摘要
目的探讨RNA干扰阻滞胸腺素β4表达对TGF-β1诱导人肾小管上皮-肌成纤维细胞转分化(EMT)的抑制作用。方法构建能表达针对胸腺素β4的小干扰RNA(siRNA)的重组腺病毒载体,以及表达不针对任何已知mRNA的siRNA的阴性对照载体,分别感染人肾小管上皮细胞株(HKC),得到胸腺素β4表达受抑制的HKC-siTβ4和胸腺素β4表达未受影响的HKC-siC。根据用TGF-β1处理HKC、HKC-siTβ4和HKC-siC的情况,将培养的细胞分为4组:正常HKC组(C组),TGF-β1处理HKC组(T+C组),TGF-β1处理HKC-siTβ4组(T+siTβ4组)和TGF-β1处理HKC-siC组(T+siC组)。48h后,分别采用RT-PCR和Western blotting检测各组胸腺素β4、α-平滑肌肌动蛋白(α-SMA)和E-钙黏素表达,并分析胸腺素β4和α-SMA表达量的相关性。结果C组胸腺素β4表达弱阳性,α-SMA表达阴性,E-钙黏素表达强阳性;T+C组胸腺素β4和α-SMA表达强阳性,E-钙黏素表达较C组减弱(P<0.01);T+siTβ4组胸腺素β4和α-SMA表达弱阳性,表达量显著低于T+C组(P<0.01),E-钙黏素显著高于T+C组(P<0.01);T+siC组胸腺素β4、α-SMA和E-钙黏素表达与T+C组相比均无显著差异(P>0.05)。胸腺素β4和α-SMA表达呈显著正相关。结论RNAi阻滞胸腺素β4表达能有效地抑制TGF-β1诱导EMT,提示胸腺素β4是介导TGF-β1诱导EMT的重要分子。
Objective To investigate the inhibitory effects of RNA interference （RNAi） targeting thymosin β1 on human renal tubu lar epithelial-myofibroblast transdifferentiation （EMT） induced by transforming growth factor-β1 （TGF-β1） in vitro. Methods Recombi nant adenovirus packaging of vectors capable of producing small interfering RNA （siRNA） targeting thymosin β1 or producing siRNA that does not match any known human coding mRNA was designed, constructed, and infected into human renal tubular epithelial cells line （HKC）. Suppressed HKC-siTβ1 cells expressing thymosin β1 or uninfluenced HKC-siC cells expressing thymosin β1 were selected. Based on TGF-β1 treatment, cells cultured in vitro were divided into four groups： normal HKC （group C）, HKC treated with TGF-β1 （group T＋C）, HKC-siTβ1 treated with TGF-β1 （group T＋siTβ1 ）, and HKC sic treated with TGF -β1 （group T＋siC）. 48 hours after treatment, the levels of thymosin β1, alphwsmooth muscle actin （ccSMA） and E-cadherin expression were determined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. The relationship between the expressions of thymosin β1 and α-SMA was analyzed. Results The expression of thymosin β1 was weakly positive and that of α-SMA was negative, while that of E cadherin was strongly posi tive in group C. Both thymosin β1 and ccSMA expressions were strongly positive, and E cadherin expression was weaker in group T＋C than that in group C （P〈0.01）. Compared with group T＋C, both thymosin β1 and α-SMA expression were positive but significantly de creased, and E-cadherin expression was significantly enhanced in group T＋siTβ1 （P〈0.01）. No significant difference was found in thymo sin β1, α-SMA and E-cadherin expressions between group T＋siC and T＋C group （P〉0. 05）. There was a positive correlation between the thymosin β1 expression and α-SMA expression. Conclusions RNAi targeting thymosin β1 can efficiently inhibit EMT induced by TGF-β1, implying that thymosin β1 is essential to EMT induced by TGF-β1.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第9期1092-1095,共4页
Medical Journal of Chinese People's Liberation Army
基金
重庆市自然科学基金资助项目(2006135096)
