和侵襲能力。HPV E7可能通過轉(zhuǎn)錄因子E2F1激活NCAPH的轉(zhuǎn)錄從而參與宮頸致癌過程。
[Abstract]:Background: cervical cancer is one of the most common gynecologic tumors in the world. The persistent infection of Human papillomavirus (HPV) is its primary factor..HPV is a double chain ring DNA virus, which encodes early protein E6. E7 is the key protein that causes malignant transformation of cervix cells. However, the infection only of HPV can not lead to cells. Malignant transformation, it is only a series of changes in the molecular signaling pathway in the host cell induced by.HPV infection of the cervix epithelium after the first carcinogenic process of the cervix, which eventually leads to the malignant transformation of the cells. However, the mechanism of the occurrence and development of cervical cancer in HPV still remains to be clarified. The subunit H (Non-SMC Condensin I Complex SubunitH, NCAPH), a subunit of chromosome structure maintaining protein, mainly participates in the transformation of the interphase nucleus chromatin into a highly spiral nuclear chromosome. Previous studies have shown that NCAPH is associated with the poor prognosis of non-small cell lung cancer, which suggests that NCAPH may be associated with the development of tumor, but the mechanism is unknown. The objective of this study is to preliminarily clarify the expression of NCAPH in cervical cancer and the possible mechanism of cervical cancer cell growth through in vitro experiments. The regulatory relationship between HPVE7 and NCAPH is preliminarily revealed. Methods: 1) 10 fresh tissue specimens of normal cervix and 22 cases of cervical cancer were collected, and the total RNA in the tissue was extracted and reverse transcriptase to cDNA. The total RNA in three cervical cancer cell lines was extracted and reverse transcriptive to cDNA. The expression of NCAPH was detected by real time fluorescence quantitative PCR, and the expression difference in normal cervix and cervical cancer was compared. 65 normal cervix tissues and 149 paraffin specimens of cervical cancer were collected, and the expression of NCAPH protein was detected by immunohistochemical method. Difference in expression of NCAPH protein in cervical and cervical cancer; 2) cultured cervical cancer cell lines HeLa and SiHa, transient transfection to NCAPH siRNA, CCK-8 kits and flat clones to detect the effect of NCAPH on cell proliferation, and the effect of NCAPH on cell migration and invasion by Transwell migration and invasion test; 3) HeLa and HeLa. SiHa, transfection of NCAPH siRNA, using Western Blot and real-time fluorescent quantitative PCR to detect the changes in the expression of related proteins; 4) use JASPAR and PROMO software to predict the transcription factors of the NCAPH promoter region, select the highest transcriptional factor E2F1, and interfere and overexpress the transcription factors in HeLa and SiHa. 5) connect the NCAPH gene promoter sequence to the pGL3-Basic vector, construct the overexpression vector of the transcription factor E2F1, then co transfect the 293T cell, and use the double luciferase reporter detection system to verify whether the transcription factor E2F1 can combine the NCAPH promoter region, activate the NCAPH gene transcription, and 6) to cultivate the RPE1 E7 of the high expression of HPV16E7. The effect of E7 overexpression on the expression of NCAPH and transcription factor E2F1, and the interference of E7 in HeLa and SiHa, to detect the expression of E2F1 in NCAPH and transcription factors in HeLa and SiHa, and to interfere with the expression of the transcription factor E2F1 in RPE1E7 cells. Results: 1) 1) in 3 kinds of cervical cancer Compared with normal cervical tissue, the expression level of NCAPH in cervical cancer tissues was significantly higher than that of normal cervical tissues; 2) CCK-8. The experimental results showed that after interfering with the expression of NCAPH in cervical cancer cells, the proliferation ability of the cells decreased significantly and the clone formation energy decreased significantly; and 3) interfered with the expression of NCAPH in cervical cancer cells. The cell migration and invasion ability were significantly inhibited, the expression level of epithelial related protein ZO-1 increased significantly, while the protein expression level of interrelated leaf related Vimentin, Snail decreased significantly; 4) after interfering with the expression of NCAPH in cervical cancer cells, the total AKT in the cell was not obviously changed, and the expression of AKT protein phosphorylation of serine site was expressed. Significant decrease; 5) transcription factor E2F1 can bind to the promoter region of NCAPH and increase the transcription of NCAPH. The key carcinogenic protein E7 of HPV can regulate the expression of NCAPH downstream by regulating E2F1. Conclusion: NCAPH may participate in the development of cervical cancer as a new oncogene. It can significantly promote the proliferation of cervical cancer cells. Over promotion of EMT enhances the migration and invasion of cervical cancer cells..HPV E7 may activate transcription of NCAPH through transcription factor E2F1 and participate in cervical carcinogenesis.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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