Study The Role Of Wnt Signaling Pathway Involved In The Lung Resident Mesenchymal Stem Cell Differentiation And Intervention Mechanisms Of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis(IPF)is a devastating lung disease of unknown cause,characterized by pulmonary interstitial fibrosis.There was no significant benefit in clinical treatment,and patients have a median survival of less than 3 years after diagnosis.IPF occurs in middle-aged and elderly adults(median age at diagnosis 66 years,range 55-75 years),is limited to the lungs,and is associated with a histopathological or radiological pattern typical of usual interstitial pneumonia.Some studies have demonstrated that Wnt/β-catenin signaling pathway takes an indispensable part in the genesis of IPF and the activation of Wnt/β-catenin pathway promotes the progression of IPF.In this study,we determined the role of Wnt/β-Catenin Signaling in pulmonary fibrosis.Moreover,resent study suggested that lung resident mesenchymal stem cells(LR-MSCs)played an important role in the progression of IPF.Therefore,in this study we study the role of Wnt signal pathway involved in the LR-MSCs differentiation and intervention mechanisms of IPF.At first,our aim was to explore the roles of Wnt/β-catenin signaling on cell proliferation and differentiation of resident mesenchymal stem cells(LR-MSCs).We have successfully isolated LR-MSCs.In order to clarify the regulatory mechanisms of Wnt/β-catenin signaling in the differentiation of LR-MSCs,we measured LR-MSCs following indirect co-culture with alveolar epithelial type II(ATII)cells,confirming that blocked Wnt/β-catenin pathway may promote the differentiation of LR-MSCs.While,activated Wnt/β-catenin signaling induces myofibroblast differentiation.In conclusion,our study demonstrated that Wnt/β-catenin signaling may be an essential mechanism underlying the regulation of differentiation of LR-MSCs.Next,we determined the therapeutic efficacy of canonical inhibitors(DKK-1,XAV939 and NSC668036)of Wnt/β-catenin signaling in bleomycin-induced pulmonary fibrosis murine model.We found that the inhibitors significantly suppressed accumulation of collagen that can inhibit pulmonary fibrogenesis.Because fibrotic lung exhibit abesrrant activation of Wnt/β-catenin signaling,our data collectively suggest that inhibition of Wnt/β-catenin signaling a may be an effective approach to the treatment of fibrotic lung diseases.In the end,considering that the differentiation of myofibroblast is crucial in pulmonary fibrosis,we examined gene expression patterns during the transforming growth factor(TGF)-β1 induced myofibroblast differentiation of LR-MSCs by Affymetrix GeneChip Mouse Gene 1.0 ST Arrays.We identified that Wnt8b as the genes that were overexpressed during the differentiation of LR-MSCs by comparing the expression of the Wnt proteins.Moreover,we verified the microarray results in vitro and in vivo.Our study demonstrated that Wnt8b may be an essential point to underlying the regulation of differentiation of LR-MSCs and the process of murine pulmonary fibrosis.We confirm that down-regulated Wnt8b by the lentiviral vectors could inhibit the fibroblast differentiation of LR-MSCs and pulmonary fibrosis.Above all,this study is useful to find out the effective target of intervening the progression of IPF,and provide a new way for clinical treatment.This thesis is divided into three parts:Part Ⅰ Roles of Wnt/p-catenin signaling in the diffientiation of lung resident mesenchymal stem cellsObjectiveIsolation of lung resident mesenchymal stem cells(LR-MSCs)and identification.We established the primary mouse lung resident mesenchymal stem cell separation and purification methods,and phenotype identified the primary LR-MSCs.To investigate the regulatory role of Wnt/β-catenin signaling pathway in LR-MSCs bidirectional differentiation.And we employed a new method of physics to monitor the bidirectional differentiation of LR-MSCs continuously and sensitively.Methods1.Primary mouse LR-MSCs were obtained by MACS.3-6 generations of LR-MSCs were identified by flow cytometry.2.Indirect co-culture was established to induce differentiation of LR-MSCs into ATII cells by using cell culture inserts(0.4μm PET,4.5 cm2,Millipore).LR-MSCs and ATII cells were each plated at 105 cells/ml.LR-MSCs were plated in the lower chamber and ATII cells were plated in the upper chamber.On days 7 and 14,inserts were removed and LR-MSCs were harvested for SERS test,Western blotting and immunocytochemistry analysis.3.To induce differentiation of LR-MSCs into fibroblasts,LR-MSCs were incubated with 10 ng/ml TGF-β and 10 ng/ml Wnt3a.It has already been confirmed that TGF-β signaling contributes to activation of the canonical Wnt pathway.On days 3,7 and 14,LR-MSCs were harvested for the SERS test,Western blotting and immunocytochemistry analysis.4.We isolated the LR-MSCs from mouse lung and used a new set of intracellular SERS probes to label the LR-MSCs that were next co-cultured with ATII cells and incubated with TGF-β for differentiating into alveolar epithelial cells and fibroblasts,respectively.Furthermore,HIV-derived Tat peptide(TAT)-conjugated AuNSs as cellular SERS probes allowed us to investigate changes of cellular components during the differentiation of LR-MSCs.Results1.Primary mouse LR-MSCs demonstrated a homogeneous fibroblast-like and spindle-shaped morphology.FACS analysis and immunocytochemistry analysis demonstrated that LR-MSCs expressed Sca-1,CD44,CD 106,CD90,CD29 and ABCG-2,but not CD31,and CD45.2.LR-MSCs co-culture with ATII cells could induce LR-MSCs differentiate into ATII cells.Immunofluorescence and Western Blot results showed that the expression of epithelial markers,such as SP-C,CK18,CK19,Occludin,were significantly improved after blocked Wnt/β-catenin signaling pathway.3.Wnt3a and TGF-p both could induce LR-MSCs differentiate into fibroblast.Immunofluorescence and Western Blot results showed that the expression of fibroblast markers,such as Collagen,α-SMA,Vimentin,were significantly improved after activity Wnt/β-catenin signaling pathway by incubating with Wnt3a or TGF-β.4.We here show successful segregation of differentiated subtypes using the noninvasive method of SERS.SERS spectra were analyzed using principal component analysis(PCA),which has been demonstrated to be a highly sensitive method for distinguishing and identifying the differentiation of LR-MSCs.Conclusion We confirmed that co-culture with ATII cells and incubation with TGF-p could induce LR-MSCs to differentiate into ATII cells and fibroblasts,respectively.And we here show LR-MSCs differentiate into different cells by regulating Wnt/p-catenin signaling pathway.And we found that inhibited of Wnt/β-catenin signaling pathway could promote LR-MSCs to epithelial differentiation,activated Wnt/β-catenin signaling pathway could promote the LR-MSCs to fibroblast differentiation.Part Ⅱ Screening of the key Wnt protein in the fibroblasts differentiation of lung resident mesenchymal stem cells by mouse genome microarrayObjectiveTo explore the mechanisms of Wnt/β-catenin signaling pathway on regulating the fibroblast differentiation of LR-MSCs,and screening the key Wnt protein of fibroblast differentiation of LR-MSCs.Study the roles of Wnt8b in the LR-MSCs differentiation process,to investigate the molecular mechanisms of regulation in process of bleomycin-induced pulmonary fibrosis.Methods1.To induce differentiation of LR-MSCs into fibroblasts,LR-MSCs were incubated with 10 ng/ml TGF-β.It has already been confirmed that TGF-β signaling contributes to activation of the canonical Wnt pathway.LR-MSCs were harvested for the,Western blotting and immunocytochemistry analysis,on 7,14 days.2.We examined gene expression patterns during the transforming growth factor(TGF)-β induced myofibroblast differentiation of LR-MSCs by Affymetrix GeneChip Mouse Gene 1.0 ST Arrays.3.Real-time PCR,immunofluorescence and Western Blot was used to detect the expression of Wnt8b in the fibroblast differentiation of LR-MSCs,and bleomycin-induced pulmonary fibrosis murine model.Results1.Real-time PCR,immunofluorescence and Western Blot results showed that the expression of fibroblast markers,such as Collagen,α-SMA,Vimentin,were significantly improved after activity Wnt/p-catenin signaling pathway by incubating with TGF-P at 7 days.2.The Affymetrix GeneChip Mouse Gene 1.0 ST Arrays results show that the expression of Wnt8b were significantly improved at the beginning of fibroblast differentiation of LR-MSCs3.Real-time PCR,immunofluorescence and Western Blot results confirmed that Wnt8b was significantly upregulate during the fibroblast differentiation of LR-MSCs.Furthermore,the immunohistochemical and Western blot results demonstrated that the expression levels of Wnt8b was significantly upregulate in bleomycin-induced pulmonary fibrosis murine model.ConclusionThe activation of Wnt/β-catenin signaling pathway by incubated with TGF-P could induce LR-MSCs differentiated into fibroblasts(myofibroblasts)at the 7 days.The expression of Wnt8b was significantly up-regulate during the fibroblasts differentiation,may promoted the formation of fibroblast foci,and finally promote the development of idiopathic pulmonary fibro genesis.Therefore,our results indicated that Wnt8b may serve as a specific target for the intervention of idiopathic pulmonary fibrosis..Part Ⅲ The study of the intervention of specific blocking Wnt/β-catenin signaling pathway in idiopathic pulmonary fibrosisObjectiveTo explore the mechanisms of Wnt8b on regulating the differentiation of LR-MSCs,and study the roles of targeted inhibitor of Wnt8b to inhibited the fibroblast differentiation of LR-MSCs,and access the effect of targeted inhibitor of Wnt8b to intervene the process of idiopathic pulmonary fibrosis for the treatment of IPF in order to provide new ideas and theoretical basis for clinical treatment IPF and other lung diseases.Methods1.The Lentivirus-Wnt8b-RNAi and Lentivirus-Wnt8b were obtained from JiKai Bio-technology Co.Ltd.(Shanghai,China).Verified the role of Lentivirus-Wnt8b-RNAi and Lentivirus-Wnt8b in the regulation of Wnt8b.2.After 72 h transfection of Lentivirus-Wnt8b-RNAi at 5×107 TU/ml,LR-MSCs was incubated with 10 ng/ml TGF-β to induced the fibroblast differentiation.RT-PCR,immunofluorescence and Western Blot was used to detect the expression of Wnt8b and fibroblast markers in LR-MSCs after transfection.3.The Lentivirus-Wnt8b transfected at 5×107 TU/ml for 7 days.RT-PCR,immunofluorescence and Western Blot was used to detect the expression of β-catenin and fibroblast markers in LR-MSCs after transfection.4.The mouse were randomly divided into 4 groups.①NC group:Mouse were transfected negative control Lentivirus by airway instillation.After 7 days,the mouse were injected with 50 μl PBS by the same way.②NC+BLM group:Mouse were transfected negative control Lectivirus(2×108 TU/ml)by airway instillation.After 7 days,the mouse were injected with 50 μl bleomycin(5 mg/kg)by the same way.③RNAi+BLM group:Mouse were transfected Lentivirus-Wnt8b-RNAi(2×108 TU/ml)by airway instillation.After 7 days,the mouse were injected with 50 μl bleomycin(5 mg/kg)by the same way.④Wnt8b group:mouse were just transfected Lentivirus-Wnt8b(2×108 TU/ml)by airway instillation.Histology examination was used to determine histology structure integrity and the extent of collagen deposition in lung tissue 7 and 14 days after bleomycin injection.5.Immunofluorescence,immunohistochemistry and Western Blot was used to detect the expression of the β-catenin and fibroblast markers in lung tissue.Results1.The Lentivirus-Wnt8b-RNAi can effectively reduce the expression of Wnt8b in the process of LR-MSCs fibroblast differentiation and during the process bleomycin-induced pulmonary fibrosis;Lentivirus-Wnt8b can significantly improve Wnt8b transcription and protein level of expression.2.Real-time PCR,Western blot and immunofluorescence results show that the fibroblast markers were significantly downregulate after Lentivirus-Wnt8b-RNAi transfection during the process of fibroblast differentiation.3.Real-time PCR,Western blot and immunofluorescence results show that the fibroblast markers were significantly upregulate after Lentivirus-Wnt8b transfection.4.Lung tissue was removed after bleomycin injection for 7,14 days,and was analyzed by Western blot and immunofluorescence for detection of fibroblast markers.Histology examination was used to determine histology structure integrity and the extent of collagen deposition in lung tissue.The results show that targeted inhibitor of Wnt8b could downregulate the expression of fibroblast markers,inhibit the process of pulmonary fibrosis.ConclusionThe regulation of Wnt8b can induce LR-MSCs differentiate into fibroblast by activation the Wnt/p-catenin signaling pathway.Means Wnt8b plays an important role in idiopathic pulmonary fibrosis.This key Wnt protein is an important regulation mechanism of LR-MSCs differentiation.Targeted inhibitor of Wnt8b could effectively inhibit LR-MSCs differentiate into fibroblasts,but to some extent,intervene of the idiopathic pulmonary fibrosis.Wnt signaling pathway is highly active during the process of IPF,and highly activation of Wnt/p-catenin signaling pathway may promote the formation of fibroblast foci,which contributed to pulmonary fibrogenesis.