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目的探讨特异性小分子干扰RNA对晚期糖基化终产物受体(RAGE)介导肝星状细胞(HSC)激活和胶原生成的影响。方法构建RAGE特异性siRNA表达载体,经脂质体转染入HSC-T6细胞,以空白和转染非特异性siRNA表达载体p GCsi-C为对照.分别用实时荧光定量PCR和Western印迹法检测各组HSC-T6细胞RAGE、α平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原基因及蛋白的表达。结果与空白对照组和pGCsi-C组相比,转染特异性siRNA表达载体pGCsi-R1的HSC-T6细胞RAGE mRNA和相对分子质量50×10~3、46×10~3的RAGE蛋白表达分别下调79.65%、78.04%(F= 26.005,P<0.01,56.09%、54.93%(F=365.185,P<0.01)和63.67%、59.67%(F=386.07.P<0.01).同时α-SMA mRNA和蛋白、Ⅰ型胶原mRNA和蛋白的表达也受到明显抑制,分别为空白对照绀和pGCsi-C组的57.53%、65.50%(F=20.913,P<0.05)、58.18%、62.80%(F=56.592,P<0.05)、71.16%、74.23%(F=18.091,P<0.05)和71.91%、76.09%(F=17.299,P<0.05)。结论RAGE特异性siRNA可在细胞内稳定表达,并能有效抑制HSC的激活和Ⅰ型胶原生成。
Objective To investigate the effect of specific small interfering RNA on the activation of advanced hepatic stellate cells (HSC) and collagen production mediated by advanced glycation end-products receptor (RAGE). Methods The RAGE-specific siRNA expression vector was constructed and transfected into HSC-T6 cells by lipofectamine 2000. The blank and transfected non-specific siRNA expression vector p GCsi-C were used as controls.Results The expression of RAGE specific siRNA was detected by real-time fluorescence quantitative PCR and Western blot respectively The expression of RAGE, α-smooth muscle actin (α-SMA) and type Ⅰ collagen gene and protein in HSC-T6 cells were determined. Results Compared with the blank control group and pGCsi-C group, RAGE mRNA and RAGE protein of HSC-T6 cells transfected with pGCsi-R1 specific siRNA expression vector and RAGE protein with relative molecular mass of 50 × 10 ~ 3,46 × 10 ~ 3 (F = 36.005, P <0.01, 56.09%, 54.93% (F = 365.185, P <0.01) and 63.67%, 59.67% (P <0.05), 58.18% and 62.80% (F = 20.913, P <0.05) in the control group and the pGCsi-C group were significantly lower than those in the control group 56.592), 71.16%, 74.23% (F = 18.091, P <0.05) and 71.91%, 76.09% (F = 17.299, P <0.05) .Conclusion RAGE-specific siRNA can be stably expressed in cells Can effectively inhibit HSC activation and type Ⅰ collagen production.