The Study Of DcR3 Interfered Pulmonary Fibrosis Animal Model And Transfected Human Fibroblast Cells

Generally pulmonary fibrosis is the disease that toxicology,spontaneous immune response,drug side effects,infection,severe trauma,such as the stimulation of different factors cause lung inflammation,alveolar injury sustained,repeated extracellular matrix damage,repair,reconstruction and the excessive deposition,resulting in changes to the structure of normal lung tissue, loss of function.Pulmonary fibrosis in interstitial lung disease are all the end-result,interstitial lung disease and respiratory diseases at one of the disease,so the occurrence of pulmonary fibrosis,the development of relations between the categories of the disease are reviewed.The incidence of pulmonary fibrosis rate had an upward trend year after year,so far no effective drug to reverse the natural process of pulmonary fibrosis and its end result.Usually the occurrence of pulmonary fibrosis process is divided into two phases,the initial stage alveolitis:pathogenic factors cause epithelial injury, subepithelial basement membrane damage,inflammatory cells,immune cell activation,secretion of various inflammatory factors;pulmonary fibrosis phases: it is the role of inflammatory cytokines from first phase that the fibroblast rais,differentiate,proliferate,collagen and matrix hyperplasia and pulmonary fibrosis happened.This study will adopt the model of pulmonary fibrosis in rats to observed at different stages of inflammation in rat model of OcR3 on the extent of the impact of pulmonary fibrosis,explore the effect of DcR3 on alveolar macrophages,peripheral blood lymphocytes and human fibroblasts cells,which revealed the role of DcR3 in the pathogenesis of interstitial lung disease,in order to lay the foundation for the treatment of pulmonary fibrosis.First experimens:we solute bleomycin(5mg/kg)to copy pulmonary fibrosis animal model.Division of laboratory animals:normal control group(C group): from modeling to give intravenous normal saline 1ml every day;bleomycin group (BL group):from model-making give the tail vein injection 1ml saline every day; bleomycin+ DcR3 group(BL+DcR3 group):Since the beginning the model were given DcR3 protein(3.33μg/kg) 1mlin tail vein injection,one time/day,2 weeks.Each group separately at 2W,4W,6W,8W rats were killed 5,the collection of BALF cells for detection expression of TNF-α,TGF-βmRNA by RT-PCR and cell count tests; Detect supernatant lavage fluid collection TNF-α,TGF-βcontent;frozen left the organization to detect hydroxyproline;right lung were submerged in 10% paraformaldehyde solution,paraffin-embedded,stained sections in order to organize staining.The results showed:normal control rats lively active and healthy;lung tissue at each time point in lung tissue were pink,smooth and uniform surface without hemorrhage edema;light microscope clearly shows that the structure of rat lung, the observation points were not significant change,Masson staining of collagen deposition not green;the extent of alveolitis and pulmonary fibrosis at various time points no significant change;BALF cells in the total number of fluctuations at 6-7×10~6/ml;there is micro-TNF-α,TGF-βmRNA expression and micro-hydroxyproline.Bleomycin rats slow motion,shortness of breath,eat less, make them apathetic,curled,decreased body weight,dark matte fur,high mortality; lung tissue of two weeks show significant congestion and edema,bleeding points, 4 week shows that the lung surface smooth,pale,small nodules and lung hardness increase can be seen,6 weeks shows gray or brown dense fibrous tissue surrounding lungs.Histopathology shows alveolar septal thickening,accompanied by macrophages,a small amount of neutrophils and other inflammatory cell infiltration at 2 weeks;shows part alveolar structure of damage,a significant thickening of alveolar walls,infiltration of the alveolar cavity extract,an increase in fibroblasts on weeks and a large number green collagen can be seen within the alveolar septal by Masson staining;6 weeks marked alveolar structural damage,alveolar wall thickening,an increasing number of fibroblasts,pulmonary fibrosis scar formation,capillary cavity obliteration and a large number green collagen deposition can be seen within the alveolar septalby Masson staining; a large number green collagen deposition can be seen at8 weeks by Masson staining. Pathological assessment:BL group showed that the extent of alveolitis at 2W demonstrated at the most,then gradually reduced,compared with the control group were significantly different(p＜0.05);BL group gradually increase the degree of pulmonary fibrosis,at each time point with the control group compared with significant difference(p＜0.05).BL group the total number of cells in BALF in general higher than control groups,especially at 2-4W there was a significant difference(p＜0.01),6-8W compared with the control group no significant difference(p＞0.05).BL groups the expression of TNF-α,TGF-βmRNA was significantly increased,compared with the control group were significantly different(p＜0.01).BL group gradually increased content of hydroxyproline,6W to peaked,at different time points compared with the control group were significantly different(p＜0.05).BL+DcR3 groups:bleomycin injected rats with DcR3 increased activity,eating restore,restore breathing,body weight gradually increase,after 4 weeks no significant difference between the control group.Lung tissue can be seen at 2 weeks with a small amount of edema,bleeding spots;4 weeks lungs pink color, white can be seen surrounding individual lung nodules;lungs on 6 weeks was soft, pink color,only seen a small amount of surrounding lung nodules scattered. Histopathology:interval no significant thickening,a small amount of alveolar macrophages,neutrophils infiltration and a small number of interstitial fibroblasts at 2 weeks,even a small amount of green collagen can be seen by Masson staining;the number of early inflammatory cells such as neutrophils significantly reduced,alveolar exudate little cavity,fibroblasts and collagen matrix had no significant increase at 4 weeks,occasionally see a small amount of green staining of collagen deposition and fibroblasts spindle by Masson staining;6 weeks no inflammatory cell infiltration,occasional green collagen deposition,there was no an increase than 4 weeks;the change of 8 weeks was similar to 6 weeks,we can see most of the normal alveolar structure.Pathological assessment:BL+DcR3 group the extent of alveolitis manifested as a gradual reducing,the degree of inflammation were lower than the BL group,there was significant difference(p＜0.05);BL+DcR3 group the extent of pulmonary fibrosis had no significant to increase compared with the control group,there was significant difference(p＜0.05),compared with the BL group there was significantly different(p＜0.01).BL+DcR3 group the total number of BALF cells at 2W highest then dropped significantly,even it was lower than the control group at 8W,the difference was significant(p＜0.05),there was no significant difference(p＞0.05) compared with the control group on other times,there were significantly different(p＜0.05) compared with the BL group at 2W,4W,8W;The inflammatory cytokines TNF-α,TGF-βshowed decreasing expression with DcR3 treatment.The hydroxyproline content of BL+DcR3 Group compared with the control group were no significant difference (p＞0.05),compared with the BL group were significant difference(p＜0.05).The result of this study indicate DcR3 inhibits early inflammatory response.Second experiments:at 2W rats were killed 5 to prepare alveolar macrophages and peripheral blood lymphocytes,to detect the ability of peripheral blood lymphocyte proliferation(MTT),to decte the percentage of CD69~+ lymphocytes and percentage of apoptosis by flow cytometry,to detecte alveolar macrophage proliferation capacity(MTT)and percentage of apoptosis of macrophages by flow cytometry,to detecte the content of TNF-α,TGF-βo in macrophage culture supernatant in vitro.MTT test results showed:BL group the proliferation ability of alveolar macrophage decreased,interventions of DcR3 to promote the alveolar macrophage proliferation.BL group the proliferation ablility of peripheral blood lymphocytes decreased,intervention of DcR3 enhance the ability of peripheral blood lymphocyte proliferation.BL group the proportion of CD69~+ lymphocytes was 29.11±0.02%,BL+DcR3 group the proportion of CD69~+ lymphocytes was 59.89±0.03%,DcR3 enhanced the ability of peripheral blood lymphocyte activation. Macrophages in vitro after 48 hours,detected supernatant TNF-α,TGF-βcontent: Two cytokines of BL groups were higher than C groups,the values of cytokines of BL+DcR3 group was highter than control and BL groups,that was DcR3 enhanced secretiong function of the alveolar macrophage slightly.Flow cytometry results showed that:BL group the apoptosis percentage of macrophages and lymphocytes were higher than control group,there was significant difference(p＜0.05);BL+ DcR3 group the percentage of apoptosis of macrophages and lymphocyte were lower than the BL group,there was significant difference(p＜0.01),and the percentage of apoptosis of macrophage than the control group,there was a significant difference(p＜0.01).That was the bleomycin model of pulmonary fibrosis in rat, the percentage of apoptosis alveolar macrophages and peripheral blood lymphocytes ware increased,DcR3 to reduce the apoptosis.Third experiments:we constructed DcR3 eukaryotic expression vector throught over-PCR method,transfected HLF cells with lipofectamine,then detected the typeⅣcollagen content of HLF cells by Western blot,detected HLFcells proliferation throught MTT,the percentage of apoptosis of cells was detected by Flow cytometry.The results were:pvax1-DcR3 constructed eukaryotic expression vector successfully and transfected into HLF cells;MTT assay results showed that DcR3 transfected HLF cells,enhance the capacity of the cell proliferation, compared with-the control transfection group and blank group,there was significant difference(P＜0.01).Detected by Western blot and image analysis, after DcR3 transfected human lung fibroblasts,the cells transfected with the expression of ColⅣwas significantly increased,beginning from 12h to 72h, compared with the control group were statistically significant differences (P＜0.01),that was the expression of DcR3 enhanced the ability of collagen synthesis in HLF.The apoptosis percentage of transfection group was significantly lower than the control group and blank control group(P＜0.05),that was DcR3 inhibits fibroblast apoptosis.Combining the results of three experiments,the following conclusions can be drawn:in the bleomycin pulmonary fibrosis animal model,DcR3 inhibite the early application of inflammatory response;DcR3 inhibite alveolar macrophages and peripheral blood lymphocyte apoptosis;DcR3 promote peripheral blood lymphocyte proliferation and activation capacity;DcR3 promote alveolar macrophage proliferation and secretory function;DcR3 inhibited apoptosis of HLF transfected with DcR3 and promoted cell proliferation,enhanced the ability of collagen synthesis in HLF;the role of DcR3 in body rather than single,may have a two-sided effect;It is really regrettable that the imbalance between the more, that is,early secretion of DcR3 show anti-inflammatory effect,once the inflammation expanded,excessive secretion,then instead promoted the occurrence of pulmonary fibrosis.The study not only improved the pathogenesis of pulmonary fibrosis,but also has great significance to the clinical treatment.