【摘要】 背景与目的:T-bet(TboxexpressedinTcells)是一种特异性调控Th1淋巴细胞的核转录因子,它广泛调控多种免疫细胞(如Th1、NK、CD8+、DC、B细胞等)的生物学活性。本研究观察转染小鼠T-bet基因(pcDNA3.0-mT-bet)对小鼠巨噬细胞Raw264.7生物学功能的影响。方法:构建含T-bet的真核表达载体pcDNA3.0-mT-bet,通过双酶切鉴定及测序、PCR鉴定其准确性,RT-PCR及Westernblot检测其在Raw264.7细胞中的表达。以PBS、pcDNA3.0空载体作为对照,将其瞬时转染入Raw264.7细胞,48h后观察其对细胞周期、细胞表面分子MHCⅠ/Ⅱ表达的影响,以及对FITC-右旋糖酐(FITC-dextran)的吞噬能力、一氧化氮(nitricoxide,NO)分泌水平及T-bet对小鼠白血病细胞L1210的杀伤活性。结果:载体pcDNA3.0-mT-bet构建成功并在Raw264.7细胞中表达;T-bet对Raw264.7细胞周期没有影响(P>0.05);经T-bet修饰后,细胞表面MHCⅠ类分子的表达(24.8±0.6)高于空载体组(20.8±0.7),有统计学意义(P<0.05);但两组间MHCⅡ类分子的表达无显著性差异(P>0.05)。T-bet修饰组吞噬Dextran的平均荧光强度(32.8±0.8)高于空载体组(28.2±0.4),有统计学意义(P<0.05)。在无脂多糖(lipopolysaccharide,LPS)刺激时,T-bet组NO的产生量[(1.7±0.6)pmol]高于空载体组,两组相比差异有显著性(P<0.05);经10μg/mlLPS持续刺激20h后,T-bet组NO的产生量[(15.6±1.6)pmol]显著高于空载体组[(10.5±1.3)pmol](P<0.05)。pcDNA3.0-mT-bet对L1210细胞的体外杀伤活性[(51.9±3.5)%]明显高于空载体组[(35.6±2.1)%],差异有显著性(P<0.05)。结论:T-bet可诱导Raw264.7细胞表达MHCⅠ类分子,增强其对Dextran的吞噬能力,促进其分泌NO,增加其对L1210细胞的杀伤活性,但对其细胞周期及MHCⅡ类分子的表达无影响。更多还原

【Abstract】 BACKGROUND & OBJECTIVE: T-bet (T box expressed in T cells), a Th1-specific T box transcription factor, controls many kinds of immune cells, such as Th1, NK, CD8+, dendritic cells, and B cells. This study was to explore potential effects of T-bet gene on biological functions of mouse macrophage Raw264.7 cells in vitro. METHODS: The eukaryotic expression vector carrying mouse T-bet (pcDNA3.0-mT-bet) was constructed and identified by consequence analysis, double restrictive endonucleases digestion and polymerase chain reaction (PCR). The gene expression in Raw264.7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. PBS, pcDNA3.0, and pcDNA3.0-mT-bet were transiently transfected into Raw264.7 cells respectively; cell cycle, MHC I/Ⅱexpression levels, phagocytic activity of FITC-dextran, nitric oxide (NO) secretion level, and the cytotoxicity of Raw264.7 cells to mouse leukemia cell line L1210 were evaluated 48 hours after transfection. RESULTS: Eukarytic expression vector which could express T-bet protein in Raw264.7 cells was successfully constructed. There was no difference in cell cycle between pcDNA3.0 group and pcDNA3.0-mT-bet group. There was significant difference in MHC I expression level between pcDNA3.0 group (20.8±0.7) and pcDNA3.0-mT-bet group (24.8±0.6, P<0.05), but not in MHCⅡ expression level; there was also difference in mean fluorescence intensity of phagocytized dextran between pcDNA3.0 group(28.2±0.4) and pcDNA3.0-mT-bet group (32.8±0.8, P<0.05); there was also significant difference in NO secretion level between pcDNA3 group(0 pmol) and pcDNA3.0-mT-bet group [(1.7±0.6) pmol, P<0.05] without lipopolysaccharide (LPS) stimulation; meanwhile, significant difference was also observed between pcDNA3.0 group[(10.5 ±1.3) pmol] and pcDNA3.0-mT-bet group [(15.6±1.6) pmol, P<0.05] under the stimulation of LPS (10 μg/ml) for 20 h; there was also difference in cytotoxicity of Raw264.7 cells to L1210 cells in vitro between pcDNA3 group[(35.6±2.1)%] and pcDNA3.0-mT-bet group [(51.9±3.5)%, P<0.05]. CONCLUSION: T-bet up-regulates MHC Ⅰ expression and NO secretion level in Raw264.7 cells, increases their cytotoxicity to L1210 cells, but has no influences on the cell cycle and MHCⅡ expression.更多还原