Distribution Of Bone Marrow Mesenchyal Stem Cells In Rats Treated By Silica And The Antagonism Of The Cells On Pulmonary Fibrosis

Silicosis is a diffuse pulmonary fibrosis of systemic disease caused by long-term inhale of free silicon dioxide,which is pathologically characterized by diffuse pulmonary fibrosis.Silicosis is the most serious occupational disease for laborers,which has the highest incidence rates,and there is no effective treatment now.Bone marrow mesenchymal stem cells(BMSCs)as an ideal target for cell-and gene-therapy,have most possible application foreground and provide a new train of thought for the treatment of silicosis.In this research,technologies including near-infrared(NIR)fluorescence imaging system,Micro CT for lung tissue scanning in vivo,pathological section staining technology were used,and the silicosis model was established through tracheal instillation of silica in rats in order to investigate the distribution regularities of the cells in rats and the inhibitory effect of BMSCs intervention on rat fibrosis.The research would provide innovative way for the further exploration of effective way silicosis treatment and improvement of patients' life quality.This study aimed to:(1)investigate the distribution regularities of different time points and homing of BMSCs in rats and provide experimental basis for the dosing interval of subsequent experimental secondary injection;(2)explore the assessment of antagonistic effect of bone marrow mesenchymal stem cells on pulmonary fibrosis in silicosis rat model by Micro CT.Part I Objective: Acorrding to rat bone marrow adherence method,the rat BMSCs were isolated,cultured and amplified.Methods: BMSCs from rats were isolated,cultured and purified by the whole bone marrow adherence method.Bone marrow cells suspension fluid were washed out and were moved in a petri dish.Isolated cells cultivated were grown at 37? under 5% CO2.Suspension cells were removed by changing the culture medium.Start to passage when cells grow to the bottom of the area of 80% to 90%.BMSCs at passage 3 were used for cell morphological observation,identification of the surface makers and the capacity of multi-directional differentiation.Result:(1)The cells grew rapidly after subculturing of original generation cultured BMSCs,which were a class of fusiform or spindle-shape cells and showed typical swirl morphology;(2)Cells expressed CD90+(97.1%),CD44+(99.1%),CD45-(2.1%),CD11b-(4.5%)by flow cytometer and these features conformed to the characteristics of cell surface markers.(3)After a period of time of adipocyte inducing,the BMSCs showed accumulation of lipid droplets which were red under oil red O staining and the shape of lipid droplets were like bead.Mineralized nodules were orange under alizarin red staining.Part II Objective: To elucidate whether the images that distribution and lung tissue tropism of intravenously injected BMSCs in rats were observed according to NIR imaging system and provide experimental basis for the time point of subsequent experimental secondary injection Methods: A total of 72 adult female rats(SPF species,200-240g)were randomly classified into four experimental groups(n=18):(1)control group instilled saline and the tail vein injected saline;(2)BMSCs group instilled saline and the tail vein injected BMSCs;(3)silica+BMSCs group instilled silica and the tail vein injected BMSCs;(4)silica+DiR group instilled saline and the tail vein injected DiR dyes diluents.In control and BMSCs groups,each rat received 1 ml of saline(0.9%)by intratracheal administration.In intervention and DiR groups,each rat was intratracheally given 1 ml(50mg/ml)of silica suspension(containing 5000 U penicillin).Passage 3 to 5 of cells derived from the thighbone of male rats and the cells were stained by DiR for the tail intravenous injection.24 h after intratracheal instillation,each rat in the silica+DiR group was injected DiR suspended in saline at a concentration of 5?g /ml by caudal vein.And each rat in BMSCs and silica+BMSCs groups was injected BMSCs suspended in saline at a concentration of 2×106/ml by caudal vein.The 1 ml of saline was also injected by caudal vein to the rats in the control group to match the other groups.To evaluate the duration and distribution of the signal intensity of the injected BMSCs in vivo,BMSCs were injected into the caudal vein and detected at 1h,6h,24 h,3d,15 d,30d,until there was no detectable signal.The organs(lung,liver,spleen,heart,double kidney)were removed and placed in a petri dish after obtaining the images.And then NIR fluorescence measurements were taken at 1h,6h,24 h,3d,15 d,30d for the detection of the duration and distribution of the injected BMSCs in the organs.Finally the results were verified by PCR and immunofluorescence.Result:(1)The DiR dye had no effect on survival and proliferation of BMSCs.(2)NIR fluorescent imaging showed the tissue distribution regularities of systemically injected BMSCs-DiR and the third day after the first tail vein injection was determined as the second administration time.(3)BMSCs can home to silica-injured lung tissue in pulmonary fibrosis rats.Part III Objective: To explore Micro CT is able to evaluate pulmonary fibrosis and reflect antagonistic effect of BMSCs in silicosis rat model.Methods: Sixty Wistar female rats were randomly divided into three groups,n=20.Silica group and silica+BMSCs group were intratracheally given silica suspension(50mg/ml).After silica instillation,silica+BMSCs group were injected BMSCs by caudal vein.Other groups were injected saline.Respectively,rat lung received computed tomography(CT)scan after silica instillation.The rats' lungs were fixed and HE stained for pathology observation after rats were sacrificed.Result:(1)Compared with the control group,at 7,15,30 day,silica group show that inflammatory cell infiltration,scattered silicon nodules and enlarged silicon nodules respectively by pathological section.And shadow of heighten patchy density in Pulmonary hilar,round or round cell nodules with high density and high density patching shadows were observed by CT at 7,15,30 day.(2)Compared with the silica group,silica+BMSCs group reveal that the inflammatory exudates decreased pathologically at the 7 day after silica instillation,but these results can't be detected on CT.At the 15,30 day,pathologically,silicon nodules decreased and CT indicate that high density area was smaller.Conclusions:(1)BMSCs were successfully isolated,identified,purified and expanded by establishing BMSCs whole bone marrow adherent culture method.;(2)NIR fluorescence imaging technique provides significant advantages to visualize cells distribution.It was found that BMSCs could specifically migrate to silica-injured lung tissue,and the third day after the first tail vein injection as the second administration time;(3)BMSCs could antagonize the silicosis in rats.Micro CT can evaluate the degree of silicosis induced by silica and reflect the antagonistic effect of BMSCs on pulmonary fibrosis.