BEZ235體外抑制宮頸癌增殖與遷移作用研究及Skp2過(guò)表達(dá)的臨床意義
發(fā)布時(shí)間：2018-08-20 12:37
【摘要】：研究背景：宮頸癌作為女性第二大惡性腫瘤,發(fā)病率僅次于乳腺癌。BEZ235是磷脂酰肌醇-3(PI3K)及其下游因子哺乳動(dòng)物雷帕毒素蛋白(mammalian target of rapamycin, mTOR)的雙重靶向抑制劑,已表現(xiàn)出抑制多種腫瘤的作用,但在宮頸癌組織中的作用報(bào)道較少。S期激酶相關(guān)蛋白2(Skp2)是在調(diào)控細(xì)胞周期中發(fā)揮重要作用的蛋白,并參與細(xì)胞的增殖及凋亡。目前已發(fā)現(xiàn)Skp2蛋白在多種惡性腫瘤中呈過(guò)表達(dá)且導(dǎo)致腫瘤患者的不良預(yù)后。文獻(xiàn)報(bào)道,Skp2對(duì)腫瘤發(fā)生發(fā)展的影響受PI3K信號(hào)通路的調(diào)控。在多種惡性腫瘤中,PI3K信號(hào)通路均可在轉(zhuǎn)錄和翻譯前、后水平上對(duì)Skp2蛋白進(jìn)行調(diào)控。 研究目的：探討宮頸癌細(xì)胞中PI3K抑制劑BEZ235通過(guò)下調(diào)Skp2影響宮頸癌細(xì)胞的增殖及遷移能力；探討Skp2蛋白在宮頸鱗狀細(xì)胞癌組織中表達(dá)的臨床病理學(xué)意義。 材料和方法：第一部分：宮頸癌Hela、C33A、SiHa細(xì)胞的傳代培養(yǎng)；噻唑藍(lán)(MTT)實(shí)驗(yàn)檢測(cè)BEZ235對(duì)細(xì)胞生長(zhǎng)及增殖情況的影響,并繪制生長(zhǎng)曲線(xiàn),篩選藥物濃度；Hela和C33A細(xì)胞中加入0.1μM、0.4μM的BEZ235藥物,對(duì)照組加入等體積的DMSO,作用48h后,提取RNA及蛋白,通過(guò)RT-PCR法檢測(cè)宮頸癌細(xì)胞中Skp2mRNA的變化,并用Western-blot法檢測(cè)宮頸癌細(xì)胞中Skp2、Ezrin、Six1蛋白的變化；劃痕實(shí)驗(yàn)法檢測(cè)BEZ235對(duì)Hela細(xì)胞遷移能力的影響。第二部分：免疫組化方法檢測(cè)在25例正常宮頸上皮組織、84例宮頸上皮內(nèi)瘤變(CIN)和163例宮頸鱗狀細(xì)胞癌(SCC)中Skp2蛋白的表達(dá)情況；RT-PCR法檢測(cè)人乳頭瘤病毒(HPV)在宮頸癌不同組織中的表達(dá)情況；結(jié)合宮頸癌HPV 感染情況、臨床分期等生物學(xué)特點(diǎn)檢驗(yàn)Skp2蛋白在宮頸癌組織表達(dá)的臨床病理學(xué)意義；統(tǒng)計(jì)學(xué)分析Skp2蛋白表達(dá)對(duì)患者生存時(shí)間的影響。 結(jié)果：第一部分：MTT實(shí)驗(yàn)結(jié)果表明,用BEZ235阻斷PI3K信號(hào)通路可抑制宮頸癌細(xì)胞Hela、C33A和SiHa的增殖；RT-PCR和Western blot結(jié)果表明,宮頸癌Hela和C33A細(xì)胞中Skp2mRNA和Slp2、Ezrin、Sixl蛋白的表達(dá)水平均隨BEZ235的濃度(0.1μM,0.4μM)增高而降低；劃痕試驗(yàn)結(jié)果顯示,對(duì)宮頸癌Hela細(xì)胞進(jìn)行BEZ235藥物處理24h、48h后,細(xì)胞移動(dòng)距離較對(duì)照組明顯縮短。第二部分：RT-PCR檢測(cè)HPV mRNA在14例宮頸癌組織中有11例呈陽(yáng)性表達(dá)；Skp2在CIN-1、CIN-2和CIN-3中的陽(yáng)性率依次增高,且均高于正常組織(P均0.01)；在宮頸鱗狀細(xì)胞癌中Skp2蛋白呈明顯的彌漫性強(qiáng)陽(yáng)性染色,其陽(yáng)性率顯著高于正常宮頸上皮組織(P0.01)；Skp2蛋白表達(dá)與FIGO分期及高危型HPV感染關(guān)系密切(P0.05)；Skp2蛋白陽(yáng)性表達(dá)的宮頸鱗狀細(xì)胞癌患者無(wú)瘤生存率及總生存率明顯低于陰性表達(dá)的患者(P均0.01)。 結(jié)論：1.PI3K抑制劑BEZ235通過(guò)下調(diào)Skp2蛋白抑制宮頸癌細(xì)胞增殖與遷移能力；2.Skp2蛋白過(guò)表達(dá)可能是預(yù)示宮頸鱗狀細(xì)胞癌患者的不良預(yù)后的檢測(cè)指標(biāo),Skp2蛋白檢測(cè)可作為宮頸鱗狀細(xì)胞癌增殖指數(shù)測(cè)定及預(yù)后評(píng)估的有效分子標(biāo)志物。
[Abstract]:Background: as the second largest malignant tumor in women, cervical cancer is a double targeting inhibitor of phosphatidylinositol -3 (PI3K) and its downstream mammal rapa toxin (mammalian target of rapamycin, mTOR). Although it has been shown to inhibit many kinds of tumors, the role of S phase kinase associated protein 2 (Skp2) in cervical carcinoma is less reported, which plays an important role in the regulation of cell cycle, and participates in cell proliferation and apoptosis. It has been found that Skp2 protein is overexpressed in many malignant tumors and leads to poor prognosis of tumor patients. The effect of Skp2 on tumor development is regulated by PI3K signaling pathway. PI3K signaling pathway can regulate Skp2 protein before and after translation in many kinds of malignant tumors. Objective: to investigate the effect of PI3K inhibitor BEZ235 on the proliferation and migration of cervical cancer cells by down-regulating Skp2, and to explore the clinicopathological significance of the expression of Skp2 protein in cervical squamous cell carcinoma. Materials and methods: the first part: the passage culture of HelaA33AnSiHa cell line of cervical cancer, the effect of BEZ235 on cell growth and proliferation was detected by thiazolyl (MTT) assay, and the growth curve was drawn, and the drug concentration was screened by adding 0.4 渭 M BEZ235 drug to Hela and C33A cells. The control group was treated with DMSO-containing the same volume for 48h, then RNA and protein were extracted, the changes of Skp2mRNA in cervical cancer cells were detected by RT-PCR method, and the changes of Skp2Ezrin1 protein in cervical cancer cells were detected by Western-blot assay. The effect of BEZ235 on the migration of Hela cells was detected by scratch test. The second part: immunohistochemical method was used to detect the expression of Skp2 protein in 84 cases of cervical intraepithelial neoplasia (CIN) and 163 cases of cervical squamous cell carcinoma (SCC) in 25 cases of normal cervical epithelial tissue and 163 cases of cervical squamous cell carcinoma. The expression of human papillomavirus (HPV) in different tissues of cervical cancer was detected by RT-PCR, and the clinicopathological significance of the expression of Skp2 protein in cervical carcinoma was examined by combining with the biological characteristics of cervical cancer HPV infection and clinical staging. The effect of Skp2 protein expression on survival time was analyzed statistically. Results: the results of the first part of the experiment showed that blocking the PI3K signaling pathway with BEZ235 could inhibit the proliferation of HelaC33A and SiHa cells by RT-PCR and Western blot. The expression level of Skp2mRNA and Slp2Ezrininosin Sixl protein in cervical cancer Hela and C33A cells decreased with the increase of BEZ235 concentration (0.1 渭 M, 0.4 渭 M), and the results of scratch test showed that the cell migration distance of cervical cancer Hela cells treated with BEZ235 for 24 h or 48 h was significantly shorter than that of the control group. In the second part, the positive rates of Skp2 in CIN-1 and CIN-2 and CIN-3 were increased in 11 of 14 cases of cervical carcinoma by RT-PCR, and were higher than those in normal tissues (P0.01). The positive rate of Skp2 protein in cervical squamous cell carcinoma was significantly higher than that in normal cervical epithelium (P0.01). The expression of Skp2 protein was closely related to FIGO stage and high risk HPV infection (P0.05). The tumor-free survival rate and overall survival rate of cervical squamous cell carcinoma patients with positive expression of Skp2 protein were significantly lower than those with negative expression (P0.01). Conclusion 1. PI3K inhibitor BEZ235 inhibits the proliferation and migration of cervical cancer cells by down-regulating Skp2 protein. Overexpression of 2.Skp2 protein may be an index to predict the poor prognosis of patients with cervical squamous cell carcinoma. The detection of Skp2 protein can be used as an effective molecular marker for the detection of proliferation index and prognosis evaluation of cervical squamous cell carcinoma.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33
本文編號(hào)：2193634
[Abstract]:Background: as the second largest malignant tumor in women, cervical cancer is a double targeting inhibitor of phosphatidylinositol -3 (PI3K) and its downstream mammal rapa toxin (mammalian target of rapamycin, mTOR). Although it has been shown to inhibit many kinds of tumors, the role of S phase kinase associated protein 2 (Skp2) in cervical carcinoma is less reported, which plays an important role in the regulation of cell cycle, and participates in cell proliferation and apoptosis. It has been found that Skp2 protein is overexpressed in many malignant tumors and leads to poor prognosis of tumor patients. The effect of Skp2 on tumor development is regulated by PI3K signaling pathway. PI3K signaling pathway can regulate Skp2 protein before and after translation in many kinds of malignant tumors. Objective: to investigate the effect of PI3K inhibitor BEZ235 on the proliferation and migration of cervical cancer cells by down-regulating Skp2, and to explore the clinicopathological significance of the expression of Skp2 protein in cervical squamous cell carcinoma. Materials and methods: the first part: the passage culture of HelaA33AnSiHa cell line of cervical cancer, the effect of BEZ235 on cell growth and proliferation was detected by thiazolyl (MTT) assay, and the growth curve was drawn, and the drug concentration was screened by adding 0.4 渭 M BEZ235 drug to Hela and C33A cells. The control group was treated with DMSO-containing the same volume for 48h, then RNA and protein were extracted, the changes of Skp2mRNA in cervical cancer cells were detected by RT-PCR method, and the changes of Skp2Ezrin1 protein in cervical cancer cells were detected by Western-blot assay. The effect of BEZ235 on the migration of Hela cells was detected by scratch test. The second part: immunohistochemical method was used to detect the expression of Skp2 protein in 84 cases of cervical intraepithelial neoplasia (CIN) and 163 cases of cervical squamous cell carcinoma (SCC) in 25 cases of normal cervical epithelial tissue and 163 cases of cervical squamous cell carcinoma. The expression of human papillomavirus (HPV) in different tissues of cervical cancer was detected by RT-PCR, and the clinicopathological significance of the expression of Skp2 protein in cervical carcinoma was examined by combining with the biological characteristics of cervical cancer HPV infection and clinical staging. The effect of Skp2 protein expression on survival time was analyzed statistically. Results: the results of the first part of the experiment showed that blocking the PI3K signaling pathway with BEZ235 could inhibit the proliferation of HelaC33A and SiHa cells by RT-PCR and Western blot. The expression level of Skp2mRNA and Slp2Ezrininosin Sixl protein in cervical cancer Hela and C33A cells decreased with the increase of BEZ235 concentration (0.1 渭 M, 0.4 渭 M), and the results of scratch test showed that the cell migration distance of cervical cancer Hela cells treated with BEZ235 for 24 h or 48 h was significantly shorter than that of the control group. In the second part, the positive rates of Skp2 in CIN-1 and CIN-2 and CIN-3 were increased in 11 of 14 cases of cervical carcinoma by RT-PCR, and were higher than those in normal tissues (P0.01). The positive rate of Skp2 protein in cervical squamous cell carcinoma was significantly higher than that in normal cervical epithelium (P0.01). The expression of Skp2 protein was closely related to FIGO stage and high risk HPV infection (P0.05). The tumor-free survival rate and overall survival rate of cervical squamous cell carcinoma patients with positive expression of Skp2 protein were significantly lower than those with negative expression (P0.01). Conclusion 1. PI3K inhibitor BEZ235 inhibits the proliferation and migration of cervical cancer cells by down-regulating Skp2 protein. Overexpression of 2.Skp2 protein may be an index to predict the poor prognosis of patients with cervical squamous cell carcinoma. The detection of Skp2 protein can be used as an effective molecular marker for the detection of proliferation index and prognosis evaluation of cervical squamous cell carcinoma.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 黃秀蘭;崔國(guó)輝;周克元;;PI3K-Akt信號(hào)通路與腫瘤細(xì)胞凋亡關(guān)系的研究進(jìn)展[J];癌癥;2008年03期
2 魏麗惠;;宮頸病變?cè)\斷和治療中的過(guò)度與不足[J];中國(guó)婦產(chǎn)科臨床雜志;2009年02期
3 周傳香;癌基因Skp2與腫瘤關(guān)系研究進(jìn)展[J];國(guó)外醫(yī)學(xué).口腔醫(yī)學(xué)分冊(cè);2003年06期
4 高冬梅,何春年;Skp2研究進(jìn)展及其與婦科腫瘤的關(guān)系[J];國(guó)外醫(yī)學(xué).婦產(chǎn)科學(xué)分冊(cè);2005年04期
5 廖丹梅;黃明春;;HPV感染與子宮頸癌的研究現(xiàn)狀[J];廣西醫(yī)學(xué);2006年03期
6 張芳;韓娜;蔡晶;方黎;張中冕;;BEZ235對(duì)A549細(xì)胞增殖及akt、mek、erk mRNA表達(dá)的影響[J];鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2013年04期
7 許朝;姜藻;;PI3K/Akt信號(hào)通路抑制劑對(duì)胃癌細(xì)胞SGC7901中Skp2和NAG-1表達(dá)的影響及其效應(yīng)[J];中國(guó)全科醫(yī)學(xué);2010年14期
8 解智慧;孫秀華;柯丹;張洪開(kāi);于愛(ài)鳴;;肺癌中PI3K/AKT信號(hào)通路對(duì)Skp2調(diào)控機(jī)制的研究[J];山東醫(yī)藥;2010年08期
9 傅奕;方征宇;王麗影;李增霞;楊勇;查錫良;;PI3K信號(hào)通路通過(guò)Skp2、p27調(diào)節(jié)肝癌細(xì)胞的增殖[J];生物化學(xué)與生物物理進(jìn)展;2008年11期
10 陳觀(guān)娣;錢(qián)德英;李志剛;歐陽(yáng)云雁;;高危型人乳頭瘤病毒-DNA檢測(cè)在宮頸鱗狀上皮內(nèi)高度病變篩查中的價(jià)值[J];實(shí)用醫(yī)學(xué)雜志;2009年09期
，本文編號(hào)：2193634
本文鏈接：http://sikaile.net/yixuelunwen/fuchankeerkelunwen/2193634.html
最近更新
教材專(zhuān)著
