本文選題：神經(jīng)膠質(zhì)瘤　切入點(diǎn)：Notch信號(hào)通路　出處：《福建醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 1、探討Notch1受體和其下游信號(hào)分子Hes1、Hes5在神經(jīng)膠質(zhì)瘤中表達(dá)特點(diǎn)和相互關(guān)系; 2、構(gòu)建Hes1、Hes5基因RNAi慢病毒載體,篩選獲得Hes1、Hes5沉默的神經(jīng)膠質(zhì)瘤細(xì)胞株; 3、觀察Hes1、Hes5基因沉默對(duì)神經(jīng)膠質(zhì)細(xì)胞U251增殖的影響,初步探討Notch-Hes信號(hào)通路調(diào)控神經(jīng)膠質(zhì)瘤增殖的可能機(jī)制。 方法: 1、應(yīng)用免疫組織化學(xué)方法檢測(cè)人腦星形細(xì)胞瘤和正常人腦組織中Notch1和Hes1、Hes5表達(dá)情況; 2、設(shè)計(jì)、合成針對(duì)靶基因Hes1、Hes5的shRNA編碼序列,將其克隆到pENTR /U6 RNAi入門(mén)載體,經(jīng)瞬時(shí)轉(zhuǎn)染篩選獲得有效的靶序列。將含有Hes1、Hes5有效干擾序列的入門(mén)載體與pLenti6/BLOCK-iT?-DEST目的載體通過(guò)Gateway LR重組構(gòu)建出相應(yīng)的shRNA慢病毒表達(dá)載體,經(jīng)293FT細(xì)胞包裝獲得慢病毒顆粒。通過(guò)慢病毒轉(zhuǎn)導(dǎo)U251細(xì)胞后,篩選獲得Hes1、Hes5基因沉默的U251細(xì)胞株; 3、采用MTT法,平板克隆形成實(shí)驗(yàn),流式細(xì)胞術(shù)檢測(cè)Hes1、Hes5基因沉默對(duì)U251細(xì)胞增殖及細(xì)胞周期的影響。 結(jié)果: 1、人腦星形細(xì)胞瘤中Notch1、Hes1和Hes5的表達(dá)均顯著強(qiáng)于正常腦組織;在不同病理分級(jí)的星形細(xì)胞瘤中,Ⅱ～Ⅲ級(jí)組Notch1和Hes1的表達(dá)水平顯著高于Ⅳ級(jí)星形細(xì)胞瘤組,Hes5的表達(dá)在兩組間無(wú)顯著性差異。星形細(xì)胞瘤中Notch1與Hes1的表達(dá)有較好的一致性,與Hes5的表達(dá)一致性較差。 2、成功篩選出Hes1、Hes5基因特異、有效的RNA干擾靶點(diǎn),構(gòu)建了針對(duì)Hes1、Hes5基因特異性shRNA慢病毒表達(dá)載體,并在293FT細(xì)胞中包裝獲得慢病毒顆粒,病毒滴度分別為8.4×104TU/ml、7.2×104TU/ml。 3、建立了Hes1、Hes5基因穩(wěn)定沉默的神經(jīng)膠質(zhì)瘤U251細(xì)胞株。 4、沉默Hes1或Hes5均能明顯抑制U251細(xì)胞增殖及克隆形成能力。 結(jié)論: 1、Notch1表達(dá)與星形細(xì)胞瘤的發(fā)生有關(guān),與星形細(xì)胞瘤的病理分級(jí)無(wú)關(guān)。Notch1可能主要是通過(guò)下游分子Hes1而非Hes5發(fā)揮作用。 2、構(gòu)建的Hes1、Hes5特異性RNAi慢病毒表達(dá)載體能有效抑制膠質(zhì)瘤細(xì)胞U251中Hes1、Hes5基因的表達(dá),可作為研究Notch-Hes信號(hào)通路及其與膠質(zhì)瘤關(guān)系的重要技術(shù)手段。 3、Hes1、Hes5與膠質(zhì)瘤細(xì)胞的增殖密切相關(guān),在體外Hes1、Hes5特異性RNAi能夠抑制膠質(zhì)瘤細(xì)胞U251的增殖能力。
[Abstract]:Objective:. 1. To investigate the expression of Notch1 receptor and its downstream signal molecule Hes1, Hes5, in gliomas. 2. The RNAi lentivirus vector of Hes1 hes5 gene was constructed, and the silencing glioma cell line of Hes1 hes5 was obtained. 3. To observe the effect of Hes1 Hes5 gene silencing on the proliferation of glial cell U251, and to explore the possible mechanism of Notch-Hes signaling pathway regulating glioma proliferation. Methods:. 1. The expression of Notch1 and Hes5 in human astrocytoma and normal human brain tissues were detected by immunohistochemical method. 2. Design and synthesize the shRNA coding sequence for Hes1 gene Hes5, and clone it into pENTR / U6 RNAi entry vector, and obtain the effective target sequence by transient transfection screening. The entry vector containing Hes1hes5 effective interference sequence and pLenti6% BLOCK-iTT? ShRNA lentivirus expression vector was constructed by recombinant Gateway LR. Lentivirus particles were obtained by packaging 293FT cells. After lentivirus was transduced into U251 cells, the U251 cell lines with Hes1Hes5 gene silencing were screened. 3. The effect of Hes1 Hes5 gene silencing on the proliferation and cell cycle of U251 cells was detected by MTT assay and plate clone formation assay. Results:. 1. The expression of Notch1Hes1 and Hes5 in human astrocytoma was significantly higher than that in normal brain tissue. The expression of Notch1 and Hes1 in grade 鈪，

本文編號(hào)：1598277