| 【Background and Objective】: Pulmonary fibrosis(PF)is a group of diseases with chronic progressive exacerbation and high mortality.Currently,clinical approved drugs for the treatment of pulmonary fibrosis can slow down the symptoms of patients,whereas hardly improve their survival and quality of life.Stem cell therapy is an emerging therapeutic modality for the treatment of various inflammatory and degenerative diseases,particularly the use of mesenchymal stem cells(MSCs).After in vitro expansion and allogeneic infusion,MSCs are recruited to the site of injury,and promote epithelial tissue repair,which exhibits powerful immunomodulatory properties.IL-1 expression levels in the lungs have been found to correlate with the development of pulmonary fibrosis in rodents exposed to bleomycin or radiation and to be upregulated in the fibroproliferative regions of the lungs in patients with idiopathic pulmonary fibrosis.IL-1Ra produced by MSCs protects mice from bleomycin-induced lung injury by blocking the production and/or activity of the major pro-inflammatory cytokines TNF-α and IL-1α in lung tissue.In this study,IL-1Ra gene overexpression vector was constructed,and human umbilical cord mesenchymal stem cells(h UCMSCs)with IL-1Ra gene overexpression were established.To investigate whether overexpression of IL-1Ra enhance the therapeutic effect,h UC-MSCs overexpressing IL-1Ra were constructed for the treatment of bleomycin-induced pulmonary fibrosis mice.This study provides a new idea for the study of anti-fibrosis,which is beneficial for the treatment and prevention and control of pulmonary fibrosis.Since there are obvious anatomical differences between mouse and human lungs,and the development process of human pulmonary fibrosis disease is very different from that of mice.This study induced differentiation of human embryonic stem cells(h ESCs)to form 3D structured human lung organoids in vitro for later establishment of an in vitro model of human pulmonary fibrosis.The results facilitate the study of the pathogenesis of pulmonary fibrosis and the development of drug therapy.【Materials and Methods】: The gene overexpression vector of IL-1Ra was constructed by genetic engineering technology and introduced into h UC-MSCs,and the overexpression of IL-1Ra in the cells was verified by Real-time PCR.C57BL/6 mice were randomly divided into four groups: control group(PBS+PBS),model group(BLM+PBS),stem cell treatment group(BLM+MSCs)and overexpressing IL-1Ra gene stem cell treatment group(BLM+IL-1Ra-MSCs).After mice were anesthetized,5 mg/kg of PBS was dripped into their trachea in the blank control group and equal volume of bleomycin was given to the rest of the groups,respectively.After 24 h of modeling,the stem cell treatment group and overexpressing IL-1Ra gene stem cell treatment group were injected with corresponding stem cell suspension 5 × 105 /200ul/each via tail vein,and the control and model groups were given equal volume of sterile PBS.the survival rate,mental status and body weight changes of mice were observed,and lung tissue samples were collected 21 days after modeling for HE and Masson staining.Ashcroft score,hydroxyproline content assay,lung tissue α-SMA,Collagen Ⅰ expression assay were performed to assess the degree of lung fibrosis in each group of mice.Finally,human embryonic stem cells were subjected to in vitro definitive endodermal differentiation,anterior foregut endodermal differentiation and lung organoids differentiation,and the related markers of pulmonary organoids were detected by Real-time PCR and immunofluorescence staining.【Results】:(1)Real-time PCR results showed that the m RNA expression of IL-1Ra was significantly increased in the transfected group of h UC-MSCs,and the difference was statistically significant(P<0.05).(2)The survival curve and body weight of mice showed that the survival rate and body weight of mice in the model group were decreased significantly compared with the control group,and the difference was statistically significant(P<0.05).Both the MSCs treatment group and the IL-1Ra-MSCs treatment group improved the survival rate and increased the body weight of mice with pulmonary fibrosis(P<0.05).Compared with the MSCs treatment group,the IL-1Ra-MSCs treatment group showed more significant increase in survival rate and body weight.(3)HE and Masson staining results showed that the control mice had normal lung tissue structure,and the model mice had disorganized lung tissue structure,severe alveolar destruction,significant thickening of lung septa,and a large number of collagen fibers stained blue,and a significant increase in Ashcroft score compared with the control mice,with statistically significant differences(P<0.05).The treatment of both MSCs and IL-1Ra-MSCs improved the degree of alveolar damage and fibrosis in mice with pulmonary fibrosis,and decreased the Ashcroft score,the difference was statistically significant(P<0.05),and the reduction was more obvious in the IL-1RaMSCs treatment group compared with the MSCs treatment group.(4)Hydroxyproline detection results showed that compared with the control group,the content of hydroxyproline in the model group was significantly increased,and the difference was statistically significant(P<0.05).MSCs and IL-1Ra-MSCs both reduced the content of hydroxyproline in mice with pulmonary fibrosis(P<0.05).The hydroxyproline content in IL-1Ra-MSCs treatment group decreased more significantly than that in MSCs treatment group,and the difference was statistically significant(P<0.05).(5)Real-time PCR results showed that the m RNA expressions of α-SMA,Col1a1 and Col1a2 in lung tissue of mice in model group were significantly increased compared with those in control group,with statistical significance(P<0.05).The m RNA expression levels of α-SMA,Col1a1 and Col1a2 in MSCs treatment group and IL-1Ra-MSCs treatment group were significantly decreased compared with model group(P<0.05).The m RNA expressions of α-SMA and Col1a1 in IL-1Ra-MSCs treated group were decreased more significantly than those in MSCs treated group(P<0.05).(6)Western blot results showed that the expressions of α-SMA and Col1 agen Ⅰ protein in lung tissue of model group were significantly increased compared with that of control group,and the difference was statistically significant(P<0.05).The expression levels of α-SMA and Col1 agen Ⅰ protein in MSCs and IL-1Ra-MSCs treated groups were significantly decreased compared with model group(P<0.05).The α-SMA protein expression in IL-1Ra-MSCs treated group was more significantly decreased than that in MSCs treated group(P<0.05).(7)Immunofluorescence results showed that the fluorescence expression of α-SMA in the lung tissue of mice in the model group was significantly increased compared with that in the control group,and the fluorescence expression of α-SMA in the lung tissue of mice in the MSCs treatment group was decreased,while the fluorescence expression of α-SMA in the IL-1Ra-MSCs treatment group was decreased more significantly.(8)Real-time PCR results showed that lung organoids inducing differentiation of D21-D31 highly expressed NKX2.1,P63,SOX9 and PDPN genes,while lung organoids inducing differentiation of D41 highly expressed SPC,HOPX and SCGB1A1 genes and other pulmonary organoid related markers(P<0.05).(9)Immunofluorescence results showed that SPC markers were highly expressed in lung organoids induced by differentiation of D41.【Conclusion】:(1)In this study,a mouse model of pulmonary fibrosis was successfully established.(2)Overexpressing IL-1Ra gene could enhance the therapeutic effects of h UCMSCs in mice with bleomycin-induced pulmonary fibrosis.(3)In this study,h ESCs were successfully induced to differentiate in vitro to form 3D structured human lung-like organs,which will help to establish an in vitro model of human pulmonary fibrosis at a later stage. |
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