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作 者:段文娟[1] 余学清[1] 窦献蕊[1] 杨琼琼[1] 李晓艳[1]
机构地区:[1]中山大学附属第一医院肾内科,广东广州510080
出 处:《中国病理生理杂志》2006年第10期1873-1878,共6页Chinese Journal of Pathophysiology
基 金:中华人民共和国卫生部临床重点基金资助项目(2004-468);广州市临床重点项目资助(2004Z2-E004)
摘 要:目的:探讨外源性转入并上调表达抑制性信号蛋白Sm ad7对TGF-β1作用下大鼠腹膜间皮细胞Sm ad2表达的影响。方法:通过脂质体介导的方法,将表达Sm ad7的重组质粒(PCDNA3-Sm ad7)转染培养的大鼠腹膜间皮细胞,分别培养于不同浓度TGF-β1培养液(0、1.25、2.5和10μg/L),用RT-PCR及W estern b lotting的方法检测不同时间(0、5、15、30、60和120 m in)Sm ad2、Sm ad7表达的水平。结果:正常间皮细胞Sm ad2 mRNA和蛋白在TGF-β1刺激后5 m in开始表达,呈时间依赖性,在30 m in达到高峰,而后逐渐减弱;Sm ad7 mRNA和蛋白也在TGF-β1刺激后5 m in可见表达,但此后逐渐减弱,30 m in达到最低,60 m in又开始逐渐增强。Sm ad2和Sm ad7mRNA和蛋白,随TGF-β1浓度的增高而表达增强。转染后大鼠腹膜间皮细胞可见Sm ad7 mRNA和蛋白表达显著上调,并可持续高表达。转染后的腹膜间皮细胞在TGF-β1刺激下,Sm ad2 mRNA及蛋白的表达均低下,刺激0、5、15、30、60和120 m in后Sm ad2 mRNA表达分别低33%、56%、67%、71%、63%和57%(P<0.05)。Sm ad2蛋白表达分别低78%、89%、89%、88%和76%(P<0.05)。结论:上调表达抑制性信号蛋白Sm ad7可显著抑制腹膜间皮细胞中受体调控信号蛋白Sm ad2的表达和活性,提示Sm ad7可能对TGF-β1起反向调控作用。AIM: To investigate the role of Smad7 in the Smad2 expression induced by transforming growth factor-β1 (TGF-β1 ) in rat peritoneal mesothelial cells (PMCs). METHODS: Rat PMCs were cultured at different doses of TGF -β1 (0,1.25,2. 5,10 μg/L) for different time (0,5,15,30,60,120 min). PCDNA3 -Smad7 was then transfected into cultured rat PMCs by lipofectamine, and the cells were stimulated like the above. Endogenous Smad2 and Smad7 expression was evaluated by RT- PCR and Western blotting. RESULTS: TGF-β1 induced increase in Smad2 mRNA and protein expression at 5 min, peaked at 30 min, and declined to baseline levels at 120 min, which was in a time -dependent manner. TGF-β1 also induced Smad7 mRNA expression at 5 min, and then declined, down to the lowest at 30 min, but at 60 min it increased again. Smad2, Smad7 mRNA and protein expression induced by TGF-β1 were also dose - dependent. After transfection, overexpressions of Smad7 mRNA and protein in rat PMCs were observed, which did not decline with time. The expression of Smad2 mRNA significantly decreased by 33%, 56%, 67%, 71%, 63% and 57% (P 〈 0. 05) , the expression of Smad2 protein declined by 78% ,89% ,89% ,88% and 76% (P 〈0. 05) respectively at 0, 5, 15, 30, 60 and 120 min. CONCLUSION: Overexpression of Smad7 inhibits Smad2 gene and protein expression in peritoneal mesothelial cells. Smad7 may be a negative regulator of TGF -β1 signaling.
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