本文選題：穩(wěn)定過表達(dá)　切入點(diǎn)：NF-k　出處：《中山大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版)》2016年02期 　論文類型：期刊論文

【摘要】：【目的】研究過表達(dá)整合素鏈接激酶(ILK)對(duì)膠質(zhì)瘤細(xì)胞上皮間質(zhì)轉(zhuǎn)化(EMT)的調(diào)控作用及初步機(jī)制。【方法】項(xiàng)目組前期實(shí)驗(yàn)已經(jīng)成功構(gòu)建了重組質(zhì)粒p EGFP-C1-ILK,并將其轉(zhuǎn)染給SHG-44膠質(zhì)瘤細(xì)胞,通過G418篩選出了穩(wěn)定轉(zhuǎn)染的細(xì)胞株。實(shí)驗(yàn)分組如下:SHG-44(空白對(duì)照組)、p EGFP-C1(空載組),及p EGFP-C1-ILK(穩(wěn)轉(zhuǎn)組),先檢測(cè)各組中ILK的表達(dá)情況(RNA及蛋白質(zhì)水平)及侵襲能力,再檢測(cè)過表達(dá)ILK后對(duì)EMT標(biāo)記物的影響,最后再分別應(yīng)用NF-κB通路的特異性阻斷劑BAY11-7028和RNA沉默的方法阻斷核因子NF-κB通路,Western blot方法檢測(cè)上皮間質(zhì)轉(zhuǎn)化標(biāo)記物Ecadherin在阻斷前及阻斷后的表達(dá)情況,初步探討NF-k B通路在過表達(dá)ILK的膠質(zhì)瘤細(xì)胞中對(duì)EMT的調(diào)控作用�！窘Y(jié)果】穩(wěn)轉(zhuǎn)組中ILK明顯過表達(dá),同時(shí)侵襲能力增強(qiáng)(Transwell小室透膜細(xì)胞數(shù)在對(duì)照組、空載組和穩(wěn)轉(zhuǎn)組分別為:92,87,229)。Western blot檢測(cè)EMT標(biāo)記物蛋白的表達(dá):穩(wěn)定轉(zhuǎn)染組中snail,slug,twist,vimentin的表達(dá)較對(duì)照組及空載組的表達(dá)明顯增高,而E-cadherin的表達(dá)則在穩(wěn)定轉(zhuǎn)染組中明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。當(dāng)分別用NF-k B特異性阻斷劑BAY11-7028及p65 si RNA的方法阻斷該通路后,穩(wěn)定轉(zhuǎn)染組中E-cadherin蛋白表達(dá)明顯升高�！窘Y(jié)論】膠質(zhì)瘤中過表達(dá)ILK可使侵襲能力增強(qiáng),同時(shí)可下調(diào)E-cadherin,上調(diào)vimentin及Snail,Slug,Twist的表達(dá),可能通過此機(jī)制促進(jìn)腦膠質(zhì)瘤細(xì)胞的上皮間質(zhì)轉(zhuǎn)化,NF-k B通路可能參與、調(diào)控該進(jìn)程。
[Abstract]:[objective] to study the regulation and mechanism of overexpression of integrin linked kinase (ILK) on epithelial interstitial transformation of glioma cells. [methods] Recombinant plasmid pEGFP-C1-ILK has been successfully constructed and transfected into SHG-44 glioma cells. The stable transfected cell lines were selected by G418. The experimental groups were as follows: SHG-44 (blank control group, pEGFP-C1-ILK) and invasion ability. The effects of ILK expression on EMT markers were also detected. Finally, BAY11-7028 and RNA silencing of NF- 魏 B pathway were used to detect the expression of Ecadherin before and after blocking NF-魏 B pathway by Western blot. To investigate the role of NF-k B pathway in the regulation of EMT in glioma cells overexpression of ILK. [results] the expression of ILK was significantly overexpressed in the stable transfer group, and the invasiveness of ILK was enhanced in the control group, and the number of transwell permeability cells was increased in the control group. The expression of EMT marker protein in the stable transfection group was significantly higher than that in the control group and the no-load group, but the expression of E-cadherin in the stable transfection group was significantly lower than that in the stable transfection group, and the expression of E-cadherin in the stable transfection group was significantly lower than that in the control group and the no-load group, and the expression of E-cadherin in the stable transfection group was significantly lower than that in the control group. The expression of E-cadherin protein increased significantly in stable transfection group after blocking the pathway with NF-k B specific blocker BAY11-7028 and p65si RNA respectively. [conclusion] overexpression of ILK in glioma can enhance the invasiveness. At the same time, E-cadherin could be down-regulated and the expression of vimentin and Sluger-Twist could be upregulated, which may promote the epithelial interstitial transformation of glioma cells and the NF-kB pathway may be involved in the regulation of this process.
【作者單位】： 遼寧醫(yī)學(xué)院附屬第一醫(yī)院神經(jīng)外科;遼寧醫(yī)學(xué)院附屬第一醫(yī)院兒科;
【基金】：遼寧省博士啟動(dòng)基金(201501101)
【分類號(hào)】：R739.41

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