本文選題：分化抑制因子 + β-catenin　； 參考：《醫(yī)學(xué)研究生學(xué)報(bào)》2017年05期

【摘要】：目的分化抑制因子3(Id3)參與腫瘤發(fā)生、細(xì)胞增殖和凋亡等過(guò)程;β-catenin是導(dǎo)致腫瘤發(fā)生的關(guān)鍵。文中探討Id3與β-catenin在不同腫瘤細(xì)胞中的表達(dá)及Id3對(duì)β-catenin的調(diào)控作用。方法采用Trizol法提取各腫瘤細(xì)胞總RNA,運(yùn)用實(shí)時(shí)熒光定量PCR技術(shù)(qRT-PCR)分析腫瘤細(xì)胞中Id3和β-catenin的相對(duì)表達(dá)量;用非脂質(zhì)體轉(zhuǎn)染技術(shù)將含人Id3基因的真核表達(dá)載體p EGFP/Id3分別導(dǎo)入SW-480、人肺腺癌細(xì)胞A549和人肺腺癌細(xì)胞耐順鉑株A549/DDP 3種細(xì)胞,熒光顯微鏡觀察細(xì)胞內(nèi)EGFP-Id3融合蛋白的表達(dá)情況;qRT-PCR技術(shù)分析轉(zhuǎn)染后細(xì)胞內(nèi)Id3及β-catenin mRNA的表達(dá)水平;Western blot分析轉(zhuǎn)染后細(xì)胞內(nèi)Id3及β-catenin蛋白的表達(dá)水平。結(jié)果 Id3在腸癌SW-480及HT-29細(xì)胞中表達(dá)量最低,明顯低于A549及其他腫瘤細(xì)胞(P0.05);在鼻咽癌CNE、5-8F細(xì)胞中的表達(dá)量顯著高于其他腫瘤細(xì)胞組(P0.05)。Id3表達(dá)量最低的腸癌SW-480中β-catenin與其他組細(xì)胞相比含量最高(P0.05),Id3表達(dá)較低的胃癌AGS細(xì)胞及腸癌HT-29細(xì)胞β-catenin表達(dá)次之,其他腫瘤細(xì)胞如H446、A549、SPC-A-1、A549/DDP、SK-MES-1細(xì)胞中β-catenin均呈低表達(dá),而Id3表達(dá)量較高的CNE、5-8F等腫瘤細(xì)胞中β-catenin的含量相對(duì)極低或幾乎不表達(dá),且與其他組細(xì)胞相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。熒光顯微鏡觀察發(fā)現(xiàn),轉(zhuǎn)染Id3/p EGFP的細(xì)胞體積縮小,細(xì)胞膜皺縮,折光度消失,而轉(zhuǎn)染空載體p EGFP后,大部分細(xì)胞未見(jiàn)上述變化。與對(duì)照組相比,Id3/p EGFP組A549、A549/DDI、SW-480細(xì)胞轉(zhuǎn)染后Id3 mRNA表達(dá)水平均有顯著增高(1.24±0.12 vs 193.12±2.80,1.09±0.11 vs 188.30±2.60,0.92±0.29 vs 19.08±0.59,P0.01)。與對(duì)照組比較,β-catenin mRNA在Id3過(guò)表達(dá)的腸癌SW-480細(xì)胞中表達(dá)明顯下調(diào)(0.98±0.05 vs 0.32±0.03,P0.01);而在A549和A549/DDP細(xì)胞中,Id3轉(zhuǎn)染后β-catenin表達(dá)水平差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。Western blot檢測(cè)結(jié)果顯示,與對(duì)照組比較,Id3過(guò)表達(dá)后可明顯下調(diào)腸癌細(xì)胞SW-480中β-catenin的表達(dá)水平,而肺癌細(xì)胞A549和A549/DDP中β-catenin的表達(dá)水平則無(wú)明顯變化。結(jié)論 Id3和β-catenin在不同的腫瘤細(xì)胞中有不同的表達(dá)水平,提示β-catenin在腸癌細(xì)胞中的異常高表達(dá)是引起腸癌的重要因素之一;外源性轉(zhuǎn)染Id3基因抑制腸癌SW-480細(xì)胞中β-catenin的表達(dá),有望為腸癌靶基因治療提供新思路。
[Abstract]:Objective differentiation inhibitory factor 3 (Id3) is involved in tumorigenesis, cell proliferation and apoptosis, and 尾 -catenin is the key to tumorigenesis. To investigate the expression of Id3 and 尾 -catenin in different tumor cells and the regulatory effect of Id3 on 尾 -catenin. Methods the total RNAs of tumor cells were extracted by Trizol method. The relative expressions of Id3 and 尾 -catenin in tumor cells were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). The eukaryotic expression vector pEGFP / Id3 containing human Id3 gene was transfected into SW-480 cells by non-liposome transfection technique. The expression of EGFP-Id3 fusion protein was observed by fluorescence microscope and the expression level of Id3 and 尾 -catenin mRNA in transfected cells was analyzed by qRT-PCR. The expression level of Id3 and 尾 -catenin protein in transfected cells was analyzed by Western blot. Results Id3 expression was the lowest in SW-480 and HT-29 cells. The expression of 尾 -catenin in CNE5-8F cells of nasopharyngeal carcinoma was significantly higher than that of other cancer cells (P0.05) .Id3 expression was the lowest in colorectal cancer SW-480, and the highest 尾 -catenin expression was found in cancer cells with lower expression of AGS than other cancer cells (P0.05), and the expression of 尾 -catenin was significantly higher than that of other cancer cells (P0.05), and the expression of 尾 -catenin was significantly higher than that of other cancer cells (P0.05). The expression of 尾 -catenin in HT-29 cells was the second. The expression of 尾 -catenin in other tumor cells such as H446A549SPC-A-1A549 / DDPnSK-MES-1 cells was low, while the expression of 尾 -catenin in CNE5-8F cells with high Id3 expression was very low or almost non-expressed, and there was significant difference compared with other groups (P0.05). Fluorescence microscopy showed that the cells transfected with Id3 / p EGFP decreased in volume, the cell membrane shrank, and the diopter disappeared. However, after transfection with empty vector pEGFP, most of the cells did not show the above changes. Compared with the control group, the expression of Id3 mRNA in A549 / DDIP-SW-480 cells was significantly higher than that in the control group (1.24 鹵0.12 vs 193.12 鹵2.800.09 鹵0.11 vs 188.30 鹵2.600.92 鹵0.29 vs 19.08 鹵0.59P0.01), and the expression of Id3 mRNA was significantly higher than that in the control group (1.24 鹵0.12 vs 193.12 鹵2.80,0.09 鹵0.11 vs 188.30 鹵2.600.92 鹵0.29 vs 19.08 鹵0.59P0.01). Compared with the control group, 尾 -catenin mRNA was significantly down-regulated in Id3 overexpressed SW-480 cells (0.98 鹵0.05 vs 0.32 鹵0.03P0.01), but there was no significant difference in 尾 -catenin expression in A549 and A549-DDP cells after transfection (P0.05). Compared with the control group, the expression of 尾 -catenin in SW-480 cells was significantly down-regulated, but the expression level of 尾 -catenin in A549 and A549 / DDP cells was not significantly changed. Conclusion there are different expression levels of Id3 and 尾 -catenin in different tumor cells, suggesting that the abnormal overexpression of 尾 -catenin in intestinal cancer cells is one of the important factors leading to colorectal cancer, and exogenous transfection of Id3 gene inhibits the expression of 尾 -catenin in SW-480 cells. It is expected to provide a new idea for target gene therapy of colorectal cancer.
【作者單位】： 南方醫(yī)科大學(xué)金陵醫(yī)院(南京軍區(qū)南京總醫(yī)院)中心實(shí)驗(yàn)科;
【基金】：國(guó)家自然科學(xué)基金(81171652)
【分類(lèi)號(hào)】：R730.2

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