本文選題：整合素連接激酶 + 核糖核酸酶抑制因子　； 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的研究整合素連接激酶(ILK)與核糖核酸酶抑制因子(RI)蛋白的相互作用,探討兩者相互作用對(duì)膀胱癌EJ細(xì)胞的ILK信號(hào)通路及EMT影響的分子機(jī)制。方法1.根據(jù)ILK基因序列設(shè)計(jì)引物,經(jīng)PCR法擴(kuò)增獲得ILK基因片段后,分別連接到真核表達(dá)載體pEYFP-N1和pCMV-3xflag-CMVTM-10上。通過(guò)酶切和DNA測(cè)序鑒定載體,并分別命名為p EYFP-N1-ILK and pCMV-3×Flag-ILK。將pCMV-3×Flag-ILK載體和對(duì)照空白載體,以及LV5-RNH1和LV5NC分別穩(wěn)定轉(zhuǎn)染EJ細(xì)胞株,命名為EJ-ILK,EJ-FLAG,EJ-RI和EJ-LV5,經(jīng)Western blot和細(xì)胞免疫熒光檢測(cè)蛋白過(guò)表達(dá)情況。2.在EJ和HEK293細(xì)胞內(nèi)進(jìn)行ILK與RI的免疫共沉淀實(shí)驗(yàn),熒光共振能量轉(zhuǎn)移實(shí)驗(yàn)和免疫熒光共定位實(shí)驗(yàn),用以研究體內(nèi)的相互作用。在細(xì)胞外應(yīng)用GST pulldown實(shí)驗(yàn)研究ILK與RI在體外的相互作用。3.CCK8檢測(cè)各組細(xì)胞增殖能力;流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期;細(xì)胞免疫熒光檢測(cè)蛋白ri,ilk,p-akt和p-gsk3β表達(dá)水平;小管形成實(shí)驗(yàn)檢測(cè)ilk和ri相互作用對(duì)體外血管生成的影響。3.收集的穩(wěn)定表達(dá)細(xì)胞株ej-ilk,ej-flag,ej-ri,ej-lv5和ej,用westernblot檢測(cè)ilk與ri相互作用對(duì)ej細(xì)胞emt及ilk信號(hào)通路的影響。5.構(gòu)建balb/c裸鼠移植瘤模型,觀察移植瘤生長(zhǎng),腫瘤微血管生長(zhǎng)以及肺轉(zhuǎn)移情況;應(yīng)用免疫組化和組織免疫熒光檢測(cè)腫瘤組織中emt相關(guān)蛋白及ilk信號(hào)通路關(guān)鍵蛋白的表達(dá)水平。另外,在臨床人膀胱癌及癌旁組織標(biāo)本中同時(shí)檢測(cè)了ilk及ri蛋白的表達(dá)。結(jié)果1.測(cè)序結(jié)果顯示:重組真核表達(dá)質(zhì)粒peyfp-n1-ilk和pcmv-3×flag-ilk構(gòu)建正確。細(xì)胞免疫熒光和westernblot結(jié)果顯示ilk和ri在ej細(xì)胞中成功過(guò)表達(dá)。2.實(shí)驗(yàn)結(jié)果顯示:ilk與ri在體內(nèi)和體外均存在相互作用。4.cck8結(jié)果顯示ej-ilk細(xì)胞增值能力較對(duì)照組明顯增強(qiáng),而ej-ri細(xì)胞則明顯減弱;流式細(xì)胞分析結(jié)果表明ej-ri細(xì)胞與對(duì)照組相比,明顯阻滯于s期;小管形成實(shí)驗(yàn)提示ri和ej相互作用能夠抑制體外血管的形成。3.通過(guò)檢測(cè)各組穩(wěn)定表達(dá)細(xì)胞株,顯示emt相關(guān)蛋白和ilk信號(hào)通路的一些關(guān)鍵靶蛋白呈現(xiàn)顯著的變化。5.balb/c裸鼠注射各組細(xì)胞后,與2個(gè)對(duì)照組相比,ej-ilk組瘤重明顯增加;而EJ-RI組與對(duì)照組比較,瘤重明顯降低。組織免疫熒光和HE染色結(jié)果提示EJ-ILK組微血管明顯增加,EJ-RI組則明顯減少。肺組織HE染色實(shí)驗(yàn)表明,EJ-ILK組有明顯肺轉(zhuǎn)移,而EJ-RI組未見(jiàn)明顯轉(zhuǎn)移。免疫組織化學(xué)和免疫熒光檢測(cè)結(jié)果顯示:EJ-ILK組中EMT及ILK信號(hào)通路蛋白變化明顯;EJ-RI組中EMT相關(guān)蛋白和ILK通路關(guān)鍵蛋白與對(duì)照組比較有顯著變化,且與EJ-ILK組成反相關(guān)。結(jié)論1.ILK和RI在體內(nèi)外均存在相互作用。2.過(guò)表達(dá)ILK促進(jìn)細(xì)胞增殖,EMT以及轉(zhuǎn)移。3.上調(diào)RI抑制ILK信號(hào)通路以及EMT。4.上調(diào)RI抑制血管生成,腫瘤形成以及轉(zhuǎn)移。
[Abstract]:Objective to study the interaction between integrin linked kinase (ILK) and ribonuclease inhibitor (Ri) protein, and to explore the effect of integrin linked kinase (ILK) and ribonuclease inhibitor (Ri) on the ilk signaling pathway and the molecular mechanism of EMT in EJ cells of bladder cancer. Method 1. The ilk gene fragment was amplified by PCR and cloned into eukaryotic expression vectors pEYFP-N1 and pCMV-3xflag-CMVTM-10, respectively. The vector was identified by restriction endonuclease digestion and DNA sequencing and named as pEYFP-N1-ilk and pCMV-3 脳 Flag-ILK. The pCMV-3 脳 Flag-ilk vector and the control vector, LV5-RNH1 and LV5NC were stably transfected into EJ cell line, named EJ-ILKPER-FLAGN EJ-RI and EJ-LV5, respectively. The overexpression of protein was detected by Western blot and cellular immunofluorescence. The immunoprecipitation of ilk and RI, fluorescence resonance energy transfer and immunofluorescence co-localization were carried out in EJ and HEK293 cells to study the interaction in vivo. The interaction of ilk and RI in vitro was studied by pulldown assay in vitro. 3. The proliferative ability of each group was detected by CCK8, the cell cycle was detected by flow cytometry, and the expression of rifikophane p-akt and p-gsk3 尾 were detected by cellular immunofluorescence. The effect of ilk and ri interaction on angiogenesis in vitro was detected by tubulogenesis assay. The stable expression cell lines ej-ilkine ej-flagej-rij-lv5 and ejj-lv5 were collected. The effects of ilk and ri interaction on emt and ilk signaling pathway were detected by westernblot. The balb/c xenograft tumor model was established to observe the tumor growth, tumor microvessel growth and lung metastasis. The expression of emt related proteins and key proteins of ilk signaling pathway in tumor tissues were detected by immunohistochemistry and tissue immunofluorescence. In addition, the expression of ilk and ri protein were detected in clinical specimens of bladder cancer and adjacent tissues. Result 1. Sequencing results showed that the recombinant eukaryotic expression plasmids peyfp-n1-ilk and pcmv-3 脳 flag-ilk were constructed correctly. The results of cellular immunofluorescence and westernblot showed that ilk and ri were overexpressed in EJ cells. The results showed that there was interaction between in vivo and in vitro. 4. Cck8 results showed that the proliferation of ej-ilk cells was significantly higher than that of the control group, while that of ej-ri cells was significantly decreased, and the flow cytometry analysis showed that ej-ri cells were significantly higher than those of the control group. The results showed that the interaction of ri and EJ could inhibit the formation of blood vessels in vitro. By detecting the stable expression cell lines of each group, it was found that emt related protein and some key target proteins of ilk signal pathway showed significant changes. 5. After injected with each group of cells, the tumor weight of the control group was significantly increased compared with that of the two control groups. The tumor weight in EJB-RI group was significantly lower than that in control group. The results of tissue immunofluorescence and HE staining showed that the increase of microvessels in EJB-ilk group was significantly decreased in EJ- RI group. Lung tissue HE staining showed that there were significant lung metastasis in EJ-ilk group, but no obvious metastasis in EJ-RI group. The changes of EMT and ilk signaling pathway proteins in the EJ-ILK group were significantly different from those in the control group, and were inversely correlated with the composition of EJ-ilk in the EJ-RI group. Ilk and RI have interaction in vivo and in vitro. 2. Overexpression of ilk promotes proliferation and metastasis of EMT. Upregulation of RI inhibits ilk signaling pathway and EMT.4. Upregulation of RI inhibits angiogenesis, tumor formation and metastasis.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R737.14

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