【摘要】：目的:原發(fā)的成人腦腫瘤中膠質(zhì)瘤是最常見的類型。它侵襲性強,與周圍組織界限不清晰。因此,常規(guī)的治療手段如手術治療和放射治療對它作用都很有限。不但如此,膠質(zhì)瘤的耐藥性也導致其化學治療收獲甚微。因此,膠質(zhì)瘤患者需要更有效的治療方案,而這些治療方案的建立需要我們找到新的分子靶點。核黃素轉(zhuǎn)運基因2(RFT2)是SLC52家族的一員,它在核黃素的代謝中起著重要的作用。最近,一些研究已經(jīng)證實,RFT2在許多人類腫瘤(食管鱗狀細胞癌、胃癌、宮頸癌)的發(fā)生發(fā)展中十分重要。本研究旨在探究RFT2在人類膠質(zhì)瘤中的表達情況及其對膠質(zhì)瘤細胞生物學特性的作用,進而為膠質(zhì)瘤的分子治療提供新的靶點。方法:為了檢測RFT2在正常腦組織和人類腦膠質(zhì)瘤組織中的表達水平,我們對120例不同級別的膠質(zhì)瘤組織、24例正常腦組織進行了免疫組織化學染色。利用RFT2-siRNA-3抑制膠質(zhì)瘤細胞系LN308和LN229中RFT2的表達。使用MTT、平板克隆形成實驗、流式細胞術、Transwell實驗、Western blot和裸鼠皮下荷瘤模型從體內(nèi)和體外兩方面來進行膠質(zhì)瘤細胞增殖、遷移、侵襲等表型的實驗評估。結(jié)果:1、與正常腦組織相比RFT2在膠質(zhì)瘤組織中是高表達的。而且,膠質(zhì)瘤中RFT2的表達水平與WHO分級呈正相關。2、與對照組和無義序列轉(zhuǎn)染組相比,敲低LN308和LN229細胞RFT2的表達后細胞增殖被明顯抑制(P0.01),細胞集落形成率明顯下降(P0.01)。敲低RFT2可有效抑制移植瘤的體內(nèi)生長(P0.001)。3、敲低RFT2后LN308和LN229細胞阻滯在G1/G0期(P0.05)。在LN308和LN229細胞中敲低RFT2可以明顯升高其凋亡細胞的比例(P0.05)。各組中細胞周期和凋亡相關調(diào)節(jié)蛋白(p21、p27、Bcl-2、Bax)的表達都明顯改變。4、敲低RFT2后LN308和LN229細胞的遷移和侵襲能力受到抑制,其相關調(diào)節(jié)蛋白MMP-2和MMP-9的表達水平也相應下降。結(jié)論:本研究發(fā)現(xiàn),膠質(zhì)瘤組織中RFT2呈高表達,它的表達水平與膠質(zhì)瘤WHO分級呈正相關。敲低RFT2促進了LN308和LN229細胞的細胞周期阻滯、細胞凋亡,抑制了細胞增殖、遷移和侵襲的能力。不但如此,裸鼠荷瘤模型也進一步更好地證明了敲低RFT2后膠質(zhì)瘤細胞的增殖受到抑制。這些結(jié)果提示,RFT2可以成為膠質(zhì)瘤分子治療的新靶點。
[Abstract]:Objective: gliomas are the most common type of primary adult brain tumors. It is aggressive, and its boundaries with surrounding tissues are not clear. As a result, conventional treatments such as surgery and radiotherapy have limited effects on it. Moreover, the drug resistance of gliomas leads to little success in chemotherapy. Therefore, glioma patients need more effective treatment protocols, and the establishment of these protocols requires us to find new molecular targets. Riboflavin transporter gene 2 (RFT2) is a member of SLC52 family and plays an important role in riboflavin metabolism. Recently, some studies have confirmed that RFT2 plays an important role in the development of many human tumors (esophageal squamous cell carcinoma, gastric cancer, cervical cancer). The purpose of this study was to investigate the expression of RFT2 in human glioma and its effect on the biological characteristics of glioma cells, and to provide a new target for the molecular therapy of glioma. Methods: in order to detect the expression of RFT2 in normal brain tissues and human glioma tissues, immunohistochemical staining was performed in 120 gliomas of different grades and 24 normal brain tissues. The expression of RFT2 in glioma cell line LN308 and LN229 was inhibited by RFT2-siRNA-3. The phenotypes of glioma cell proliferation, migration and invasion were evaluated in vivo and in vitro by MTT, plate clone formation assay, flow cytometry, Transwell assay, Western blot and nude mice subcutaneous tumor model. Results: 1. The expression of RFT2 in glioma was higher than that in normal brain tissue. Moreover, the expression of RFT2 in gliomas was positively correlated with the WHO grade. 2Compared with the control group and the nonsense sequence transfection group, the proliferation of LN308 and LN229 cells was significantly inhibited after the expression of RFT2 was lowered (P0.01). The colony formation rate decreased significantly (P0.01). Knockout of RFT2 could effectively inhibit the growth of transplanted tumor in vivo (P0.001). 3. LN308 and LN229 cells were blocked in G1/G0 phase after knock down RFT2 (P0.05). Knockout of RFT2 in LN308 and LN229 cells significantly increased the percentage of apoptotic cells (P0.05). The cell cycle and the expression of apoptosis-related regulatory protein (p21, p27, Bcl-2, Bax) were significantly changed in each group. 4. The migration and invasion of LN308 and LN229 cells were inhibited after knockout with RFT2. The expression levels of MMP-2 and MMP-9 were also decreased. Conclusion: high expression of RFT2 was found in glioma tissues, and its expression level was positively correlated with WHO grade of gliomas. Knockout of RFT2 promoted cell cycle arrest, apoptosis and cell proliferation, migration and invasion of LN308 and LN229 cells. In addition, the nude mice tumor-bearing model further demonstrated that the proliferation of glioma cells was inhibited after knockout of RFT2. These results suggest that RFT2 may be a new target for glioma molecular therapy.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

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