摘要
目的探讨转化生长因子β1（TGF—β1）小分子干扰RNA（siRNA）体外转染新生鼠肺成纤维细胞的干预效果，研究TGF—β1 siRNA的最佳干预剂量及维持时间。方法首先采用化学合成TGF—β1 siRNA对新生鼠肺成纤维细胞进行干预，通过实时定量PCR检测技术筛选出有效干扰片段；然后构建TGF-β1 siRNA腺病毒载体，体外转染新生鼠肺成纤维细胞，半定量PCR检测体外转染后TGF—β1基因的抑制效果，确定其最佳干预剂量及维持时间。结果（1）化学合成siRNA抑制TGF-β1基因表达的效果：siRNA剂量为20pmol，转染后24h检测抑制效果，第2、3、4条siRNA均有不同效果，其中第3条siRNA抑制效率〉70％；（2）TGF—β1 siRNA腺病毒载体转染细胞，观察20、50μl不同剂量转染后24h抑制情况，并与20μl阴性和空白对照比较，结果显示20、50μl均有明显抑制效果；观察转染后24、48、72h的抑制效果，比较显示转染后24h有明显抑制效果，72h抑制效果最强，说明随着转染时间延长，抑制作用越明显。结论TGF—β1 siRNA腺病毒载体体外转染新生鼠肺成纤维细胞对TGF-β1的表达均有明显抑制效果。
Objective To investigate the effect of transforming growth factor β1 （TGF-β1） small interfering RNA （siRNA） interference in transfecting neonatal rat＇s lung fibroblasts in vitro, and explore the best dose and maintenance time of TGF-β1 siRNA interference. Methods Neonatal rat＇ s lung fibroblasts cells were interfered with chemosynthesised TGF-β1 siRNA, effective siRNA was selected with real-time quantitative PCR, and then adenovirus vector for TGF-β1 siRNA was constructed to transfect neonatal rat＇ s lung fibroblasts. TGF-β1 mRNA expression was analyzed after transfection in vitro with real-time quantitative PCR to determine the best dose and maintenance time of TGF-β1siRNA interference. Results （ 1 ） After chemosynthesised siRNA interference for 24 h with 20 pmol, the second, third and forth siRNA all had different effect, and inhibition efficiency of the third siRNA was more than 70%. （2） TGF-β1 siRNA adenovirus vector was used to transfect neonatal rat＇ s lung fibroblasts with 20 μl and 50 μl for 24 h, and then the inhibition effect was observed and compared with negative control group with 20 μl and blank group. The inhibition effect was obvious at 20 μl and 50 μl . The inhibition effect was observed with 20 μl for 24, 48,72 h respectively, it showed that transfection for 24 h had strong inhibition effect, and inhibition effect of transfection increased more and more obviously for 48 h and 72 h. Conclusions The inhibition effect of TGF-β1 expression was obvious with TGF-β1 siRNA adenovirus vector in transfecting neonatal rat＇ s lung fibroblast cells in vitro.
出处
《中华生物医学工程杂志》
CAS
2008年第2期96-100,共5页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金（30571967）
关键词
RNA
小分子干扰
成纤维细胞
转化生长因子Β
转染
肺
RNA, small interfering
Fibroblasts
Transforming growth factor beta
Transfection
Lung
