| 摘要: |
| 目的：筛选能高效干扰大鼠Toll样受体4（Toll-like receptor 4，TLR4）mRNA的最佳小分子干扰RNA（small interfering RNA,siRNA）序列。方法：克隆大鼠TLR4基因全长，将TLR4基因与含增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）的质粒pEGFP-C1重组，构建pEGFP-rTLR4，化学合成法合成3对干扰大鼠TLR4的siRNA后，将3对siRNA、阴性对照siRNA和干扰EGFP的siRNA分别与pEGFP-rTLR4经Lipofectamine2000共转染HEK-293细胞株，通过倒置相差显微镜和流式细胞仪观察EGFP的荧光强度。结果：与阴性对照组相比，3对针对TLR4的siRNA及针对EGFP的siRNA均明显抑制EGFP的荧光表达(P<0.05)。其中尤以siRNA2(核苷酸序列为5′-GTC TCA GAT ATC TAG ATC T-3′，位于TLR4基因序列的1 352～1 370位)的抑制效果最强，干扰效率>75%。结论：成功筛选出体外可高效干扰大鼠TLR4 mRNA的siRNA片段。 |
| 关键词: 小分子干扰RNA Toll样受体4 PCR 化学合成法 |
| DOI：10.3724/SP.J.1008.2008.00048 |
| 投稿时间：2007-09-05 |
| 基金项目: |
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| Screening for siRNA sequence targeting rat Toll-like receptor 4 mRNA in vitro |
| BIAN Jin-jun1, ZHU Ke-ming1, WAN Xiao-jian1, DENG Xiao-ming1*，HUANG Sheng-dong2,YUAN Yang2, GONG De-jun2 |
| (1.Department of Anesthesiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; 2.Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433) |
| Abstract: |
| Objective：To screen for an optimized siRNA sequence targeting rat Toll-like receptor 4 (TLR4) in vitro. Methods: The full length gene of rat TLR4 was cloned and inserted into pEGFP-C1 plasmid to construct pEGFP-rTLR4. Three pairs of siRNAs targeting rTLR4 were chemically synthesized and were co-transfected with pEGFP-rTLR4 into HEK-293 cells via Lipofectamine2000. Cells were also co-transfected with siRNA targeting EGFP and negative control siRNA. The expression of EGFP was observed under inverted fluorescene microscope and flow cytometry. Results: Compared with the negative control group, 3 pairs of siRNAs targeting TLR4 and one pair of siRNA targeting EGFP significantly suppressed the EGFP expression (P<0.05); the inhibitory effect of siRNA2（gene sequence:5′-GTC TCA GAT ATC TAG ATC T-3′,19 bp, 1 352-1 370）was the strongest one, with an interference efficiency over 75%.Conclusion: We have successfully obtained the siRNA sequence targeting TLR4 mRNA, which can efficiently suppress the expression of rat TLR4 mRNA in vitro. |
| Key words: small interfering RNA Toll like receptor 4 PCR chemical synthesis |
