Study On Mechanism Of Retinoic Acid On Differentiation Of Primary Fetal Alveolar Epithelial Type Ⅱ Cells

Chapter One The Primary Culture of Fetal Alveolar Epithelial Type Ⅱ Cells and Identification of its TransdifferentiationObjective To explore the method of isolation, purification and culture of primary fetal alveolar epithelial type Ⅱ cell (fAECII) and observe the growth condition and transdifferentiation of fAECII. Methods Fetal rat lung tissues were extracted from pregnant SD rats ( 19 days),and then fAECII were isolated and purified by trypsin and collagenas type IV successively digestive method, differential centrifugation and IgG immune adherence method and adhesion method. After fAECII were cultured for 24, 48,72,96 hours, the morphological, cell ultrastructure and the expression of protein SP-C as well as AQP5 were respectively observed by inverted microscope, transmission electron microscope,immunofluorescence under a confocal microscope. The fAECIIs were identified by transmission electron microscope and laser confocal microscopy. Results Under inverted microscope, fAECII could be observed adhere to the bottom of the culture dishs after cultured for 8 hours in insular growth pattern. As culture time passed, cell size as well as cell number were increasing, with obviously nucleolus, nuclear membrane and bridge connection.Cultured 96 h, cells appeared to be severely vacuolation degeneration and pseudopodium.Under laser confocal microscope, with the extended culture time, the expression level of SP-C green fluorescence in cytoplasm was highest and then decreased; the expression level of AQP5 red fluorescence in cell membrane gradually increased. Both green fluorescence and red fluorescence were detected at 72h. Under transmission electron microscope, there were characteristic ultrastructures of fAECIIs with microvilli and lamellar bodies at 24 h. Only microvilli or lamellar bodies were observed at 72h. No microvilli and lamellar bodies were observed at 96h. Conclusion fAECII were isolated and purified by trypsin and collagenas type IV successively digestive method, differential centrifugation and IgG immune adherence method and adhesion method can provide basic for follow-up studies with highly activity and purification. Both electron microscope and immunofluorescence could identify the cells.Chapter Two The changes of Wnt7b /β-catenin signaling pathway molecules in the differentiation of fetal alveolar epithelial type ⅡcellsObjective To investigate the changes of Wnt7b/β-catenin signaling pathway molecules in the differentiation of fetal rat alveolar epithelial type Ⅱ cells (fAECIIs). Methods Fetal rat lung tissues were extracted from pregnant SD rats (19 days), and then fAECII were isolated and purified. After fAECIIs were cultured for 48, 72, 96 hours,the morphological changes of fAECII and the expression of β-catenin were respectively observed by inverted microscope and immunofluorescence. The mRNA expressions of Wnt7b, cyclin D1, pulmonary surfactant C ( SP-C) and aquaporin 5 ( AQP5) were detected by real-time quantitative PCR. The protein expressions of cyclin D1, nucleus β-catenin, SP-C and AQP5 were measured by Western blotting. Results P-catenin was expressed in cell membrane when fAECII were cultured for 48 hours; the expression of β-catenin decreased in cell membrane while enhanced in cytoplasm and nucleus at 72 hours; whole-cell β-catenin expression was lowered at 96 hours. The expressions of Wnt7b and cyclin D1 mRNA were significantly raised at 72 hours and reduced obviously at 96 hours compared with 48 hours. SP-C mRNA expression level went down gradually with the extended culture time, and AQP5 mRNA level went up gradually. The protein expressions of cyclinD1 and nucleus β-catenin were significantly enhanced at 72 hours and weakened at 96 hours compared with 48 hours. SP-C protein expression was down-regulated with the prolonged culture time, and AQP5 protein expression was up-regulated. Conclusion Wnt7b/p-catenin signaling pathway may play an important role in fAECII transdifferentiation.Chapter Three Study on Mechanism of Retinoic Acid on Differentiation of Primary Fetal Alveolar Epithelial Type ⅡCellsObjective The aim of this study was to observe the effect of RA on the transdifferentiation of primary fetal alveolar epithelial type Ⅱ cells (fAECIIs), and to explore the possible signaling pathway participating in regulating transdifferentiation process. Methods Primary fAECIIs were isolated and purified from fetal rats at 19 d of gestation by differential centrifugation and adhesion methods. After been cultured 36 hours, fAECIIs were randomly divided into DMSO groups, RA groups and BMS493+RA groups, another 24, 48, 72 h were cultured. Cell proliferation and viability were detected by using 4, 5-Dimethylthiazol-2-y1-2,5-diphenyltetrazolium bromide (MTT). The mRNA expressions of Wnt7b, pulmonary surfactant and aquaporin 5 were detected by real-time quantitative PCR, and the protein expressions of cyclin D1, nucleus β-catenin,SP-C and AQP5 were measured by Western blotting. The expression of β-catenin was also observed byimmunofluorescence. Results Cell proliferation and viability: RA promoted fAECIIs proliferation, in 24, 48, 72 h, compared with DMSO groups. Compared with RA group, the attenuation effect of BMS493 on RA proliferation was observed at concentrations as low as 10-9M on 24 h; 10-8 and 10-7M BMS493 inhibited the RA proliferation on 48 h; the maximal inhibitory effect was observed when cells were exposed to 10-8 and 10-7 M BMS493 on 72 h. It was shown that cell proliferation and viability of fAECIIs exposure to 10-8M BMS493 was higher than DMSO groups. Compared with DMSO groups,the fAECIIs were larger,more obvious extense and better in RA groups; the size and extension were less large and obvious in RA+BMS493 groups. The fAECIIs transdifferentiation into AECIs: Compared with RA group, RA significantly decreased the expression of SP-C mRNA and protein SP-C on 24 h and 48 h while enhanced the expression of SP-C mRNA and protein on 72 h; RA up-regulated the expressions of AQP5 mRNA and AQP5 protein. In RA + BMS493 groups, the changes of SP-C and AQP5 protein and mRNA expression levels were partially suppressed. Signaling pathway molecules: RA promoted the expression of mRNA, nucleus β-catenin protein and cyclinDl protein, and elevated the expression of β-catenin in the cytoplasm and nucleus compared with DMSO groups. In RA + BMS493 groups, the expression of mRNA, nucleusβ-catenin protein and cyclinDl protein were partially suppressed by pan-RAR antagonist BMS493. Conclusion Retinoic acids may promotes primary fetal alveolar epithelial type Ⅱcell transdifferentiation through Wnt7b/β-catenin signaling pathway.