Influence Of RNAi Silencing Smad3 On The Functions Of Keloid Fibroblasts

PART ONEOptimization of parameters in transfecting primary cultured keloid fibroblasts in vitro by electroporationObjective: Enhanced green fluorescent protein (pEGFP) gene was transfected into primary cultured keloid fibroblasts by means of electroporation in order to investigate the effect of parameters such as voltage, pulse length, the dose of DNA and observing the time-period after transfection.Methods: pEGFP -N1 was amplified in E. coli, and purified by high purity kit. Primary cultured human fibroblasts, which were initially obtained from the keloids, were cultured in vitro and transferred with pEGFP -N1 by means of electroporation with different voltages, pulse lengths and the doses of DNA. Transient expression of pEGFP -N1 and survival rate of cells were observed under fluorescence microscope. The transfer efficiency was evaluated by flow cytometry. Results: The transfer efficiency of EGPF and survival rate of cell were the highest after transferring under the electric conditions of 250v/cm, 1 pulse, 150μs and 6μg DNA. There was significant expression of EGFP at 24h after transfection. The transfection efficiency of pEGFP -N1 into primary cultured human fibroblasts reached 32.77% at 48h.Conclusion: A higher transfection efficiency of target genes can be obtained by means of electroporation with suitable electric conditions. We propose that electroporation is a useful method for transfecting target genes to primary cultured human keloid-derived fibroblasts.PART TWO The parametric optimization in transfecting fibroblast by the method mediated by liposomeObjective: To introduce the enhancement type green fluorescent protein gene eukaryotic expression vector pEGFP-N1 plasmid to the primary cultural human being keloid-derived fibroblasts by the method mediated by liposome to probe the influencing parameters of exogenous gene transfection efficiency, such as DNA dose, liposome concentration, transfection duration and so on.Methods: To amplify plasmid pEGFP-N1 in colibacilli and choose different DNA dose, liposome concentration and transfection duration. Then plasmid pEGFP-N1 was transfected to the primary cultural human keloid-derived fibroblasts by liposome. Transient expression of pEGFP-N1 and cell survival condition were observed at the different time-periods after transfection.Results: pEGFP -N1 plasmid DNA was added to liposome in the proportion of 1:1 when Bottle Shop cells was covered to 70%-80% and cell population was about 1-2x109cells/L. Fibroblast was transfected for 4 hours without blood serum. The transfection was obvious 24 hours after transfection and to the most (38.61%) 48 hours after transfection and the cytoactivity was rather high.Conclusion: On the condition of proper liposome concentration, fairly short transfection duration, the primary cultural human keloid-derived fibroblast transfected by the method mediated by liposome can make destination gene obtain higher transfection efficiency and the least cell death. So the method mediated by liposome is a fairly ideal transfection way to research biological behaviour of the primary cultural human keloid-derived fibroblasts. PART THREEThe influence of SiRNA-Smad3 on smad3 gene expression of keloid fibroblastObjective: To probe the influence of SiRNA-Smad3 on smad3 gene expression of keloid fibroblast.Methods: Three pairs of chemically synthetical SiRNA-Smad3 (Y1,Y2,Y3)were adoped to transfect the cultured human keloid fibroblast (KFB)separately by the interfere technology of micromolecule RNA and carrier of positive ion liposome. At the same time, KFBs without transfection of SiRNA-Smad3 were used as control, so as to interfere the expression of Smad3 and to impede signal transduction of endogenous TGF-β/Smads; The effectiveness that SiRNA-Smad3 interfered with expression of Smad3 was detected by the way of RT-polymerase chain reaction (RT-PCR) and Western Blotting; The change condition of Dmad3's expression position in KFB before and after Smad3's interference was examined by immunofluorescence chemical method.Results: RNAi technology could effectively interfere with expression of Smad3MRNA and protein in KFB. The expression of Smad3MRNA determined by RT-PCR was weakest in the group of Y1. The result was coincident by Western Blot and RT-PCR. The interference of SiRNA-Smad3 could decrease the expression of Smad3 in cytoplasm and reduce its transfer from cytoplasm to cell nucleus obviously.Conclusion: SiRNA-Smad3 transfecting human keloid fibroblasts can restrain expression of Smad3MRNA and protein specifically and decrease the quantity of expression of Smad3 in cytoplasm and reduce its transfer from cytoplasm to cell nucleus obviously.PART FOURThe influence of transforming growth factor (TGF)-β/Smads signal on proliferation and apoptosis of keloid fibroblastsObjective: To research the regulating effectiveness of (TGF)-β/Smads signal to proliferation and apoptosis of keloid fibroblasts (KFB). Methods: SiRNA-Smad3 was used to transfect the primary cultured human keloid fibroblasts by liposome method. The proliferative condition of KFB was detected by 4 methyl thiazolyl tetrazolium method on condition that endogenous TGF-β/Smads signal transduction has been impeded and KFB has been stimulated by exogenous TGF-β1. The cell cycle and apoptosis condition in two conditions were determined by flow cytometry. Results: Not only has proliferation of KFB been restrained but also it could induce apoptosis if endogenous TGF-β/Smads signal transduction has been hindered. Exogenous TGF-β1 in higher Concentration could enhence proliferation of KFB but could not induce apoptosis.Conclusion: TGF-β/Smads signal can regulate the proliferation and apoptosis of KFB at the certain concentration range.PART FIVEThe influence of TGF-β/Smads signal on expression of MMP-3 in the human keloid fibroblastObjective: To study the regulating effectiveness of (TGF)-β/Smads signal on expression of MMP-3 in the human keloid fibroblasts (KFB). Methods: SiRNA-Smad3 was used to transfect the primary cultured human keloid fibroblasts by liposome method. Expression condition of MMP-3 in KFB was detected by immunohistochemistry on condition that endogenous TGF-β/Smads signal transduction has been impeded and KFB has been stimulated by exogenous TGF-β.Results: The expression of MMP-3 in keloid fibroblast was strengthened when endogenous TGF-β/Smads signal transduction has been hindered. The exogenous TGF-β1 higher concentration could restrain expression of MMP-3 in keloid fibroblasts.Conclusion: Smad3 can restrain expression of MMP-3 in keloid fibroblasts by mediating TGF-β1 signal.