| Objects: an in vitro model of RNAi the expression of leptin on cultured human hypertrophic scar fibroblasts TGF-beta 1 expression,cell proliferation and type I collagen synthesis,aimed at the prevention effect of down-regulation of leptin on pathological scar.Methods:(1)Primary cell culture: under sterile conditions,the surgical excision of hypertrophic scar and keloid tissue containing penicillin and streptomycin into DMEM medium rinse,fibroblasts cultured by digestive method,3--8 cells for experimental study.(2)The CCK8 cell proliferation assay: cells in the logarithmic growth phase cells transfected with reference to transfection reagent instructions,will be placed in the incubator culture plates incubated with the corresponding time(0,24,48 hours);adding 10 ul CCK-8 solution into each hole of the culture plate in 4 hours incubation incubator incubation,using enzyme marker measured at 450 nm.(3)The Real Time PCR test mRNA cell: cells in the logarithmic growth phase of cells transfected with transfection reagent according to instructions,48 hours after transfection,Trizol method to extract total RNA for the synthesis of cDNA reverse transcription kit according to the instructions,the synthesis of cDNA after PCR amplification,influence of siRNA on detection of leptin TGF-beta 1,collagen type I mRNA.(4)The expression of the Western blot determination of leptin,TGF-beta 1,collagen type I.(5)By using the SPSS17.0 statistical software package,numerical values are mean standard deviation((?) ± S)said,with t test count data between groups.Results:(1)Real Time PCR results: after gene transfection,the expression of leptin mRNA in hypertrophic scar transfected group and negative control group were 0.72 ± 0.17,1.01 ± 0.16,leptin relative expression of mRNA in transfected group than negative control group decreased significantly(P < 0.05),the expression of leptin mRNA in keloid scar transfected group and negative control group were 0.48 ± 0.15,1.01± 0.21,leptin relative expression of mRNA in transfected group than negative control group decreased significantly(P < 0.05);the expression of TGF beta 1 mRNA in hypertrophic scar transfected group and negative control group were 0.64 ± 0.07,1.01 ± 0.19,transfected group TGF beta 1 mRNA relative expression compared to negative control group decreased significantly(P < 0.05),the expression of TGF beta 1 mRNA in keloid transfected group and negative control group were 0.63 ± 0.17,1.02 ± 0.29,transfected group TGF beta 1 mRNA relative expression compared to negative control group decreased significantly(P < 0.05);the expression of type I collagen mRNA in hypertrophic scar transfected group and negative control group were 0.72 ± 0.15,1.00 ± 0.11,transfected collagen type I mRNA relative expression compared to negative control group decreased significantly(P < 0.05),the expression of type I collagen mRNA in keloid scar transfected group and negative control group were 0.57 ± 0.09,1.02 ± 0.29,transfected collagen type I mRNA relative expression compared to negative control group decreased significantly(P < 0.05).(2)Western blot results: after gene transfection,the protein gray value ratio of leptin,TGF-beta1,type I collagen and GAPDH in hypertrophic scar were less than those of the negative control group,the difference was statistically significant(P < 0.01);after gene transfection,the protein gray value ratio of leptin,TGF-beta1,type I collagen and GAPDH in keloid scar were less than those of the negative control group,the difference was statistically significant(P < 0.01).Conclusion: hypertrophic scar and keloid fibroblasts can be used as the target cells of leptin siRNA transfection,and the leptin gene may be an important gene for the formation of pathological scar. |
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