| Objectives In order to find a new therapeutic strategy for silicosis,the effects of BMSCsmi RNA-101 transplantation on silicosis in rats and its related mechanisms were observed by transfection of mi RNA-101-overexpressed BMSCs.Methods 1 Animal model preparation and grouping:BMSCs donor animals: 15 healthy male juvenile SD rats of(Specific pathogen free.SPF)grade without specific pathogen,aged about three weeks and weighing 90 to 110 g,they were cultured in vitro with BMSCs and passed on to the third generation for follow-up experiment.The stable BMSCs(BMSCsmi RNA-101)cells over expressing mi RNA-101 were constructed with lentiviral vector and cultured until the optimal transfection efficiency was obtained.BMSCs receptor animals: SPF grade healthy adult male SD rats were selected.Forty rats,weighing 210 to220 g,were randomly divided into 4 groups: control group,Si O2 model group,BMSCs treatment group and BMSCsmi RNA-101 treatment group,with 10 rats in each group.The rat model of silicosis was established by injecting sterilized sodium chloride and silica(Si O2)dust suspension(50.0g/L)into non-exposed tracheal intubation,and the control group was injected with the same amount of sterilized saline.Within 24 hours after the establishment of the model,BMSCs transfected with mi RNA-101 and DMEM suspension1.0ml(2×106/L),BMSCs and DMEM suspension 1.0ml(2×106/L)intervened.The rats were killed on day 3 day,7 day,14 day,28 day after the establishment of the model.2Detection index and method: Identification of phenotypic CD45,CD90 of BMSCs cells by flow cytometry.The transfection efficiency of over-expressed BMSCsmi RNA-101 cells was observed by fluorescence inverted microscope.The morphological changes of lung tissue in rats were observed by hematoxylin-eosin(HE)staining.The expression and distribution of TNF-? and IL-6 protein were detected by immunohistochemistry.The semiquantitative expression of TNF-?,IL-6,Beclin-1,LC-3,type I collagen and type ? collagen was detected by Western blot.The experimental data were built by Excel.Using SPSS19.0software was used to analyze the variance of the data,and the results were measured by(x ąs)showed that there was a significant difference between the two groups(P < 0.05).Results 1 The results of BMSCs culture showed that the primary BMSCs cells adhered gradually to the wall 24 hours after inoculation,and almost completely adhered to the wall within 48 hours.The refraction of the cells was strong,and the proliferation of the cells was mainly composed of dispersed clones.The third generation BMSCs microscopic observation was similar to fibroblast-like,with "fish tail stria" shaped colony distribution,clear nucleus and uniform cytoplasm.2 Flow cytometry(FCM)showed that CD90 was94.23%,CD45 was 6.95%.The BMSCs markers were almost non-expressed,which was consistent with the characteristics of BMSCs cells.3 The results of BMSCs and viral transfection efficiency: when transfected with MOI = 5pfu/cell,the cells continued to grow in a honeycomb-like manner,indicating that the transfection titer had little effect on the growth and proliferation of the cells.After 72 h of transfection,the cells were transfected with mi RNA-101 for 72h.The transfection efficiency was 97.36% under fluorescence microscope,and the transfection efficiency reached the peak.4 The morphological changes of lung tissue: In the control group,the alveolar structure was clear and normal,the alveolar wall was thin and the shape was good.In the Si O2 model group,the alveolar septum was significantly widened,the alveolar structure was destroyed and collapsed,and the alveolar epithelium was thickened.The pathological changes of silicosis model group in BMSCs treatment group were slightly improved,the alveolar structure was still destroyed seriously,and fibrocyte granuloma was gradually formed.In mi RNA-101 BMSCs treatment group,loose cellular nodules and congestion and edema of alveolar wall were occasionally seen in the silicosis model group.It was found that the infiltration of inflammatory cells decreased obviously.5 Immunohistochemistry: In the control group,the alveolar structure was clear and normal,the alveolar wall was thin and the shape was good.In the Si O2 model group,the alveolar septum was obviously widened and thickened,and fibrocyte granuloma,a large number of inflammatory cells infiltrated,pulmonary interstitial consolidation and congestion were observed in the Si O2 model group.In the silicosis model group,the degree of lesion was slightly improved,the alveolar structure was still seriously destroyed and gradually formed fibrocyte granuloma.In the mi RNA-101 BMSCs treatment group,loose cellular nodules were occasionally seen,while in the BMSCs treatment group,the degree of lesion was slightly improved,and the alveolar structure was still seriously destroyed.Pulmonary alveolar wall congestion and edema,found that inflammatory cell infiltration significantly reduced.6 The results of western blotting: In the control group,there were only a few basal expressions of TNF-?,IL-6,Beclin-1,LC-3,type I collagen and collagen type ?,and there was no significant difference at 3d,7d,14 d,28d.The expression of each protein in the Si O2 model group was significantly higher than that in the control group(P < 0.05).The expression of each protein in BMSCs treatment group was significantly lower than that in Si O2 model group at the same time point(P < 0.05).The expression of each protein in mi RNA-101 BMSCs treatment group was significantly lower than that in Si O2 model group at 3d,7d,14 d,28d(P< 0.05).The expression of each protein in mi RNA-101 BMSCs group was more obvious than that in BMSCs group at the corresponding time point(P < 0.05).The expression of each protein in Si O2 model group,BMSCs treatment group and mi RNA-101 BMSCs treatment group was similar in general,and all increased gradually with the prolongation of time 3 days later,reached a peak at 14 days,and began to decrease dynamically.Conclusions The therapeutic effects of lentivirus-transfected BMSCs transplantation with over-expression of mi RNA-101 on silicosis rats was significantly better than onlyf BMSCs transplantation.mi RNA-101 BMSCs could inhibit the excessive activation of autophagy in the lung tissue after dust exposure,and could significantly reduce the degree of chronic inflammation and fibrosis.Reduce collagen deposition,delay the pathological process of silicosis fibrosis,provide laboratory basis for the combination of silicosis stem cells and gene therapy,and open up a new strategy for silicosis therapy.Figure18;Table6;Reference 113... |
PDF Full Text Request