Objectives Small interfering RNA (siRNA) methodology suppresses specific gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. siRNA technology is also a promising tool for gene therapy of various diseases. Lipofectamine RNAiMAX, a new transfection reagent, has been confirmed high efficiency in delivering siRNA into mesenchymal stem cells and coronary vascular smooth muscle cells. Cardiac fibroblasts (CFs) are the most prevalent cell type in the heart and play a key role in regulating normalmyocardial function and in the adverse myocardial remodelling that occurs with hypertension, myocardialinfarction and heart failure. Myocardial fibrosis refers to excessive accumulation of collagen fibres in the myocardial extracellular matrix, apparent elevated collagen content, or changes in collagen composition. Its main feature is the proliferation of CFs and the secretion of large amounts of collagen. However, it is still unclear that siRNA could high efficiency delivering into CFs by Lipofectamine RNAiMAX. Therefore, this study was performed to investigate siRNA transfection in cultured CFs by Lipofectamine RNAiMAX.
Methods CFs cultured in 24-well format. 24 hours later, cells were transfected with positive control siRNA (green fluorescent dye-labelled siRNA) using Lipofectamine RNAiMax. The transfection efficiency, fluorescence intensity and intracellular localization of fluorescent siRNA were monitored in live cells using fluorescence microscopy at different time-points after transfection. The transfection efficiency was calculated by counting the percentage of green fluorescent protein expressing cells in the total cells.
Results Fluorescence expression analysis, cell shape and counts were obtained by fluorescence inverted microscopy in bright field and the corresponding green fluorescent dark field. The morphology of CFs was flat, spindle-shaped cell with multiple processes emanating from the main cell body. It was showed that green fluorescent protein could clearly observe in the cytoplasm and nuclei of CFs at 6 h, 12 h, 24 h, 48 h and 72 h after transfection. These results suggested that positive control siRNA (green fluorescent dye-labelled siRNA) could successfully transfect into CFs. The cell number was counted at 12 h, 24 h, 48 h and 72 h after transfection, respectively. Total number of cells reduced gradually, but transfection efficiency was not obviously reduced. The average of siRNA transfection efficiency was 93.68%.
Conclusions The present study suggested that siRNA could be efficiently transfect into cultured CFs by Lipofectamine RNAiMAX.
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