The Effects And Molecular Mechanisms Of Retinoic Acid On AEC Ⅱ In Hyperoxic Environments And The Existence Of Wnt Signaling Pathways

Part I:Primary Culture and Identification of Alveolar type ⅡEpithelial Cells in Fetal Rats.Objective:To study the method of isolation,purification and culture of alveolar typeⅡ epithelial cells(AECⅡ)in fetal mice at 19 days gestation.Methods:Fetal lung tissue was extracted by laparotomy from 19 days of naturally conceived SD rats.AECⅡ was isolated and purified by trypsin and type Ⅳ collagenase digestion,differential centrifugation method,differential adhesion method and IgG immune adhesion and so on.AECⅡ cells were cultured for 24h,48h,72h and 96h.The ultrastructure of cells was observed under transmission electron microscope.The expression of sp-c and aquaporin 5 was observed under immunofluorescence-laser confocal microscopy.In this study,alveolar type Ⅱ epithelial cells were identified by transmission electron and immunofluorescence-laser confocal assay.Results:Under inverted microscope:The adherent cells began to appear after cultured for 8 hours in insular growth pattern.The morphology of the cells was island distribution with obvious nucleus and nucleoli.As culture time passed,single cells were polygonal in shape and larger in size than before and the number of cells increased.The cells were connected to each other and spread out at the bottom of the bottle like paving stones.Cultured 96h,the refraction of the cells began to decreas and cellular pseudopod appeared.Under laser confocal microscope:SP-C labeled with green fluorescent protein was first observed in the cytoplasm at 24 hours.And the expression level of SP-C protein in cytoplasm was highest.With time goes,the protein express gradually weakened.AQP-5 labeled with red fluorescent protein was expressed at 48h and then gradually enhanced.Under transmission electron microscope:The characteristic microvilli and lamellar bodies of AECⅡ were observed in cells cultured for 24h.After 72 hours of cell culture,the cells were transformed into an intermediate state that we could observed the microvilli and lamellar bodies in the cytoplasm.Conclusion:Alveolar type Ⅱ epithelial cells were obtained by trypsin,type Ⅳcollagenase combined digestion method,differential centrifugation,differential adhesion and immune adhesion method.The cells are of high purity and activity,which can be applied in subsequent experimental studies.Fluoroscopic electron microscopy and immunofluorescence were used to identify AECII.Part Ⅱ:The effect and mechanism of retinoic acid on the damage of AECⅡ caused by hyperoxia.Objective:To study the effect of retinoic acid intervention on AECⅡ cells under high oxygen exposure and to explore whether the mechanism is related to the activation of classical Wnt signaling pathway.Methods:AECⅡ cells were isolated and purified from 19 days pregnant rats.And the cells were identified by electron microscopy.According to the principle of random grouping,the cells were divided into the following three groups:1.Air group(the cells were cultured in incubators with an environment of 5%CO2 and 21%O2);2.High-oxygen group(the cells were cultured in an airtight oxygen chamber with 5%CO2 and 95%O2);3.High-oxygen+RA group(10-6mol/L retinoic acid was pre-added into the cell culture medium,and the culture conditions were the same as the high-oxygen group).The three groups of cells were placed in the incubator for further culture for 24 hours.The growth and morphological changes of cells were observed under an optical microscope.Cell apoptosis was detected by flow cytometry.The expressions of Wnt7b and bet-catenin mRNA in Wnt pathway were detected by real-time quantitative PCR,and the expression levels of Wnt7b and bet-catenin in Wnt pathway were detected by Western blot.Results:Compared with the air group,AECII cells were cultured for 24h in the high-oxygen group showed that significant changes in intercellular space and decreased refraction.Some cells were dewalled.After RA intervention,the morphology of the cells was similar to that of the air group,and the intercellular space was widened and vacuoles could still be seen.However,compared with the high-oxygen group,there were more adherent cells.Flow cytometry showed that the number of AECII double labeled with AnnexinV(+)PI(-)and AnnexinV(+)PI(+)in the high-oxygen group was higher than that in the air group.RA can significantly down-regulate apoptotic cells(P<0.05).PCR results showed that the expression levels of Wnt7b and-catenin mRNA in the high-oxygen group were decreased after 24 hours of culture,while the expression levels were increased after RA intervention.Western blot was further improved to find that the expressions of Wnt7b and-catenin proteins in the cells of the high-oxygen group also decreased correspondingly.And the expression levels of Wnt7b and-catenin increased after RA was added,but all of them were lower than those in the air group.The above results suggest that the protective effect of RA on hyperoxic lung injury may be related to the activation of the classical Wnt pathway.Conclusion:RA can reduce the high oxygen induced AEC Ⅱ cell damage.During this process,the changes of Wnt pathway suggest that it can participate in the protective effect by activating the Wnt signaling pathway.