| Objectives Vitro silicosis model was established on MRC-5 that were stimulated by supernatant medium of AM from patients with silicosis.The IGFBP5 gene in MRC-5 was silenced by RNA interference.The expression and content of collagen I and III in MRC-5cells were detected by ICC and ELISA.To explore the relationship between IGFBP5 gene and silicosis.Methods 1 To establishment the vitro silicosis model:AM from bronchoalveolar lavage fluid of stage II silicosis patients were obtained from Emergency management department pneumoconiosis rehabilitation center.The supernatant obtained by stimulating alveolar macrophages with SiO2(50 g/ ml)after 18 h in vitro culture.The vitro silicosis model was established by co-incubation of supernatant and MRC-5.2 IGFBP5 interference vector construction and the selection of the best interference vector:IGFBP5 interference vector and negative vector were constructed by Shanghai Gemma.Interference vectors and negative vector were transfected into MRC-5.Use RT-PCR and and Western blot to screen the highest interference vector after 48 h culture.3 The experimental groups:control group(C),stimulation group(S),transfection group(T)and negative transfection group(N).4Expression and content of collagen I and III in MRC-5:The expression and content of collagen I and III were detected by ICC and ELISA after MRC-5 culture for 12 h,24h,36 h,48h and 60 h in each group.Results 1 Large number of activated AM were obtained from the lavage solution.The supernatant was obtained after the secondary stimulation of silica dust.2 The supernatant can effectively stimulate the MRC-5 cells to produce collagen I and III.3 Using the confocal to observe vector:LipofectamineTM2000=2ug:3ul was The highest efficiency of cell transfection l after interference plasmid.4 IGFBP5 mRNA and protein expression were detected by RT-PCR and Western blot.All the four plasmids can silence IGFBP5 gene.The expression of IGFBP5 mRNA and protein in MRC-5 cells transfected with interfering plasmids were lower than those in the group(C).The expression of protein in the four groups was statistically significant compared with that in the group(N)and the group(C),Rt-pcr method(F=156.478,P<0.05),western-blot method(F=967.144,P<0.05),Vector A had the most significant effect,and the protein of IGFBP5 had the best effect.5 The expression and content of collagen I and III in MRC-5 by ICC and ELISA.The expression and content of collagen I and III in MRC-5 in group(C)were the lowest at the same time point.The expression and content of collagen I and III in MRC-5 were the highest in group(S)and group(N),and there was no difference between the two groups(P > 0.05).The expression and content of collagen I and III in MRC-5 in group(T)were significantly lower than those in group(S)and group(N),and higher than those in group(C),with statistically significant difference in pantwise comparison(P<0.05).The expression and content of collagen I and III in MRC-5 at 12 h,24h,36 h,48h and 60 h were compared at different time points in the same group.The expression and content of collagen I reached the peak at 36 h and that of collagen III reached the peak at 48 h.Conclusions Transfection of IGFBP5-siRNA into MRC-5 cells using RNA interference technology can effectively inhibit the expression of IGFBP5 gene.,The expression of collagen I and III in MRC-5 cells stimulated by AM supernatant were decreased after silencing the up-regulated gene IGFBP5.Figure10;Table7;Reference 77... |
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