本文關(guān)鍵詞： 腎母細(xì)胞瘤 astrocyte elevated gene-1 慢病毒 RNA干擾　出處：《山東大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景和目的 腎母細(xì)胞瘤,又稱(chēng)Wilms瘤,約占所有兒童腫瘤的比例為8%,是兒童比較常見(jiàn)的惡性腫瘤,同時(shí)也是兒童泌尿系統(tǒng)中最常見(jiàn)的惡性腫瘤。近些年來(lái),雖然經(jīng)過(guò)手術(shù)、化療、放療等綜合治療,腎母細(xì)胞瘤的生存率有所提高,但仍有部分患兒由于復(fù)發(fā)、轉(zhuǎn)移以及對(duì)化療藥物的不敏感導(dǎo)致治療效果不佳而死亡。因此,探討腎母細(xì)胞瘤的發(fā)生發(fā)展機(jī)制,尋找腎母細(xì)胞瘤的分子生物學(xué)標(biāo)記物,具有重要的意義。 Astrocyte elevated gene-1(AEG-1)是Su等在2002年首次克隆出來(lái)的新基因,最早發(fā)現(xiàn)于感染HIV病毒的PHFA (primary human fetal astrocytes)細(xì)胞。近年來(lái)對(duì)AEG-1的深入研究,發(fā)現(xiàn)AEG-1能激活多條信號(hào)轉(zhuǎn)導(dǎo)通路,如PI3K/AKT, NF-κB、MEK/ERK、WNT/β-catenin等,與腫瘤的增殖、侵襲、轉(zhuǎn)移、血管生成、抗凋亡、耐藥等密切相關(guān)。盡管在多種體外培養(yǎng)的腫瘤細(xì)胞如胃癌、食管癌、肝細(xì)胞肝癌、惡性神經(jīng)膠質(zhì)瘤、神經(jīng)母細(xì)胞瘤、子宮內(nèi)膜癌、乳腺癌、黑色素瘤、腎細(xì)胞癌及前列腺癌等以及多種腫瘤組織標(biāo)本中都發(fā)現(xiàn)AEG-1表達(dá)顯著升高并對(duì)其機(jī)制進(jìn)行了研究,但是,AEG-1與腎母細(xì)胞瘤中的相關(guān)性研究,國(guó)內(nèi)外均未見(jiàn)報(bào)道。 RNA干擾(RNA interfering, RNAi)技術(shù)目前已廣泛應(yīng)用于基因功能、基因表達(dá)調(diào)控、基因治療以及調(diào)控細(xì)胞行為等多個(gè)領(lǐng)域,成為重要的研究工具。由慢病毒介導(dǎo)的表達(dá)載體因其表達(dá)持久、安全性好等自身優(yōu)勢(shì),在基因治療的研究中得到了廣泛的應(yīng)用,并成為目前RNAi研究和轉(zhuǎn)基因的最佳工具之一。 基于以上研究背景,本課題研究AEG-1在腎母細(xì)胞瘤中的表達(dá)情況,及其與臨床特征的關(guān)系及對(duì)病人預(yù)后的影響；構(gòu)建靶向AEG-1特異性RNA干擾慢病毒表達(dá)載體,體外轉(zhuǎn)染人高表達(dá)AEG-1的G401細(xì)胞株,沉默其AEG-1基因的表達(dá),研究AEG-1基因被抑制后G401細(xì)胞增殖、運(yùn)動(dòng)、侵襲、周期、調(diào)亡、化療敏感性等方面的影響并檢測(cè)與之相關(guān)的蛋白水平,初步探討其可能的分子機(jī)制,為臨床尋找腎母細(xì)胞瘤基因治療的新靶點(diǎn)提供借鑒。 方法 1.AEG-1在腎母細(xì)胞瘤中的表達(dá)及其對(duì)預(yù)后的影響：選取山東省立醫(yī)院腎母細(xì)胞瘤手術(shù)切除標(biāo)本42例,應(yīng)用免疫組織化學(xué)方法檢測(cè)AEG-1的表達(dá),并分析其與臨床特征的關(guān)系及對(duì)病人預(yù)后的影響。 2.慢病毒介導(dǎo)的腎母細(xì)胞瘤細(xì)胞AEG-1基因沉默：采用實(shí)時(shí)熒光定量PCR(Real time PCR)及蛋白質(zhì)印跡(western blot)技術(shù)檢測(cè)人胚腎細(xì)胞株CCC-HEK-1和人腎Wilms瘤細(xì)胞株G401中AEG-1的表達(dá)情況。構(gòu)建針對(duì)AEG-1基因的、帶綠色熒光蛋白標(biāo)記的、由U6啟動(dòng)子驅(qū)動(dòng)的慢病毒RNAi載體,轉(zhuǎn)染G401細(xì)胞。熒光顯微鏡觀(guān)察綠色熒光蛋白的表達(dá)并確定轉(zhuǎn)染效率,通過(guò)實(shí)時(shí)熒光定量PCR、western blot分別檢測(cè)細(xì)胞轉(zhuǎn)染后AEG-1mRNA和蛋白的水平變化情況,分析并確定RNA干擾的抑制效率,最終篩選出AEG-1沉默效率較高的干擾片段。 3.AEG-1基因沉默對(duì)腎母細(xì)胞瘤細(xì)胞生物學(xué)行為的影響及分子機(jī)制研究：將篩選出的最佳沉默效果的慢病毒載體轉(zhuǎn)染G401細(xì)胞,沉默AEG-1的基因表達(dá),通過(guò)MTT法檢測(cè)AEG-1干擾后G401細(xì)胞增殖能力的變化；通過(guò)細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)AEG-1沉默后G401細(xì)胞遷移能力變化,通過(guò)transwell小室侵襲實(shí)驗(yàn)檢測(cè)AEG-1基因干擾后G401細(xì)胞的侵襲能力的變化,采用、vestern blot檢測(cè)與腫瘤細(xì)胞侵襲轉(zhuǎn)移相關(guān)的基質(zhì)金屬蛋白酶MMP2和MMP9蛋白表達(dá)的變化；應(yīng)用流式細(xì)胞技術(shù)檢測(cè)AEG-1沉默對(duì)G401細(xì)胞周期和凋亡的影響,采用western blot檢測(cè)細(xì)胞周期相關(guān)蛋白Cyclin Dl、CDK2、p21Cip1、p27Kip1以及細(xì)胞凋亡相關(guān)蛋白Bcl-2、Bax、Caspase-3的表達(dá)變化；MTT法檢測(cè)AEG-1沉默后G401細(xì)胞對(duì)化療藥物阿霉素和放線(xiàn)菌素D的敏感性變化情況,采用western blot檢測(cè)耐藥基因MDRl蛋白表達(dá)的變化。 結(jié)果 1.AEG-1在腎母細(xì)胞瘤中的表達(dá)及其對(duì)預(yù)后的影響：在納入研究的42例腎母細(xì)胞瘤中,腫瘤組織中AEG-1高表達(dá)21例(50%),而瘤周正常組織中檢測(cè)不到AEG-1表達(dá)。AEG-1的表達(dá)情況在不同臨床分期(P=0.019)、病理分型(P=0.048)、復(fù)發(fā)情況(P=0.015)的患兒之間有顯著性差異,而在不同性別(P=0.751)、年齡(P=0.354)的患兒之間無(wú)顯著性差異。AEG-1高表達(dá)組的5年DFS和OS均顯著低于低表達(dá)組(P=0.006和P=0.007)。 2.慢病毒介導(dǎo)的腎母細(xì)胞瘤細(xì)胞AEG-1基因沉默：real time PCR和westernblot檢測(cè)證實(shí)G401細(xì)胞中(?)EG-1mRNA和蛋白水平顯著高于CCC-HEK-1細(xì)胞。以AEG-1mRNA基因序列為靶目標(biāo)進(jìn)行干擾片段設(shè)計(jì),成功構(gòu)建了2對(duì)針對(duì)AEG-1基因不同位點(diǎn)的特異性RNA干擾序列,經(jīng)化學(xué)修飾后與pGCSIL-GFP慢病毒載體連接,通過(guò)轉(zhuǎn)化,挑選陽(yáng)性克隆進(jìn)行測(cè)序,鑒定干擾片斷的插入位點(diǎn)和堿基序列與設(shè)計(jì)相符；在293T細(xì)胞中包裝慢病毒,獲得高滴度慢病毒上清；利用慢病毒將AEG-1基因的RNA干擾片段轉(zhuǎn)染G401細(xì)胞,并測(cè)定轉(zhuǎn)染效率達(dá)80%以上,real time PCR和western blot檢測(cè)證實(shí)與未轉(zhuǎn)染組相比,干擾組(AEG-1RNAi-LV1和(?)EG-1RNAi-LV2)的AEG-1mRNA和蛋白水平顯著降低,其中又以AEG-1RNAi-LV1的下降更明顯,說(shuō)明在2個(gè)干擾靶點(diǎn)中,(?)EG-1RNAi-LV1的干擾效果做好,后續(xù)實(shí)驗(yàn)選用該組。 3.AEG-1基因?qū)δI母細(xì)胞瘤的生長(zhǎng)、遷移、細(xì)胞周期及耐藥性具有明顯的調(diào)節(jié)作用：細(xì)胞劃痕實(shí)驗(yàn)顯示與未轉(zhuǎn)染組相比,AEG-1干擾后細(xì)胞增殖能力不斷下降,至第5天(120h)有顯著性差異(P0.0001)；在各個(gè)時(shí)間點(diǎn)的劃痕區(qū)域增寬,且在觀(guān)察48h時(shí)有顯著性差異(P=0.0413),該結(jié)果表明下調(diào)AEG-1抑制了細(xì)胞的侵襲轉(zhuǎn)移,且有顯著性差異(P=0.0238)；細(xì)胞侵襲轉(zhuǎn)移能力的下降與MMP2和MMP9蛋白表達(dá)水平密切相關(guān),MMP2與MMP9的表達(dá)明顯下調(diào),差異顯著(P=0.0395,P=0.0213)；AEG-1具有調(diào)節(jié)細(xì)胞周期的能力,結(jié)果顯示S期細(xì)胞比例明顯減少,而G0/G1期細(xì)胞比例明顯增多(P0.001),調(diào)節(jié)細(xì)胞周期的蛋白p21Cipl和p27Kip1表達(dá)顯著增加(P0.001)；AEG-1的下調(diào)亦能誘導(dǎo)細(xì)胞的凋亡,流式細(xì)胞儀的檢測(cè)結(jié)果表明細(xì)胞調(diào)亡比例增多(P=0.0024),而調(diào)控細(xì)胞凋亡的核心蛋白Bcl-2蛋白表達(dá)水平顯著降低(P=0.024),Bax和Caspase-3蛋白顯著增加(P0.001)；AEG-1也能影響腫瘤細(xì)胞對(duì)化療藥物耐藥性的變化,結(jié)果表明AEG-1的低表達(dá)對(duì)阿霉素和放線(xiàn)菌素D的半數(shù)抑制濃度IC50均顯著降低(P0.001),MDR1蛋白表達(dá)水平顯著降低(P=0.0018)。 結(jié)論 1.AEG-1的表達(dá)情況在不同臨床分期、病理分型、復(fù)發(fā)情況的患兒之間有顯著性差異,AEG-1高表達(dá)提示預(yù)后不良。 2.G401細(xì)胞的(?)EG-1mRNA和蛋白表達(dá)水平均顯著高于對(duì)照組細(xì)胞。成功構(gòu)建2種慢病毒系統(tǒng)介導(dǎo)AEG-1RNAi載體,可穩(wěn)定表達(dá)于靶細(xì)胞G401,能下調(diào)其目的基因AEG-1的(?)RNA和蛋白表達(dá),尤其以AEG-1-RNAi-LV1沉默效果更好。 3.沉默AEG-1基因后,G401細(xì)胞的增殖能力下降；通過(guò)降低MMP2和MMP9蛋白表達(dá)水平,使G401細(xì)胞的遷移能力和侵襲能力下降；通過(guò)增加p21Cip1和p27kip1蛋白表達(dá)水平,使G401細(xì)胞阻滯于G0/G1期；通過(guò)降低Bcl-2蛋白表達(dá)水平、增加Bax和Caspase-3蛋白表達(dá)水平,使G401細(xì)胞凋亡增多；通過(guò)降低MDRl/P-gp蛋白表達(dá)水平,提高了G401細(xì)胞對(duì)阿霉素和放線(xiàn)菌素D的敏感性。
[Abstract]:Background and purpose of the study Wilms tumor , also known as Wilms tumor , occupies about 8 % of all children ' s tumors . It is the most common malignant tumor in children . Astrocyte elevated gene - 1 ( AEG - 1 ) was the first new gene cloned from Su et al . in 2002 . It was found that AEG - 1 can activate multiple signal transduction pathways , such as the proliferation , invasion , metastasis , angiogenesis , apoptosis and drug resistance . RNA interfering RNA interference ( RNAi ) technology has been widely used in many fields such as gene function , gene expression regulation , gene therapy and regulation of cell behavior . On the basis of the above research background , this topic studies the expression of AEG - 1 and its relationship with clinical features and its influence on the prognosis of patients . The expression of AEG - 1 - specific RNA interference lentivirus expression vector was constructed . The expression of AEG - 1 gene in vitro was constructed . The expression of AEG - 1 gene was studied . The expression of AEG - 1 gene was studied . The expression of AEG - 1 gene was studied . The expression of AEG - 1 gene was studied . method 1 . The expression of AEG - 1 in nephrostomas and its effect on prognosis were studied : 42 cases were resected specimens from Shandong Provincial Hospital , the expression of AEG - 1 was detected by immunohistochemical method , and its relationship with clinical features was analyzed and the effect of AEG - 1 on prognosis was analyzed . 2 . The expression of AEG - 1 gene in human embryonic kidney cell line CCC - HEK - 1 and human kidney Wilms tumor cell line G401 was detected by real - time fluorescence quantitative PCR ( Real time PCR ) and Western blot . 3 . The effect of AEG - 1 gene silencing on the biological behavior of G401 cells was studied by MTT assay . The changes of cell proliferation ability after AEG - 1 gene silencing were detected by MTT assay . The changes of expression of cyclin Dl , CDK2 , p21 Cip1 , p27Kip1 and apoptosis related protein Bcl - 2 , Bax , Caspase - 3 were detected by MTT assay . Results 1 . The expression of AEG - 1 and AEG - 1 in the tumor tissues were significantly lower than those in the low expression group ( P = 0.019 ) , pathological type ( P = 0.048 ) and recurrence ( P = 0.015 ) . The expression of AEG - 1 was significantly lower in the patients with different clinical stages ( P = 0.019 ) , age ( P = 0.048 ) and recurrence ( P = 0.015 ) . 2 . The expression of AEG - 1 mRNA and protein in G401 cells was significantly higher than that of CCC - HEK - 1 cells . The interference effect of EG - 1RNAi - LV1 is well done , and the subsequent experiment selects the group . 3 . The growth , migration , cell cycle and drug resistance of the AEG - 1 gene were significantly lower than those in the untransfected group ( P = 0.02395 , P = 0.0213 ) . Conclusion 1 . The expression of AEG - 1 was significantly different among children with different clinical stages , pathological types and recurrence , and AEG - 1 high expression suggested poor prognosis . 2 . The expression level of ( ? ) EG - 1mRNA and protein in G401 cells was significantly higher than that in the control group . Two lentivirus systems were successfully constructed to mediate the expression of AEG - 1 RNAi vector , which could downregulate the expression of RNA and protein of AEG - 1 gene . Especially , the silencing effect of AEG - 1 - RNAi - LV1 was better . 3 . After silencing the AEG - 1 gene , the proliferation ability of G401 cells decreased ; by decreasing the expression level of MMP2 and MMP 9 , G401 cells were blocked in G0 / G1 phase ; by decreasing the expression level of p21 Cip1 and p27kip1 protein , the expression level of Bax and Caspase - 3 protein was increased , and the expression level of Bax and Caspase - 3 protein was increased , and the sensitivity of G401 cells to adriamycin and actinomycin D was improved by lowering the expression level of MDRI / P - gp protein .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.11

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 沈方臻;朱靜娟;林明剛;肖文靜;王寧寧;;穩(wěn)定轉(zhuǎn)染Bcl-2 shRNA對(duì)胃癌細(xì)胞SGC-7901化療敏感性的影響[J];中華腫瘤防治雜志;2010年23期

2 李小優(yōu);吳建中;曹海霞;吳劍秋;馮繼鋒;;5-aza-CdR增強(qiáng)吉非替尼對(duì)人非小細(xì)胞肺癌細(xì)胞株H1650敏感性機(jī)制的研究[J];中華腫瘤防治雜志;2012年06期

3 劉樊;周宜;;四種基源的川貝母對(duì)非小細(xì)胞肺癌A549細(xì)胞的抑制作用[J];四川中醫(yī);2011年08期

4 柳全文;徐慧;李桂華;柳成蔭;張婷;鄒寧;張?chǎng)?;多管藻中新溴代酚的鑒定及其自由基清除活性[J];食品科學(xué);2009年17期

5 甘耀坤;陳旭健;韋敏;葉楚芳;卿莉萍;;青岡櫟果實(shí)抗癌活性的實(shí)驗(yàn)研究[J];食品科技;2010年03期

6 羅淑敏;柯長(zhǎng)洪;蔡繼業(yè);李敏娟;;漢防己甲素聯(lián)合順鉑對(duì)乳腺癌細(xì)胞的作用[J];生物技術(shù);2011年03期

7 胡敏;李少林;袁犁;王勃;;γ分泌酶抑制劑阻斷Notch1信號(hào)通路對(duì)人結(jié)腸癌耐藥細(xì)胞化療敏感性的影響[J];中國(guó)生物制品學(xué)雜志;2012年01期

8 胡婕;張茵;馬保根;翟亞萍;李玉龍;程薇;;雷公藤紅素逆轉(zhuǎn)K562/A02細(xì)胞多藥耐藥的實(shí)驗(yàn)研究[J];實(shí)用癌癥雜志;2011年03期

9 徐小方;劉斌;吳軍正;王春梅;王哲;;人黏液表皮樣癌裸鼠耐藥移植瘤模型的建立及其生物學(xué)特性[J];實(shí)用口腔醫(yī)學(xué)雜志;2010年02期

10 楊慧瑩;趙麗;郭青龍;;P-糖蛋白抑制劑的研究進(jìn)展[J];實(shí)用老年醫(yī)學(xué);2011年02期

相關(guān)會(huì)議論文 前4條

1 裴曉華;李桃花;;粉防己堿對(duì)人乳腺癌MDA-MB-435S細(xì)胞抗腫瘤作用與機(jī)制研究[A];第十二次全國(guó)中醫(yī)、中西醫(yī)結(jié)合乳房病學(xué)術(shù)會(huì)議論文集[C];2011年

2 趙瑞君;劉顏崗;程t熛，

本文編號(hào)：1465673