RTN4在胃癌發(fā)病機制中的作用

張超　周業(yè)　謝黎明

摘 要：目的 了解RTN4蛋白在胃癌發(fā)病過程中的作用，為揭示胃癌發(fā)病的分子機制提供實驗資料。方法 將pIRESneo3-RTN4真核表達(dá)載體轉(zhuǎn)染胃粘膜上皮GES1細(xì)胞，建立RTN4高表達(dá)的GES1-RTN4細(xì)胞系；通過細(xì)胞生長曲線、平皿克隆與軟瓊脂集落形成實驗、流式細(xì)胞儀，觀察RTN4高表達(dá)對GES1細(xì)胞生長與增殖、周期與凋亡的影響；利用Western-blot檢測GES1-RTN4細(xì)胞中IκBα蛋白的表達(dá)。結(jié)果 GES1-RTN4細(xì)胞的生長速度較GES1和GES1-pIRESneo3細(xì)胞加快，統(tǒng)計學(xué)意義顯著（P<0.01）；GES1-RTN4細(xì)胞較GES1和GES1-pIRESneo3細(xì)胞克隆體積大、數(shù)目多，統(tǒng)計學(xué)意義顯著（P<0.01）；GES1-RTN4細(xì)胞中S期細(xì)胞比例較GES1和GES1-pIRESneo3細(xì)胞增加，細(xì)胞增殖指數(shù)增加，細(xì)胞凋亡率降低，統(tǒng)計學(xué)意義顯著（P<0.01）；GES1-RTN4細(xì)胞中IκBα蛋白表達(dá)較GES1和GES1-pIRESneo3細(xì)胞減少，統(tǒng)計學(xué)意義顯著（P<0.01）。結(jié)論 RTN4通過活化NF-κB信號通路，提高細(xì)胞增殖指數(shù)，抑制細(xì)胞凋亡，促進(jìn)GES1細(xì)胞的增殖。

關(guān)鍵詞：GES1細(xì)胞；RTN4蛋白；細(xì)胞增殖；NF-κB信號通路

中圖分類號：R735.2 文獻(xiàn)標(biāo)識碼：A DOI：10.3969/j.issn.1006-1959.2018.22.016

文章編號：1006-1959（2018）22-0055-05

The Role of RTN4 in the Pathogenesis of Gastric Carcinoma

ZHANG Chao1，2，ZHOU Ye1，XIE Li-min3，HE Xiu-sheng1，ZHANG Zhi-wei1

（1.Hunan Provincial Key Laboratory of Tumor Cell and Molecular Pathology，Cancer Research Institute，School of Medicine，Nanhua University，Hengyang 421001，Hunan，China；

2.Department of Dermatology，Nanhua Hospital，Nanhua University，Hengyang 421001，Hunan，China；

3.the First Affiliated Hospital of Nanhua University，Hengyang 421001，Hunan，China）

Abstract： Objective To investigate the role of RTN4 protein in the pathogenesis of gastric carcinoma and to provide experimental data for revealing the molecular mechanism of gastric carcinoma.Methods The pIRESneo3-RTN4 eukaryotic expression vector was transfected into gastric mucosal epithelial GES1 cells to establish a high expression of GES1-RTN4 cell line. The cell growth curve， plate clone and soft agar colony formation assay， flow cytometry， high RTN4 expression were observed. The effects of GES1 cell growth and proliferation， cycle and apoptosis were detected by Western-blot. The expression of IκBα protein in GES1-RTN4 cells was detected by Western-blot. Results The growth rate of GES1-RTN4 cells was faster than that of GES1 and GES1-pIRESneo3 cells， which was statistically significant（P<0.01）. The GES1-RTN4 cells were larger and more numerous than GES1 and GES1-pIRESneo3 cells，the statistical significance was significant（P<0.01）； the proportion of S phase cells in GES1-RTN4 cells was higher than that of GES1 and GES1-pIRESneo3 cells， the cell proliferation index increased， and the apoptosis rate decreased， statistically significant（P<0.01）；The expression of IκBα protein in GES1-RTN4 cells was significantly lower than that in GES1 and GES1-pIRESneo3 cells，statistically significant（P<0.01）. Conclusion RTN4 can increase the cell proliferation index， inhibit cell apoptosis and promote the proliferation of GES1 cells by activating NF-κB signaling pathway.

Key words：GES1 cells；RTN4 protein；Cell proliferation；NF-κB signaling pathway

胃癌（gastric carcinoma，GC）是我國常見的十大惡性腫瘤之一，其發(fā)病率與死亡率居第2位，消化道腫瘤的第1位[1，2]?，F(xiàn)今，胃癌在我國的發(fā)病率呈現(xiàn)逐年下降與年輕化的趨勢，但仍然嚴(yán)重危害人們健康與生命[3]，其發(fā)病的分子機制仍不十分清楚。本課題組前期采用定量蛋白質(zhì)組學(xué)技術(shù)鑒定了243個胃癌相關(guān)蛋白質(zhì)，發(fā)現(xiàn)RTN4蛋白在胃粘膜癌變過程中表達(dá)上調(diào)[4]，但其在胃癌發(fā)病過程中的具體作用及機制仍不清楚，深入了解其在胃癌發(fā)病中的作用，將為闡明胃癌發(fā)病的分子機制提供理論依據(jù)。

1材料與方法

1.1材料

1.1.1細(xì)胞系和質(zhì)粒 永生化胃粘膜上皮GES1細(xì)胞購自上海中國醫(yī)學(xué)科學(xué)院細(xì)胞生物研究所，用10%小牛血清的RPMI 1640培養(yǎng)基常規(guī)培養(yǎng)。pIRESneo3-RTN4真核表達(dá)載體購自長沙佳和生物科技有限公司。

1.1.2引物 根據(jù)RTN4基因序列，采用Premier 6.0軟件設(shè)計重組RTN4和β-actin內(nèi)對照引物，由上海英駿生物技術(shù)有限公司合成RTN4引物（5'-AAAGATATCAAATATGGACTTGAA-3'；5-AAAGGATCCCTGACCCTCCC CCGTA-3'，大小2961 bp）與β-actin引物（5'-TCTACAATGAGCTGCGTGTGG-3'；5'-GGAACCGCTCATTGCCAATG-3'，大小498 bp）

1.1.3主要試劑 高保真酶、T4 DNA Ligand merase、Reverse transcription system試劑盒購自寶生物工程（大連）有限公司，限制性內(nèi)切酶HindIII和XhoI購自 Fermentas公司，一抗與二抗購自Santa Cruz公司?！?br>