The lung tissue of 20-day fetal rat was digested by
trypsin and
collagenase. AECIIs and lung fibroblasts (LFs) were isolated and purified at different centrifugal force and different adherence, then cultured. The nature of the cultures was identified by
cytokeratin staining,
vimentin staining and transmission electron micrography. For establishing
hyperoxia-exposed cell model, purified AECIIs were cultured for 24 hours after culture flasks were filled with 95%
oxygen-5% CO2 at 3 L/min for 10 min, and then sealed.
Oxygen concentrations were tested in CYS-1 digital
oxygen monitor after 24 hours of exposure. A sample was discarded if its
oxygen concentration was < 90%. Cell proliferating vitality was examined by MTT assay
after treatment with
amygdalin at various concentrations.
DNA content,
protein expression of
proliferating cell nuclear antigen (
PCNA) and
mRNA levels of SPs of AECIIs were analyzed with flow cytometric assay, Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively after 24 hours of air or
hyperoxia exposure in the presence or absence of 200 micromol/L
amygdalin.
RESULTS: Excellent yields of highly purified, culturable AECIIs could be obtained from 20-day fetal lungs. The expression of
cytokeratin in AECIIs was positive and that of
vimentin negative by immunocytochemistry. Those, however, in LFs were just opposite. Lamellar bodies in purified AECIIs were revealed by transmission electron micrography. The established
hyperoxia-exposed cell model assured the
oxygen concentrations of culture flasks more than 90%.
Amygdalin at the concentration range from 50 micromol/L to 200 micromol/L stimulated the proliferation of AECIIs in a dose-dependent manner; however, at the concentration of 400 micromol/L inhibited the proliferation of AECII. Flow cytometric analysis showed that the apoptosis rate and G0/G1 phase percentage increased significantly (P < 0.01), S phase and G2/M phase percentage decreased significantly (P < 0.01), in
hyperoxia group compared with that of air group. The apoptosis rate of air plus 200 micromol/L
amygdalin group, compared with air group, was not significantly different (P > 0.05); however, G0/G1 phase percentage decreased markedly, S phase percentage increased significantly, G2/M phase percentage did not significantly change (P > 0.05). The apoptosis rate of
hyperoxia plus 200 micromol/L
amygdalin group was not significantly different (P > 0.05) from that of
hyperoxia group, S phase and G2/M phase percentage increased significantly (P < 0.01), G0/G1 phase percentage decreased significantly (P < 0.01). Western blot analysis showed that the
protein expression levels of
PCNA in all group was significantly different, in turn,
hyperoxia group <
hyperoxia plus 200 micromol/L
amygdalin < air group < air puls 200 micromol/L
amygdalin (P < 0.01). SPs
mRNA levels were significantly decreased in
hyperoxia group, as compared with air group (P < 0.01). After
amygdalin was added, SPs
mRNA levels were elevated in air plus
amygdalin group and
hyperoxia plus
amygdalin group, as compared with
hyperoxia group (P < 0.01, P < 0.05, respectively), but compared with air group, SP
mRNA levels were not significantly elevated (P > 0.05).
CONCLUSION: AECIIs of premature rats were isolated, purified and cultured successfully.
Hyperoxia-exposed cell model was established in AECIIs of premature rat in this experiment.
Amygdalin promotes the proliferation of premature rat AECII exposed to air or
hyperoxia, the concentration of
amygdalin with the best effect was 200 micromol/L.
Hyperoxia inhibited the proliferation and decreased SPs mRNAs levels in AECIIs in vitro, which may contribute to
hyperoxia-induced
lung injury in premature rats.
Amygdalin could inhibit the changes of SPs mRNAs levels and cell proliferation of AECIIs resulted from
hyperoxia and may play partial protective role in
hyperoxia-induced premature
lung injury.