The Mechanism About Roles Of Bone Marrow Mesenchymal Stem Cells In The Repair Of Acute Lung Injury

Avian influenza(AI)is a kind of virulent infectious disease harm to livestock and human health,caused by avian influenza virus AIV).The avian influenza virus(AIV)can cross species barriers and expand its host range from avians to mammalians,even human,leading high mortality,causing high attention from the medical community and governments around the world.After infection,AIV could quickly copied in respiratory tract,which in turn leading to excessive immune response,damage alveolar epithelium and the capillary endothelial cells,the symptoms of AI are similar to the clinical picture of acute lung injury(ALI),imbalance of pro-inflammatory and anti-inflammatory responses is the main cause of acute lung injury.Clinical treatment mainly antiviral and support treatment,curative effect is not good,mortality is still extremely high.With the research of the multi-differentiation potential of stem cell,stem cells in the lung tissue rehabilitation applications have caused the attention of many scholars,there are a number of research about mesenchymal stem cells,bone marrow stem cells used in animal models on the repair the damaged lung tissue,and has opened up a new way for the treatment of ALI/ARDS.AI in this study,we first establish AI mice model and to detect the expression of chemokines and inflammatory factor in the model;then we use MSCs to the early treatment of AI,exert its immune regulating function,reduce the proinflammation factor expression,alleviate lung tissue damage,improve lung function,which provide theoretical guidance for the application of MSCs in the treatment of ALI.This thesis is divided into three parts:Part I Establishment of Avian influenza(AI)mice modelObjectiveWe established acute Avian influenza(AI)model to detect the inflammatory reaction,lung function and lung tissue of acute damage degree,explore the AI inflammation factor expression in lung tissue of miceMethods1.The AIV used in the study was H9N2(A/PH/CA/2373/98),kindly provided by Prof.Zheng Xing(Medicine school,Nanjng university).We inoculate virus to allantoic cavity of 9-day-old SPF chicken embryos,collect the allantoic fluid to obtain virus after 48 hours,then infected MDCK cells to measured TCID50.2.To get the AI lung injury models,we used diethyl ether to anesthetize the C57BL/6 mice and then administrated intranasally with H9N2 virus.We use blood gas analysis to detect arterial oxygen pressure(PaO2)and carbon dioxide partial pressure(PaCO2),Routine blood test to detect white blood cell count and Monocyte proportion,tissue biopsy of lung tissue damage,Histopathologic observation to detect lung tissue damage,1,3,7,14 days after treatment,to confirm AI model successfully established.3.We use real-time RT-PCR,and ELSIA detecting inflammation factors expression level in lung and serum:interleukin 1(IL-la),interleukin 6(IL-6),tumor necrosis factor(TNF-a)and interferon(IFN-y).1,3,7 and 14 days after treatment.Results1.After Chicken embryos amplification,the final concentration of H9N2 avian influenza virus is 1×10011.4 pfu/ml.2.Intranasal H9N2 avian influenza viruses can infect mice,results in acute lung injury,blood gas analysis showed after infection the partial pressure of arterial oxygen(PaO2)was significantly decreased,while partial pressure of arterial carbon dioxide(PaCO2)was increased significantly.Routine blood test results found that blood leukocyte counts and monocyte percentage were decreased significantly;the pathological section analysis demonstrated that after the establishment of AI model,alveolar collapse and and pulmonary interstitial edema,polymorphonuclear cells were aggregated in the lung,normal lung tissue structure were damaged seriously.3.After AIV treatment for 24 hours,the IL-1α,IL-6,TNF-α and IFN-γ expression was significantly increased in lung tissue and serum,can maintain the high expression for aroud two weeks.ConclusionAI model was successfully established via H9N2 AIV administrated intranasally,leading to pulmonary function injury.The PaO2 was significantly decreased,while PaCO2 was increased significantly;Leading to structural damage in lung tissue:lung parenchymal cell damaged and pulmonary interstitial edema,inflammatory cell infiltration;IL-1α,IL-6,TNF-α and IFN-γ expression levels significantly up-regulated after lung injury and reached peak after lung injury for 24h,and can maintain for aroud two weeks.Part Ⅱ The study on immunity regulation effect of MSCs transplantation on AIObjectiveMSCs cultured in vitro,construct recombinant adenovirus of GFP protein,transfect MSCs and detection of GFP protein.Assess the beneficial effect of MSCs of the early treatment of AI,and explore its mechanism.Methods1.Use the recombinant GFP adenovirus to transfect HEK293A cells with MOI =10 proportion,extracting virus after amplification 3 times,using TCIDSO(Tissue Culture Infection Dose 50)method for the determination of virus titer after WB identify.2.C57BL/6 bone marrow mesenchymal stem cells were purchased from Cyagen(Guangzhou)Biological Technology Co.,Ltd.,we choice of the 4th generation MSCs.3.The 4th generation of MSCs transfected with the recombinant adenovirus at MOI＝400.Transfection efficiency was observed under fluorescence microscopy and the cell growth curve was detected by the CCK8 method,finally immunofluorescence and Western Blot was used to detect the expression of GFP in MSCs after transfection.4.We use H9N2 avian influenza virus to establish AI C57BL/6 mice model,then transfected MSCs via the tail vein injection.Mice were randomly divided into 4 groups.The blank control group(Control group):untreated H9N2 avian influenza virus,also do not accept the MSCs transplantation;the control group(MSCs group):untreated H9N2 avian influenza virus intranasal,intravenous injection of MSCs-GFP;model group(AI group):the H9N2 avian influenza virus intranasal molding,tail intravenous injection of normal saline;MSC treatment group(AI+MSC group):the H9N2 avian influenza virus intranasal molding,intravenous injection of transfected MSCs-GFP.Lung tissue was removed after MSCs transplantation for 3 and 7 days,and was analyzed by flow cytometry for detection of MSCs engraftment in injured sites;Arterial blood gas analysis of oxygen partial pressure(Pa02)and carbon dioxide(PaC02),Histopathological observation of lung pathology injury,to evaluate the repair effect of MSCs on AI mice.5.Flexible Multilyte Profiling to detect Chemokine KC,MCP,MIP-1 a and MIG,inflammatory factor IL-1α,IL-6,IL-10,TNF-α and IFN-γ,express in serum vascular endothelial growth factor(VEGF)and fibroblast growth factor cytokines(bFGF),real-time PCR to detect IL-1α,IL-6,IL-10,TNF-a and IFN-y mRNA expression in lung,to determine the immune response of each mouse.Results1.Recombinant GFP adenovirus infected 293A cells,after amplified purification,we finally got 1×108.8 pfu/ml of high titer virus solution,and WB results proved that recombinant adenovirus carrying the GFP which can be applied to MSCs transfection.2.MSCs demonstrated a homogeneous fibroblast-like and spindle-shaped morphology.Recombinant adenovirus transfected MSCs at MOI = 100 did not lead to MSCs appear cytopathic effect and transfection efficiency reaching about 80%.The cell growth curve showed that adenovirus transfection(MOI = 100)had no bad effect on the proliferation of MSCs.Immunofluorescence results showed that the expression of GFP was significantly improved after transfection of MSCs,reaching peak after transfected for 48 hours and maintain high expression.3.After MSCs transplantation,blood gas analysis results show that the PaO2 of AI+MSC group mice was significantly higher than that of in AI model mice,the PaCO2 was significantly lower than that of AI model mice;Routine blood test test results:the WBC and mononuclear cells proportion of AI+MSC mice was significantly higher than that in AI model mice;Pathological examination result:The lung damage were significantly improved in AI+MSC mice.4.Flexible Multilyte Profiling results:Compare with AI mice,the expression of GM-CSF KC,MCP,MIP-1α and MIG,IL-1α,IL-6,expression of TNF-α and IFN-γwere significantly decreased,IL-10,VEGF and bFGF increased significantly,in AI+MSC mice serum;real-time PCR shown:mRNA expression of IL-1α,IL-6,the TNF-a and IFN-y were significantly decreased,IL-10 expression increased significantly in AI+MSC mice lung.ConclusionApplication of MSCs in the early treatment of AI,could alleviate the lung injury and improve lung function,by immunoregulation to decreased expression of chemokine and inflammatory factors in blood and lung tissue of AI mice,thereby reducing the excessive inflammatory response.Part Ⅲ The study on regulation of the Wnt signaling pathway for MSCs differentiationObjectiveTo explore the mechanisms of Wnt/p-catenin signaling pathway on regulating the differentiation of engraftment of MSCs,and study the roles of inhibition of Wnt/β-catenin signaling pathway for MSCs fibroblast differentiation,finally finding the key molecular targets of transplanted MSCs for the treatment of AI in order to provide new ideas and theoretical basis for clinical treatment AI and other lung diseases.Methods1.Primary mice MSCs were obtained by differential adhesion separation method.A several surface markers of 3-6 generations of MSCs were identified by flow cytometry,including CD45,CD34,CD79,CD105,CD73,CD90.2.MSCs were randomly divided into 5 groups,control group:normal cultured MSCs;MSCs + TGF-β group:add 0.5 ng/ml TGF-β in medium,to induce MSCs to fibroblast differentiation;MSCs + DKK1 groups:add 20 ng/ml of DKK1 in medium,inhibition of Wnt/β-catenin signaling pathway;MSCs + TGF-P+ DKK1 groups:medium,add 0.5 ng/ml of TGF-β,as well as 100 ng/ml DKK1,inhibition of Wnt/p-catenin signaling pathway and induced MSCs fibroblasts differentiation at the same time.3.Use 2,3,4,5 generations of MSCs,real-time PCR,immunofluorescence technology testing each MSCs in β-Catenin,TCF,GSK-3p,Axin1 and JNK,verify the activation and inhibition of Wnt signaling pathways.4.WB,real-time PCR detect the expression of fibroblast markers of MSCs in ench group:Vimentin,a-SMA and TE-7,respectively.Detection of the Wnt/β-Catenin signaling pathway activation and inhibition effects on MSCs fibroblast differentiation.Results1.MSCs demonstrated a homogeneous fibroblast-like and spindle-shaped morphology.FACS analysis demonstrated that MSCs expressed CD73、CD90(+),but not CD34,CD45 and CD79.2.Fibroblasts differentiation of MSCs can be induced by TGF-P,while the Wnt signaling pathway was activated.Immunofluorescence technology and real-time PCR testing results showed the expression of β-Catenin,TCF,GSK-3β,Axin1,JNK of TGF-β+MSCs were increased.3.Add DKK1 in MSCs fibroblasts differentiation process,can inhibition of Wnt signaling pathway.Immunofluorescence technology and real-time PCR testing results showed that compared with TGF-β+ MSCs group,MSCs + TGF-β+ DKK1 Wnt signal pathway protein:β-Catenin,TCF,GSK-3β and Axin1,JNK expression are lower.4.WB and real-time PCR detected fibroblasts marker Vimentin,a-SMA,and TE-7 of MSCs,respective.Results shown:compared with MSCs+TGF-β group,the expression of Vimentin,a-SMA and TE-7 of 4,and 5 generations of MSCs in TGF-β+ MSCs + DKK1 group,were decreased significantly.ConclusionThe activation of Wnt/β-catenin signaling pathway can promote MSCs differentiation to fibroblasts,and inhibition of Wnt/β-catenin signaling pathway can inhibit MSCs differentiation to fibroblasts.