Establishment of TIMP1 low-expression model in mesangial cells

Objective Tocompoundthree different small interference RNAs specific for TIMP-1 and establish low-expression model of TIMP-1 in mesangial cells of rats, in order toprovide experimental basis for renal diseases. Methods Three different small interference RNA sequences that were specific for TIMP-1 were designedand compounded. The nonsense siRNA markedwith green fluorescent perssad(6-carboxy-fluorescein, FAM) was usedas control groupand the three siRNA groups were transfectedintoculturedmesangial cells in rats by liposome transfection method, then the transfectedcells were collectedat 48 hours after transfection toidentify the transfection efficiency with fluorescent microscope. At the same time, the total RNA in mesangial cells was extractedwith Trizol, the TIMP-1 mRNA expression level of all four groups was detectedby Taqman probe technique. Results The fluorescent microscope revealedthat the transfection efficiency was over than 90% in nonsense siRNA group, while the PCR results demonstratedthat TIMP-1 mRNA expression level in all three groups was lower than control group(P＜ 0.01), which suggestedthat our three siRNAs hadinhibitedthe expression of TIMP-1, and siTIMP1-1 hadthe best effect for it showedthe lowest expression level. Conclusion Our study has successfully establishedthe low-expression cell model of TIMP-1 in primary MCs with siRNA transfection technique.