【摘要】：背景和目的： 腦膠質瘤是中樞神經(jīng)系統(tǒng)最常見的原發(fā)性腫瘤,由于惡性膠質瘤有侵襲性生長、增殖速度快等特征,應用目前的治療措施如外科手術切除、術后放療、化療和免疫治療等不能治愈,患者復發(fā)率很高,臨床預后甚差。造成這種現(xiàn)狀的原因,很大部位是對腦膠質瘤的病因,發(fā)病機制的認識不充分。為此,必須尋找與膠質瘤發(fā)生、發(fā)展相關的基因和信號通路,進一步深入了解膠質瘤發(fā)生發(fā)展的分子病理機制,尋找膠質瘤治療的新靶點,在此基礎上開拓膠質瘤治療的新策略和新手段。 有研究證實,Nemo-like kinase(NLK)是一種抑癌基因,在體外的惡性腫瘤細胞株中的研究發(fā)現(xiàn)它可以通過促進腫瘤細胞的凋亡而起到抑癌作用,研究表明NLK能夠促進人的結腸癌細胞DLD-1細胞株,乳腺癌細胞株和膠質瘤細胞株的凋亡[1-3]。但NLK在腦膠質瘤中的研究甚少,故可以初步認定NLK是一個膠質瘤抑癌基因。此外,為了尋找可調控NLK表達的microRNA,我們搜索了預測microRNA靶基因網(wǎng)站,包括TARGETSCAN, MIRANDA等,發(fā)現(xiàn)有很多的microRNA,包括niR-92b在NLK mRNA3'UTR上有潛在的結合位點,故本課題重點研究了miR-92b I抑制膠質瘤增殖、細胞周期進展、凋亡和侵襲的影響及其可能的作用機制。 方法和結果： 本課題研究分為以下兩個部分： 第一部分中應用Real time PCR的方法,檢測了5個膠質瘤細胞系和38例不同級別人腦膠質瘤組織中miR-92b的表達水平。結果表明miR-92b在膠質瘤組織和細胞系中的表達較正常組織上調,并與腫瘤級別呈正相關。 在課題第二部分,miR-92b Ⅰ抑制膠質瘤惡性表型的體外研究。將niR-92b Ⅰ轉染至人腦膠質瘤細胞系SNB19和LN229中。采用Real time PCR檢測轉染后膠質瘤細胞的miR-92b表達水平以鑒定抑制效果；四甲基偶氮唑藍(MTT)實驗評價細胞的增殖率；流式細胞術檢測細胞周期變化；Annexin Ⅴ法檢測細胞早期凋亡;Transwell實驗檢測細胞侵襲能力變化。Real time PCR檢測結果顯示轉染miR-92b Ⅰ后,腫瘤細胞miR-92b表達下降,miR-92b Ⅰ組細胞增殖活性降低,細胞侵襲能力明顯受到抑制,細胞周期S期縮短,阻滯在G0/G1期以及早期凋亡增加。該實驗說明miR-92b是癌微RNA(OncomiRs),可能成為膠質瘤治療的潛在靶點。 結論： miR-92b在膠質瘤中表達上調,低級別腫瘤與高級別腫瘤比較,其差異具有顯著性,但是正常組織與低級別腫瘤比較,差異沒有顯著性。轉染miR-92b Ⅰ至人膠質瘤細胞系,可抑制膠質瘤細胞的增殖活性和侵襲能力,細胞周期S期縮短,阻滯在G0/G1期,并促進細胞凋亡,由此,初步認定miR-92b為癌微RNA,可成為膠質瘤基因治療的候選靶標。這是首次有關miR-92b在人腦膠質瘤中的表達以及對膠質瘤惡性表型的影響的研究報道,為進一步認識腦膠質瘤的分子病理機制及新一類的靶向治療提供實驗依據(jù)。
[Abstract]:Background and objective: glioma is the most common primary tumor in the central nervous system. Chemotherapy and immunotherapy can not be cured, the recurrence rate is very high, the clinical prognosis is very poor. Most of the causes of this situation are the etiology of glioma, and the pathogenesis of glioma is not well understood. Therefore, it is necessary to search for genes and signal pathways related to glioma development, to further understand the molecular pathological mechanism of glioma development, and to find new targets for the treatment of glioma. On this basis, a new strategy and a new method for the treatment of glioma were developed. Some studies have confirmed that Nemo-like kinase (NLK) is a tumor suppressor gene. In vitro, it has been found that Nemo-like kinase (NLK) can inhibit cancer by promoting apoptosis of tumor cells. It has been shown that NLK can promote human colon cancer cell DLD-1 cell line. Apoptosis of breast cancer cell line and glioma cell line [1-3]. However, there are few studies of NLK in gliomas, so it can be preliminarily concluded that NLK is a tumor suppressor gene. In addition, in order to find microRNA, that can regulate the expression of NLK, we searched microRNA target gene websites, including TARGETSCAN, MIRANDA, and found that many microRNA, including niR-92b, had potential binding sites on NLK mRNA3'UTR. Therefore, this study focused on the inhibition of glioma proliferation by miR-92b I. Cell cycle progression, the effects of apoptosis and invasion and their possible mechanisms. Methods and results: the study was divided into the following two parts: in the first part, the expression of miR-92b in 5 glioma cell lines and 38 cases of human gliomas of different grades were detected by the method of Real time PCR. The results showed that the expression of miR-92b in glioma tissues and cell lines was higher than that in normal tissues, and there was a positive correlation between the expression of miR-92b and tumor grade. In the second part of the study, miR-92b 鈪，

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