Kage Hidenori, Sugimoto Keiki, Sano Atsushi, Kitagawa Hiroshi, Nagase Takahide, Ohishi Nobuya, Takai Daiya
Department of Respiratory Medicine, University of Tokyo, Tokyo, Japan.
Exp Lung Res. 2011 Apr;37(3):175-85. doi: 10.3109/01902148.2010.529985. Epub 2011 Jan 26.
Since the discovery of RNA interference, short interfering RNA (siRNA) has become a standard research tool. However, expression of siRNA in lung alveolar epithelial cells has remained a problem. Adeno-associated virus (AAV) vectors are known to have low toxicity, and AAV type 5 vectors transduce these cells efficiently. In this study, LacZ expression was higher using AAV2/5-LacZ and LA-4 cells compared with transfection of plasmid or transduction to 3T12-3 cells. The authors designed 10 different siRNAs against mouse transforming growth factor β1 (Tgfβ1), selected one with the highest knockdown efficiency, and transduced the AAV vectors carrying the short hairpin RNA (shRNA) to target cells. The AAV vectors transduced LA-4 cells 50 times more efficiently than 3T12-3 cells, and suppression of Tgfβ1 protein expression was similar, at approximately 50%. Knockdown of mRNA was only seen in LA-4 cells. Inhibition of Tgfβ1 resulted in higher number of LA-4 cells, lower number of 3T12-3 cells, and decreased procollagen expression in LA-4 cells. Higher transduction was seen in H23 cells than in H1975 cells, and low transduction was seen MH-S cells. This study shows that AAV2/5 can be used to carry shRNA and suppress gene function in lung alveolar epithelium-derived cells.
自RNA干扰被发现以来，小干扰RNA（siRNA）已成为一种标准的研究工具。然而，siRNA在肺泡上皮细胞中的表达一直是个问题。已知腺相关病毒（AAV）载体毒性低，且5型AAV载体能有效地转导这些细胞。在本研究中，与质粒转染或转导至3T12 - 3细胞相比，使用AAV2/5 - LacZ和LA - 4细胞时LacZ表达更高。作者设计了10种针对小鼠转化生长因子β1（Tgfβ1）的不同siRNA，选择了一种敲低效率最高的，并将携带短发夹RNA（shRNA）的AAV载体转导至靶细胞。AAV载体转导LA - 4细胞的效率比3T12 - 3细胞高50倍，Tgfβ1蛋白表达的抑制情况相似，约为50%。mRNA的敲低仅在LA - 4细胞中可见。Tgfβ1的抑制导致LA - 4细胞数量增加，3T12 - 3细胞数量减少，且LA - 4细胞中前胶原表达降低。H23细胞中的转导效率高于H1975细胞，而MH - S细胞中的转导效率较低。本研究表明，AAV2/5可用于携带shRNA并抑制肺泡上皮来源细胞中的基因功能。
