Effect Of RA And IL-24 On The Proliferation And Differentiation Of Primary Fetal Alveolar Epithelial Type ? Cells

Objective:To explore the method of isolation, purification and culture of primary fetal alveolar epithelial type II cell (fAECIIs) and observe the growth condition and transdifferentiation of fAECIIs; To explore and observe the effect of retinoic acid (RA) and IL-24 on fAECIIs proliferation and differentiation.Methods:Primary fAECIIs were isolated from fetal rats at 19-days of gestation and purified by trypsin and collagenas type IV combined digestive method, differential centrifugation and adhesion method and IgG immune adherence method. After culture fAECIIs for 24h,48h,72h,96h, cell growth condition, cell number, cell ultrastructure, expression of protein SP-C as well as AQP5 and expression of SP-C mRNA as well as AQP5 mRNA were respectively detected by inverted microscope, cell counting chamber, transmission electron microscope, immunofluorescence under a confocal microscope and RT-PCR. After cultured for 24h, the fAECIIs were interfered by RA, IL-24 and Anti IL-24 for 24h,48h and 72h. Cell proliferation and viability, growth state, cell cycle, cell apoptosis, expressions of SP-C mRNA as well as A QP5 mRNA and expressions of protein SP-C as well as AQP5 were respectively detected by using 4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT), inverted microscope, flow cytometry, RT-PCR and Western blot.Results:1. The fAECIIs had a purity of 97.8±2.1%. Under inverted microscope, fAECIIs was in insular growth pattern. As culture time passed, cell number was increasing and cell size was also increasing, with the appearance of dikaryocytes and bridge connection. Eventually, cells appeared to be vacuolation degeneration and apoptosis. Cell number was the highest at 72h, and then the cell number decreased. The cell number among different time points had statistical significance (P<0.01). Under transmission electron microscope, there were characteristic ultrastructure of fAECIIs that microvilli and lamellar bodies at 24h and 48h. The intermediate form cells between AECII and AECI was observed at 72h. No microvilli and lamellar bodies were observed at 96h. Under confocal microscope, 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 observed at 72h. The expression level of SP-C mRNA was highest at 48h and then decreased. As well as, the expression level of AQP5 mRNA gradually increased.2. Effect of RA on fAECIIs:When fAECIIs were treated with RA for 24h, the cell proliferation and viability did not change (P>0.05), while the proliferation and viability was significantly improved at 48h (P<0.05) and the difference was the most obvious (P<0.05) at 72h. Compared with the control group, the cell growth state was better, the cell adherence was tighter and the refraction was higher in RA group. This effect of RA was most significant at 72h. When fAECIIs were treated with RA for 24h, cell cycle did not change (P>0.05), while more cells were in G2 phase and S phase at 48h and 72h (P<0.01). Cell apoptosis did not change when cells exposure to RA for 24h; while the percentage of cells in early and late apoptosis was significantly lower than those in control group (P<0.01). RA up-regulated the expressions of AQP5 mRNA and AQP5 protein (P<0.05).The expressions of SP-C mRNA, SP-C protein was up-regulated when cells exposure to RA for 24h and 72h while it was down-regulated at 48h (all P<0.05). 3. Effect of IL-24 on fAEC?s:The expression of IL-24mRNA and IL-24 protein decreased significantly with the time (P<0.01). IL-24 significantly promoted the proliferation of fAECIIs (P<0.01). The proliferation promotion effect of IL-24 was the most obvious at the concentration of 5?g/L IL-24. Anti IL-24 stept down cells proliferation (P<0.01). The proliferation inhibition effect of anti IL-24 was the most obvious at the concentration of 20?g/L anti IL-24. The cell adherence was tighter and the refraction was higher in IL-24 group. The cell refraction was lower and cells extended more obvious in anti IL-24 group. More cells were in S phase and less cells were in G1 phase in IL-24 group. In contrary, more cells were in G1 phase and less cells were in S pahse in anti IL-24 group. The percentage of cells in early and late apoptosis in IL-24 group was significantly lower than those in control group (P<0.01) while the percentage of cells in early and late apoptosis in anti IL-24 group was significantly higher (P<0.01). IL-24 up-regulated the expressions of SP-C mRNA and SP-C protein (P<0.05), while anti IL-24 down-regulated the expressions of SP-C mRNA and SP-C protein (P<0.01). IL-24 down-regulated the expressions of AQP5 mRNA andAQP5 protein (P<0.05), while anti IL-24 up-regulated the expressions of AQP5 mRNA and AQP5 protein.Conclusion:1. fAECIIs purified by trypsin and collagenas type IV combined digestive method, differential centrifugation and adhesion method and IgG immune adherence method can provide basic for follow-up studies. Cells actively proliferate in early time (24h-48h). Then the proliferation speed decreased in the later time (48h-72h) as well as cells began to transdifferentiation and gradually to be apoptosis at 96h.2. RA promotes fAECII proliferation by improving cell viability, regulating cell cycle, and inhibiting apoptosis. In addition, RA promotes the differentiation of fAECIIs, and improves the expressions of SP-C and AQP5.3. IL-24 promotes fAECII proliferation by improving cell viability, regulating cell cycle, and inhibiting apoptosis. In addition, IL-24 inhibits the differentiation of fAECIIs.