The Mechanism Of MiRNA-26a On The Transdifferentiation Of Alveolar Epithelial Cells And The Synthesis Of Pulmonary Surfactants

Recently,due to the development of neonatal intensive care technology,more preterm infants survived.However,with their immature lungs,these preterm infants are more prone to get lung related diseases,such as respiratory distress syndrome(RDS)and bronchopulmonary dysplasia(BPD),which affect the survival rate and living quality of the preterm infants.Although antenatal corticosteroids prophylaxis and exogenous pulmonary surfactant replacement therapy have been applied,the clinical efficacy for intractable RDS is not as good as expected by conventional alternative therapies.The abnormal lung tissue repair of immature lung after damages leads to BPD,which is the main cause of infant chronic lung diseases.Therefore,exploration of the pathogenesis of RDS as well as lung injury and repair mechanisms may provide important therapeutic targets for these diseases.Type II alveolar epithelial cell(AECII),as a functional cell of lung epithelial,is a potential target for a variety of injuries.It is a necessary part of lung maturation for AECII to synthesize and secret pulmonary surfactant that plays a pivotal role in lowering surface tension and stabilizing alveolar inflation-deflation to prevent the respiratory distress syndrome.As a progenitor cell of AECI,AECII begins to tans-differentiate into type I alveolar epithelial cell(AECI)to increase lung sac-alveolar proportion for preparation of gas exchange as early as 24 weeks of pregnancy in humans.Even in mature lungs,AECII still maintains to proliferate and trans-differentiate into AECI to repair damaged lung epithelium when it is injured by various pathogenic factors.Currently,primary cultures of AECII are the main way for its function studies due to lack of ideal cell lines.Primary cultured AECII automatically trans-differentiate into AECI after 72 hours when cultured in vitro,which is a nature model of AEC? trans-differentiation study.MicroRNAs are a group of endogenous non-coding regulatory RNAs.They regulate the expression of their target genes at the post-transcriptional level and have been found to be involved in various fundamental biological processes including cell proliferation,differentiation,apoptosis and signal transduction as well as numerous pathological process,such as tumorigenesis and inflammation.Previous reported confirmed that miRNA-26a(miR-26a)differentially expressed in the lung of fetal rat in late pregnancy in the study of lung development using miRNA microarray.Bioinformatic analysis of its potential target genes had demonstrated that SMAD1 and SMAD4 are important factors for lung development.The over expression of miR-26a indicated that miR-26a inhibited the expression of SMAD1,SP-B and SP-C.Experiments below aimed to further investigate the functions of miR-26a.This paper was divided into two parts:The first part was to explore the methods of primary culture for AEC? in different stages of lung development so as to provide cell model for further study using SD rat as experimental animals.The second part was to verify the roles of miR-26a played during fetal type ? alveolar epithelial cell trans-differentiation and its possible mechanisms involved in a cell model of 19-day fetal rat's AEC?.Part ?:Primary culture of type ? alveolar epithelial cell in different stages of lung developmentObjective:To explore the methods of primary culture for type ? alveolar epithelial cell(AEC?)in different stages of lung development so as to provide cell model for further study.Methods:Lung tissues of 19-day fetal rats and 21-day fetal rats were digested with trypsin and collagenase,then the isolated AEC? was purified by different centrifugal force and differential cell attachment.Lung tissues of 1-day,3-day,7-day and 14-day postnatal rat were dispersed using Dispase or trypsin.The AEC? was purified from the crude cell mixture using differential cell attachment or centrifugation with a percoll density gradient or IgG immune sorption.Cell morphology was observed under inverted phase contrast microscope.Cell viability was identified by trypsin blue exclusion assay and the purity was determined by fluorescent immunocytochemistry.Results:1.Digestion with trypsin combined collagenase and purification by different centrifugal force and differential cell attachment could achieve good results for fetal AEC?.2.Among which the 19-day fetal rats had the best results,the quantity of cells was(38.0±3.2)×106/5 fetus meanwhile the cell viability and purity reached up to(95.0 ±2.1)%and greater than 90%respectively.For the 21-day fetal rats,that were(50.5±2.1)×106/5 fetus,(93.5± 1.5)%and around 75%-80%respectively.3.There was a significant decline for the effect of trypsin combined collagenase method used in postnatal rats.Simultaneously,cell quantity,viability and purity sharply dropped with increasing age.The purity of AECII was approximately 65%,40%,30%and 30%among the 1-day,3-day,7-day and 14-day postnatal rat separately.5.Using the improved experimental techniques of Dispase digestion,rat IgG immune adsorption and percoll gradient centrifugation for purification,there was only 10%enhancement of cell purity.Conclusions:1.Age of SD rat is a key factor in primary culture for AEC?.Cells of 19-day fetal rats are most easily purified and had high viability and purity.2.There are more difficulties in AEC? isolation and purification with further lung development.It is difficult to achieve separation and purification purposes by trypsin combined collagenase digestion and differential adhesion,especially for postnatal rats.3.Therefore,primary cultures of AEC? during late lung development remains to explore in the future.Part ?:Effects and mechanisms of miR-26a overexpression on type ? alveolar epithelial cell trans-differentiationObjective:To verify the roles of miR-26a during fetal type ? alveolar epithelial cell trans-differentiation and its possible mechanisms involved.Methods:Type ? alveolar epithelial cells of 19-day fetal rats were gained by trypsin combined collagenase digestion and differential adhesion.Cell viability and purity were identified by trypsin blue exclusion assay and fluorescent immunocytochemistry,respectively.Firstly,the expression levels of miR-26a between lung tissue and freshly isolated AECII were detected.Then the morphology changes of AECII were observed when cultured for lday,3 days,5days and 7days under inverted phase contrast microscope.Immunofluorescence staining of cells was employed to observe the expression of ABCA3(a specific marker for AECII)and T1?(a specific marker for AECI)during this procedure.The expression levels of miR-26a were detected by real-time PCR.Then we transduced the freshly isolated AECII with adenoviral vectors over expressing miR-26a and viral control respectively.At the fourth day,the transfection efficiency was assessed by the expression of GFP.Real-time PCR was used to examined the over expression of miR-26a in AECII.Immunofluorescence staining was used to detect the expression of ABCA3 and Tla.The mRNA and protein expression levels of T1? were measured by real-time PCR and western blot respectively.Its potential target genes SMAD1 and SMAD4(important downstream molecules of BMP signaling pathway)were detected by western blot in the trans-differentiation process.Results:1.There was no significant difference of miR-26a in both lung tissue and freshly isolated AECII.2.AECII gradually elongated,flattened and lost its cuboidal shape,together with the loss of AEC? features(ABCA3)and increase of AECI characteristics(Tla)indicating that AEC? trans-differentiate into AEC? cultured in vitro.3.The expression level of miR-26a in AEC? significantly decreased when cultured on D1,D3,D5 and D7.4.At 72 hours post-transduction,almost 95%cells had positive expression of GFP and the expression of miR-26a was significantly higher compared with that in cells transduced with the control virus.4.There was a significant reduction in the T1? expression but a contrary case of ABCA3 in AECII treating with adenovirus over expressing miR-26a.The mRNA and protein expression levels of Tla was dramatically decreased in cells over expressing miR-26a compared to control virus.The results above indicated that miR-26a inhibited AECII trans-differentiation.5.MiR-26a's potential target gene SMAD1 increased during the trans-differentiation process,however,there were no significant changes of SMAD4.Conclusion:This study showed that miR-26a inhibites AEC? trans-differentiation possibly by targeting SMAD1 and SMAD4 mediated by Smad pathway,which provide basis for further study on the possible mechanism of BPD and lung injury-repair.