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

【摘要】：研究背景和目的 腎母細胞瘤,又稱Wilms瘤,約占所有兒童腫瘤的比例為8%,是兒童比較常見的惡性腫瘤,同時也是兒童泌尿系統(tǒng)中最常見的惡性腫瘤。近些年來,雖然經(jīng)過手術(shù)、化療、放療等綜合治療,腎母細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,探討腎母細胞瘤的發(fā)生發(fā)展機制,尋找腎母細胞瘤的分子生物學(xué)標記物,具有重要的意義。 Astrocyte elevated gene-1(AEG-1)是Su等在2002年首次克隆出來的新基因,最早發(fā)現(xiàn)于感染HIV病毒的PHFA (primary human fetal astrocytes)細胞。近年來對AEG-1的深入研究,發(fā)現(xiàn)AEG-1能激活多條信號轉(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)的腫瘤細胞如胃癌、食管癌、肝細胞肝癌、惡性神經(jīng)膠質(zhì)瘤、神經(jīng)母細胞瘤、子宮內(nèi)膜癌、乳腺癌、黑色素瘤、腎細胞癌及前列腺癌等以及多種腫瘤組織標本中都發(fā)現(xiàn)AEG-1表達顯著升高并對其機制進行了研究,但是,AEG-1與腎母細胞瘤中的相關(guān)性研究,國內(nèi)外均未見報道。 RNA干擾(RNA interfering, RNAi)技術(shù)目前已廣泛應(yīng)用于基因功能、基因表達調(diào)控、基因治療以及調(diào)控細胞行為等多個領(lǐng)域,成為重要的研究工具。由慢病毒介導(dǎo)的表達載體因其表達持久、安全性好等自身優(yōu)勢,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在腎母細胞瘤中的表達情況,及其與臨床特征的關(guān)系及對病人預(yù)后的影響；構(gòu)建靶向AEG-1特異性RNA干擾慢病毒表達載體,體外轉(zhuǎn)染人高表達AEG-1的G401細胞株,沉默其AEG-1基因的表達,研究AEG-1基因被抑制后G401細胞增殖、運動、侵襲、周期、調(diào)亡、化療敏感性等方面的影響并檢測與之相關(guān)的蛋白水平,初步探討其可能的分子機制,為臨床尋找腎母細胞瘤基因治療的新靶點提供借鑒。 方法 1.AEG-1在腎母細胞瘤中的表達及其對預(yù)后的影響：選取山東省立醫(yī)院腎母細胞瘤手術(shù)切除標本42例,應(yīng)用免疫組織化學(xué)方法檢測AEG-1的表達,并分析其與臨床特征的關(guān)系及對病人預(yù)后的影響。 2.慢病毒介導(dǎo)的腎母細胞瘤細胞AEG-1基因沉默：采用實時熒光定量PCR(Real time PCR)及蛋白質(zhì)印跡(western blot)技術(shù)檢測人胚腎細胞株CCC-HEK-1和人腎Wilms瘤細胞株G401中AEG-1的表達情況。構(gòu)建針對AEG-1基因的、帶綠色熒光蛋白標記的、由U6啟動子驅(qū)動的慢病毒RNAi載體,轉(zhuǎn)染G401細胞。熒光顯微鏡觀察綠色熒光蛋白的表達并確定轉(zhuǎn)染效率,通過實時熒光定量PCR、western blot分別檢測細胞轉(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基因沉默對腎母細胞瘤細胞生物學(xué)行為的影響及分子機制研究：將篩選出的最佳沉默效果的慢病毒載體轉(zhuǎn)染G401細胞,沉默AEG-1的基因表達,通過MTT法檢測AEG-1干擾后G401細胞增殖能力的變化；通過細胞劃痕實驗檢測AEG-1沉默后G401細胞遷移能力變化,通過transwell小室侵襲實驗檢測AEG-1基因干擾后G401細胞的侵襲能力的變化,采用、vestern blot檢測與腫瘤細胞侵襲轉(zhuǎn)移相關(guān)的基質(zhì)金屬蛋白酶MMP2和MMP9蛋白表達的變化；應(yīng)用流式細胞技術(shù)檢測AEG-1沉默對G401細胞周期和凋亡的影響,采用western blot檢測細胞周期相關(guān)蛋白Cyclin Dl、CDK2、p21Cip1、p27Kip1以及細胞凋亡相關(guān)蛋白Bcl-2、Bax、Caspase-3的表達變化；MTT法檢測AEG-1沉默后G401細胞對化療藥物阿霉素和放線菌素D的敏感性變化情況,采用western blot檢測耐藥基因MDRl蛋白表達的變化。 結(jié)果 1.AEG-1在腎母細胞瘤中的表達及其對預(yù)后的影響：在納入研究的42例腎母細胞瘤中,腫瘤組織中AEG-1高表達21例(50%),而瘤周正常組織中檢測不到AEG-1表達。AEG-1的表達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)的患兒之間無顯著性差異。AEG-1高表達組的5年DFS和OS均顯著低于低表達組(P=0.006和P=0.007)。 2.慢病毒介導(dǎo)的腎母細胞瘤細胞AEG-1基因沉默：real time PCR和westernblot檢測證實G401細胞中(?)EG-1mRNA和蛋白水平顯著高于CCC-HEK-1細胞。以AEG-1mRNA基因序列為靶目標進行干擾片段設(shè)計,成功構(gòu)建了2對針對AEG-1基因不同位點的特異性RNA干擾序列,經(jīng)化學(xué)修飾后與pGCSIL-GFP慢病毒載體連接,通過轉(zhuǎn)化,挑選陽性克隆進行測序,鑒定干擾片斷的插入位點和堿基序列與設(shè)計相符；在293T細胞中包裝慢病毒,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細胞,并測定轉(zhuǎn)染效率達80%以上,real time PCR和western blot檢測證實與未轉(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,說明在2個干擾靶點中,(?)EG-1RNAi-LV1的干擾效果做好,后續(xù)實驗選用該組。 3.AEG-1基因?qū)δI母細胞瘤的生長、遷移、細胞周期及耐藥性具有明顯的調(diào)節(jié)作用：細胞劃痕實驗顯示與未轉(zhuǎn)染組相比,AEG-1干擾后細胞增殖能力不斷下降,至第5天(120h)有顯著性差異(P0.0001)；在各個時間點的劃痕區(qū)域增寬,且在觀察48h時有顯著性差異(P=0.0413),該結(jié)果表明下調(diào)AEG-1抑制了細胞的侵襲轉(zhuǎn)移,且有顯著性差異(P=0.0238)；細胞侵襲轉(zhuǎn)移能力的下降與MMP2和MMP9蛋白表達水平密切相關(guān),MMP2與MMP9的表達明顯下調(diào),差異顯著(P=0.0395,P=0.0213)；AEG-1具有調(diào)節(jié)細胞周期的能力,結(jié)果顯示S期細胞比例明顯減少,而G0/G1期細胞比例明顯增多(P0.001),調(diào)節(jié)細胞周期的蛋白p21Cipl和p27Kip1表達顯著增加(P0.001)；AEG-1的下調(diào)亦能誘導(dǎo)細胞的凋亡,流式細胞儀的檢測結(jié)果表明細胞調(diào)亡比例增多(P=0.0024),而調(diào)控細胞凋亡的核心蛋白Bcl-2蛋白表達水平顯著降低(P=0.024),Bax和Caspase-3蛋白顯著增加(P0.001)；AEG-1也能影響腫瘤細胞對化療藥物耐藥性的變化,結(jié)果表明AEG-1的低表達對阿霉素和放線菌素D的半數(shù)抑制濃度IC50均顯著降低(P0.001),MDR1蛋白表達水平顯著降低(P=0.0018)。 結(jié)論 1.AEG-1的表達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高表達提示預(yù)后不良。 2.G401細胞的(?)EG-1mRNA和蛋白表達水平均顯著高于對照組細胞。成功構(gòu)建2種慢病毒系統(tǒng)介導(dǎo)AEG-1RNAi載體,可穩(wěn)定表達于靶細胞G401,能下調(diào)其目的基因AEG-1的(?)RNA和蛋白表達,尤其以AEG-1-RNAi-LV1沉默效果更好。 3.沉默AEG-1基因后,G401細胞的增殖能力下降；通過降低MMP2和MMP9蛋白表達水平,使G401細胞的遷移能力和侵襲能力下降；通過增加p21Cip1和p27kip1蛋白表達水平,使G401細胞阻滯于G0/G1期；通過降低Bcl-2蛋白表達水平、增加Bax和Caspase-3蛋白表達水平,使G401細胞凋亡增多；通過降低MDRl/P-gp蛋白表達水平,提高了G401細胞對阿霉素和放線菌素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)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.11

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