摘要
目的探讨Peroxiredoxin-1(Prx-1)基因沉默对转化生长因子β1(TGF-β1)促进肺成纤维细胞合成Ⅰ和Ⅲ型胶原的影响及其作用机制。方法体外培养肺成纤维细胞分为4组:对照组(0.4%血清),TGF-β1组(5μg/L),TGF-β1+阴性转染组(5μg/L TGF-β1+阴性对照siRNA),TGF-β1+Prx-1siRNA转染组(5μg/L TGF-β1+Prx-1siRNA)。利用脂质体Lipo2000将阴性对照siRNA和Prx-1siRNA分别转染到TGF-β1+阴性转染组和TGF-β1+Prx-1siRNA转染组的肺成纤维细胞中,转染48h后用于后续实验。实时荧光定量PCR(RT-PCR)检测转染后各组的Prx-1mRNA表达水平,蛋白免疫印迹法(Western blot)检测磷酸化Akt(p-Akt)及Ⅰ和Ⅲ型胶原蛋白水平,2,7-二氯荧光素二乙酸检测活性氧(ROS)水平。结果 (1)Prx-1siRNA转染肺成纤维细胞后,TGF-β1+Prx-1siRNA转染组的Prx-1mRNA表达明显降低。(2)与对照组比较,TGF-β1组Ⅰ和Ⅲ型胶原、ROS及p-Akt蛋白水平均明显增加(0.34±0.06 vs.0.58±0.06、0.42±0.05 vs.0.56±0.06、2 988±379 vs.4 315±580和0.29±0.05 vs.0.66±0.07,差异有统计学意义(P<0.05)。与TGF-β1组比较,TGF-β1+阴性转染组Ⅰ和Ⅲ型胶原、ROS及pAkt水平无明显变化(P>0.05),但TGF-β1+Prx-1siRNA转染组Ⅰ和Ⅲ型胶原、ROS及p-Akt蛋白水平均进一步增高(0.58±0.06 vs.0.79±0.09、0.56±0.06 vs.0.77±0.08、4 315±580 vs.5 841±782和0.66±0.07 vs.0.93±0.15,差异有统计学意义(P<0.05)。结论 TGF-β1能够诱导肺成纤维细胞生成ROS,并由此促进Akt的激活和肺成纤维细胞合成Ⅰ和Ⅲ型胶原;而沉默Prx-1基因可激活ROS/Akt通路,从而有助于TGF-β1促进肺成纤维细胞合成胶原。
Objective To investigate the effect of silencing peroxiredoxin-i （Prx-1） on the expressions of collagen type Ⅰ and Ⅲ in TGF-β1-induced pulmonary fibroblasts and the possible mechanism. Methods Cultured pulmonary fibroblasts were ran- domly divided into four groups ： control group （0.4 % serum）, TGF-β1 groTap（5 μg/L）, TGF-131 -t- negative transfection group （TGF-β1＋ scramble siRNA） and TGF-β1 ＋ Prx-1 siRNA transfeetion group（TGF-β1＋ Prx-1 siRNA）. The negative control siRNA and siRNA Prx-1 were transfected into the lung {ibroblast cells transfected with TGF-β1 ＋ negative transfection group and TGF-β1 ＋ Prx-1 siRNA trans{ection group by liposome Lipo2000 respectively. After 48 h, the cells were was used for subsequent experi- ments. Real-time PCR was used to evaluate Prx-1 mRNA. Western blot was employed to detect the expressions of collagen type Ⅰ and Ⅲ ,phosphorylated Akt（p-Akt） ,and total Akt. Reactive oxygen species （ROS） were measured by DCFH-DA. Results （1）After transfection of siRNA Prx-1 into the lung {ibroblasts, Prx-1 mRNA expression was significantly decreased in the TGF-β1 ＋ Prx- 1 siRNA group. （2） Compared with control group,expressions of collagen type Ⅰ and Ⅲ ,ROS and p-Akt in TGF-β1 group were all increased0.34±0.06 vs. 0.58±0.06,0.42±0.05 vs. 0.56±0.06,2 988±379 vs. 4 315±580,0.29±0.05 vs. 0.66±0.07, （P〈0.01）. There were no differences in the levels of collagen, ROS and p-Akt between TGF-β1 group and TGF-β1 ＋ negative transfection group（P〈0. 05）. However,the levels of collagen type Ⅰ and Ⅲ ,ROS and p-Akt in TGF-β1＋Prx-1 siRNA transfection group were further higher than TGF-β1 group 0. 58±0.06 vs. 0. 79±0. 09,0. 56±0.06 vs. 0. 77±0. 08,4 315±580 vs. 5 841±782 and 0.66±0.07 vs. 0.93±0. 15, （P〈0.05）. Conclusion TGF-β1 induces pulmonary fibroblasts to generate ROS, which contributes to Akt activation and collagen type Ⅰ and Ⅲ synthesis;these changes become more obvious with the trea
出处
《重庆医学》
CAS
北大核心
2016年第29期4099-4102,共4页
Chongqing medicine
基金
国家自然科学基金资助项目(81072254)
唐山市科学技术研究与发展基金资助项目(14130275B)
