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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 investigate whether recombinant adenovirus containing Egr1 gene promoter and Smad7 cDNA expressed Smad7 protein at cellular level, which blocks the biological activity of collagen synthesis by blocking the TGFβ1 signaling pathway. Methods The recombinant adenovirus was used to infect the fibroblasts (3T6). The expression of Smad7 protein in the cells was detected by immunocytochemistry after deep X-ray irradiation. After fibroblasts were stimulated by TGFβ1, 3H-thymidine (3H) -thymidine (3H) binding assay and 3H-proline (3HPro) binding assay were used to compare the cell proliferative ability and collagen synthesis in the experimental and control groups. Scintillation counting (cpm) values are compared quantitatively. Results Immunohistochemical results showed that Smad7 protein was localized in cytoplasm. Isotope 3HPro test results showed that the experimental group cpm value of 3287, the control group cpm value of 5690, the difference between the two was statistically significant (P = 0.026). The blank group cpm value of 3625, no difference with the experimental group (P = 0.741), indicating that experimental group fibroblasts collagen synthesis was significantly lower than the control group, and the basic level. 3HTdR test results showed no significant difference in cell proliferation between groups (P = 0.312). Conclusions Smad7 protein expressed in fibroblasts by recombinant adenovirus can inhibit the biological activity of collagen synthesis by blocking the TGFβ1 signaling pathway in the cytoplasm. Its inhibition of collagen synthesis may be achieved at the transcriptional level.