本文選題：ICAT + 宮頸癌��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:研究ICAT在人宮頸癌組織中的表達情況,并探討ICAT對宮頸癌增殖和轉(zhuǎn)移的作用及其分子機制。方法:1.IHC檢測宮頸癌組織(n=41)及正常宮頸組織(n=30)中ICAT蛋白的表達,Western blot法檢測三株宮頸癌細胞系(CaSki、SiHa和He La)中ICAT的內(nèi)源性表達。2.用ICAT過表達腺病毒(AdICAT)感染低表達ICAT的SiHa細胞,ICAT小干擾RNA(siICAT)轉(zhuǎn)染高表達ICAT的CaSki細胞;分別用RT-PCR和Western blot法驗證細胞系中ICAT在轉(zhuǎn)錄水平和蛋白水平表達;MTT、流式細胞術(shù)檢測細胞的增殖能力;Transwell遷移和侵襲實驗檢測細胞遷移和侵襲能力;Western blot法檢測各組細胞增殖相關(guān)CyclinD1和轉(zhuǎn)移相關(guān)蛋白MMP9表達;Western blot法和IF檢測過表達ICAT和降低內(nèi)源性ICAT后宮頸癌細胞EMT標(biāo)志物(E-cadherin和Vimentin)的表達;提取宮頸癌細胞的胞漿和胞核蛋白,Western blot法和IF法驗證β-catenin在宮頸癌細胞的表達;IP實驗檢測ICAT對E-cadherin/β-catenin復(fù)合物的影響。3.體內(nèi)實驗分SiHa、SiHa/RFP、SiHa/ICAT三組,每組2×107個細胞,注射到裸鼠皮下,觀察瘤體生長;剝離瘤體組織,石蠟包埋,切片,he染色;ihc檢測各組瘤體ki-67、mmp9、e-cadherin和vimentin的表達。結(jié)果:1.標(biāo)本評分顯示,宮頸癌組織的icat的ihc平均分數(shù)為7.366±0.3916,而宮頸正常組織中為5.000±0.6215(p0.01);ihc結(jié)果顯示,icat主要表達于癌細胞的胞漿;rt-pcr和westernblot結(jié)果一致,三株宮頸癌細胞系中,icat在轉(zhuǎn)錄水平和蛋白水平的表達siha最低,caski最高(p0.05)。2.adicat的感染可以顯著促進siha細胞在轉(zhuǎn)錄和蛋白水平icat的表達(p0.01),siicat可以有效的干擾caski細胞在轉(zhuǎn)錄水平和蛋白水平icat的表達(p0.01);adicat感染后,與siha組和siha/rfp組相比,siha/icat組細胞的增殖明顯加快(p0.01),s期所占比例明顯增加(p0.05),穿膜細胞數(shù)增加兩倍(p0.01);siicat轉(zhuǎn)染后,與caski/sinc組相比,caski/siicat組細胞的吸光度值明顯降低(p0.01),細胞s期所占比例減低(p0.05),穿膜細胞數(shù)減少一半(p0.01);過表達icat后,siha/icat組細胞內(nèi)的e-cadherin表達降低,vimentin表達增加(p0.05);干擾掉內(nèi)源性icat表達后,caski/siicat組細胞得到了相反的結(jié)果(p0.05);β-catenin在宮頸癌細胞中主要異常表達于胞漿;過表達icat后,復(fù)合物e-cadherin/β-catenin的表達降低。3.體內(nèi)實驗結(jié)果顯示:siha/icat組瘤體明顯大于siha組和SiHa/RFP組(P0.01);HE染色顯示,SiHa/ICAT組細胞排列疏松、紊亂,核大深染;免疫組化結(jié)果顯示,SiHa/ICAT組Ki-67、MMP9和Vimentin表達均增強而E-cadherin表達降低。結(jié)論:1.ICAT在宮頸癌組織中的表達顯著高于正常組織且主要表達于癌細胞的胞漿。2.ICAT促進宮頸癌細胞的增殖、遷移、和侵襲。3.ICAT促進宮頸癌細胞的EMT,這一作用與ICAT競爭性結(jié)合β-catenin相關(guān)。
[Abstract]:Aim: to investigate the expression of ICAT in human cervical carcinoma, and to explore the role of ICAT in the proliferation and metastasis of cervical carcinoma and its molecular mechanism.Methods 1. IHC was used to detect the expression of ICAT protein in cervical carcinoma tissue (41) and normal cervix cervix (30). Western blot assay was used to detect the endogenous expression of ICAT in three cervical cancer cell lines (CaSkisiha and he La).ICAT overexpression adenovirus was used to infect SiHa cells with low expression of ICAT. Small interfering RNAs were transfected into CaSki cells with high expression of ICAT.RT-PCR and Western blot were used to verify the expression of ICAT at transcription level and protein level, respectively. Flow cytometry was used to detect cell proliferation ability. Transwell migration and invasion assay was used to detect cell migration and invasion ability. Western blot assay was used to detect cell migration and invasion.The expression of proliferation-associated CyclinD1 and metastasis associated protein MMP9 was detected by Western blot and if, and the expression of EMT markers E-cadherin and Vimentin in cervical cancer cells after endogenous ICAT was decreased.The expression of 尾 -catenin in cervical cancer cells was detected by IP assay. The effect of ICAT on E-cadherin/ 尾 -catenin complex was detected by IP assay.The mice were divided into three groups: SiHaHN / Si-RFP- SiHa- / ICAT. Each group of 2 脳 107 cells was injected into nude mice subcutaneously to observe the growth of the tumor, the tumor tissue was stripped, embedded in paraffin, and the expression of ki-67 mmp9e-cadherin and vimentin in each group was detected by HE staining.The result is 1: 1.The mean ihc score of icat in cervical cancer tissues was 7.366 鹵0.3916, while that in normal cervical tissues was 5.000 鹵0.6215p0.01p0.01ihc. The results of rt-PCR and westernblot were consistent.Expression of icat at transcriptional and protein levels in three cervical cancer cell lines the expression of icat in siha cells at the transcriptional and protein-level levels can be significantly enhanced by the infection of the lowest siha caski p0.05. 2.The expression of icat at the transcriptional and protein-level levels of caski cells can be effectively interfered with by p0.01siicat.And the expression of icat at the protein level after infection,Compared with the siha group and siha/rfp group, the proliferation of the cells increased significantly in the p0.01 / s phase, and the number of perforating cells increased twice after transfection.Compared with the caski/sinc group, the absorbance value of the cells in the caskey / siicat group was significantly lower than that in the caski/sinc group, the proportion of cells in the s phase was decreased by p0.05, the number of perforated cells was reduced by half, and the expression of e-cadherin in the cells of the icat overexpression group was decreased, and the expression of vimentin was increased by p0.05, which interfered with the endogenous icat surface.The results showed that 尾 -catenin was mainly expressed in the cytoplasm of cervical cancer cells.After overexpression of icat, the expression of e-cadherin/ 尾 -catenin decreased.The results of in vivo experiments showed that the tumor in the siha group was significantly larger than that in the siha group and the SiHa/RFP group. The results of HE staining showed that the cells in the SiHaR / ICAT group were loose, disordered and heavily stained, and the immunohistochemical results showed that the expression of Ki-67 MMP9 and Vimentin in the SiHaR / ICAT group was increased and the E-cadherin expression was decreased.Conclusion: 1. The expression of ICAT in cervical carcinoma is significantly higher than that in normal tissues and mainly expressed in the cytoplasm of cancer cells. 2. ICAT promotes the proliferation and migration of cervical cancer cells, and invasiveness. 3. ICAT promotes the EMTs of cervical cancer cells, which is related to the competitive binding of ICAT to 尾 -catenin.
【學(xué)位授予單位】：重慶醫(yī)科大學(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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1 姜亞運;ICAT對宮頸癌增殖和轉(zhuǎn)移的作用及其機制研究[D];重慶醫(yī)科大學(xué);2017年

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本文編號：1747569