Effects Of FGF-10 On Repair To Lung Injury Of Hyperoxia-induced Neonatal Mouse BPD Model

Bronchopulmonary dysplasia（BPD）is one of the most common chronic lung diseases in infants.Classic BPD was first described and defined by Northway and colleagues in1967,as a lung injury in preterm infants resulting from oxygen and mechanical ventilation.Despite major treatments advance in recent decades,the incidence of BPD is still high.However,the preventive and therapeutic options to this disease are still limited.Drug therapies are mainly palliative and of low efficacy.Therefore,a therapeutic strategy to restart alveolar growth and induce the development of alveolar structure would be a goal of therapy.FGF-10 is a paracrine factor,and mainly expressed in mesenchymal cells.It mediates mesenchymal to epithelial signaling,and plays an important role in the development and repair in various tissues and organs,especially in lung development and repair.The purpose of the study is to detect the expression of FGF-10 in lung,on establishment of hyperoxia-induced neonatal mouse BPD model,then to detect effect of FGF-10 on inflammatory cytokines,mesenchymal stem cells and type II alveolar epithelial cells,on the basic of cultured lung cells of BPD model mice,and to preliminarily discuss interaction between FGF-10 and NF-κB,to construct pCDHFGF10 vector and detcect its expression.The study will provide experimental basis for further research of FGF-10 on prevention and treatment of BPD.This study is divided into the following five parts:PartⅠ.A total of 80 Kunming mice aged 2 days were meanly and randomly assigned to the high oxygen group with oxygen concentration maintained at 60%-70%and normoxic group.Mice of hyperoxic group were placed in the self-made sealed breeding box,and Soda Lime was put at the bottom of the box,in order to adsorpt CO₂ and maintain concentration of CO₂:<0.5%.Maternal and newborn mice were put together for feeding,and maternal mice were exchanged daily to prevent maternal mice oxygen intolerance and death.Thus,we established hyperoxic-induced neonatal mouse BPD model.Mice of hyperoxic group had significantly smaller body size and poor weight growth,shaggy and rough hair,dysphoria,dyspnea,cyanosis in lip and toe at day 21.HE staining showed that lung structure of hyperoxic group mice gradually appeared typical BPD-like performance.Thickened alveolar septal,bleeding and inflammatory cell infiltration were seen at early stage,and reduced quantity of alveolar,formation of alveolar fusion,partly inflammatory atelectasis were seen at late stage.The results suggested that hyperoxic-induced BPD model was successfully established.PartⅡ.In order to study the role of FGF-10 on BPD,we first detected the expression of FGF-10 in the neonatal mouse BPD model.Mouse lungs of the hyperoxic group and the normoxic group were examined by immunohistochemistry at day 4,7,14 and 21.The results showed that the expression of FGF-10 in the lung of hyperoxia group was significantly decreased at day 21.Furthermore,we detected the expression of FGF-10 of the two groups with corresponding age by RT-PCR and Western Blot.At day 21,the level of FGF-10 mRNA was significantly decreased in hyperoxia group by RT-PCR.Western Blot detection showed that the expression of FGF-10 in hyperoxia group decreased gradually with the increase of age.The results indicated that injury induced by hyperoxia can decreased both FGF-10 mRNA and protein levels in BPD mice.PartⅢ.In order to detect the effect of exogenous FGF-10 supplementation on BPD,we divided the lung cells of mice into four groups:normoxia,normoxia+FGF-10,hyperoxia and hyperoxia+FGF-10 in vitro.We detected the expression of inflammatory cytokines,such as IL-1β,IL-6,IL-8,TNF-β,MIP-2α,TGF-βin supernatant by ELISA.And found that IL-1β,IL-6,IL-8,TNF-β,MIP-2α,TGF-βwere all significantly increased in hyperoxia group,while FGF-10 was supplemented,IL-1β,IL-6,IL-8,TNF-βand MIP-2αdecreased.The results suggest that FGF-10 can reduce the inflammatory response of cultured lung cells in vitro.To test whether FGF-10 could promote the growth of lung mesenchymal stem cells and alveolar epithelial cells type II in vitro,we tested double positive cell surface markers CD29⁺CD90⁺and single positive cell surface molecular markers CD34^-CD105⁺by flow cytometry,and found that percentage of double positive cells was the lowest in the hyperoxia group,but the percentage of double positive cells increased significantly in hyperoxia+FGF-10 group.We detected expression of SP-C by immunofluorescence,and found that expression of SP-C decreased in hyperoxia group,but was significantly higher in group hyperoxia+FGF-10.It demonstrated that FGF-10 could promote growth of lung mesenchymal stem cells and alveolar epithelial cell type II,and indicated that FGF-10 can repair the lung injury through this process.PartⅣ.It was reported that NF-κB inhibits the expression of FGF-10,while we found that FGF-10 can reduce the expression of inflammatory factors,therefore,we assume that FGF-10 could inhibit expression of NF-κB in return.We detected the expression of NF-κB p65 in lung tissue of mice in hyperoxic group by immunohistochemistry,and in mouse lung cells with FGF-10 of hyperoxia group by Western Blot.The results showed that the expression of NF-κB p65 significantly increased in hyperoxic group at day14 and day 21,expression of N F-κB p65 obviously reduced the cultured mouse lung cells with FGF-10 in vitro.These results suggested that FGF-10 can inhibit the expression of NF-κB p65,which provided theoretical support for FGF-10 to reduce the inflammatory response of lung injury.PartⅤ.To further explore the role of FGF-10 in the repair of lung injury and its mechanism,we constructed the recombinant plasmid pCDH-FGF10.Bacteria acting as the template for FGF-10 amplification by PCR,We identified specific bands,about 600bp,which were consistent with the FGF-10 base.Positive clones were sequenced,and the results matched with the sequence of FGF-10.Thus pCDHFGF10 was constructed successfully.After plasmid being transfected,the expression was seen by western blot in vitro.This laid the experimental foundation for the next experiment.In conclusion,the study confirmed our experimental ideas.Supplement of exogenous FGF-10 has a very important role on repair to lung injury of neonatal mouse BPD model,and its possible mechanism is explored,which provides a new way to explore the clinical therapeutic measures of BPD.