摘要
目的研究含Egr1基因启动子和Smad7cDNA的重组腺病毒在细胞水平表达Smad7蛋白,是否具有阻断转化生长因子β1(TGFβ1)信号传导通路从而阻断胶原合成的生物学活性。方法重组腺病毒感染成纤维细胞(3T6),经深部X线照射后通过免疫细胞化学方法检测Smad7蛋白在细胞内的表达定位。成纤维细胞再经TGFβ1刺激后通过3H胸腺嘧啶核苷(3HTdR)和3H脯氨酸(3HPro)结合法检测比较实验组和对照组细胞增殖能力和胶原合成情况,采用液体闪烁计数法测定每分钟闪烁计数(cpm)值进行定量比较。结果细胞免疫化学结果显示Smad7蛋白表达定位于细胞浆内。同位素3HPro检测结果显示实验组cpm值为3287,对照组cpm值为5690,两者差异有统计学意义(P=0.026)。空白组cpm值为3625,与实验组无差别(P=0.741),说明实验组成纤维细胞胶原合成量明显低于对照组,与基础水平相当。3HTdR检测结果显示各组间细胞增殖能力无明显差别(P=0.312)。结论通过重组腺病毒在成纤维细胞内表达的Smad7蛋白具有在细胞浆内阻断TGFβ1信号传导通路从而抑制胶原合成的生物学活性,其抑制胶原合成可能是在转录水平实现的。
Objective To study whether the expression Smad 7 protein by the recombinant adenovirus with Egr-1 promoter and Smad 7 cDNA in fibroblast cell can block the signal transduction pathway of transforming growth factor-beta1 (TGF-β1) under irradiation thereby inhibiting collagen synthesis in vitro. Methods The location of endogenous Smad 7 and exogenous Smad 7 protein in recombinant adenovirus infected fibroblast cells(3T6) were determined by immunocytochemical method. The infected 3T6 cells were irradiated and then cultured with TGF-β1 4 hours after irradiation. The activity of preventing radiation-induced fibrosis by expression Smad 7 protein was evaluated by the amount of collagen synthesis and proliferation of 3T6 cells. The amount of collagen synthesis was shown by the coruscant per minute (cmp) through the 3?H-Proline incorporation technique. Results The endogenous Smad 7 and exogenous Smad 7 protein both were located in the cytoplasm. When cultured with TGF-β1 4 hours after irradiation, the amount of collagen synthesis in the 3T6 cells infected with the recombinant adenovirus was significantly less than that in the cells without infecting adenovirus after irradiation(P=0.001), But, there was no difference in the proliferation of 3T6 cells between those with and without adenovirus infection (P= 0.312 ). Conclusions The Egr-1 promoter in the recombinant adenovirus can regulate the expression of downstream Smad 7 cDNA in 3T6 cells. The expression Smad 7 protein could block the TGF-β1 signal transduction pathway thereby inhibiting the collagen synthesis. The mechanism of inhibiting the collagen synthesis may be accomplished at the transcription level.
出处
《中华放射肿瘤学杂志》
CSCD
北大核心
2005年第4期342-346,共5页
Chinese Journal of Radiation Oncology
基金
国家自然科学基金资助项目(30170289)
