| Part one Isolation, purification, and culture of primary alveolar epithelial typeⅡcells of preterm ratsObjectiveTo investigate the method of isolation, purification and primary cell culture of alveolar epithelial typeⅡcells (AECⅡ) from preterm rats, and establishing AECⅡculture mode1 to study the effect of endothelin-1 (ET-1) on hyperoxia-injured AECⅡand its signal pathway.MethodsAECⅡwere isolated from the fetal lungs of Sprague-Dawley (SD) rats with the gestational age of 19 days. The pregnant rats were anesthetized by chloral hydrate. Uterine incision delivery was used to pick out the fetal rats and the fetal lung was isolated. Lung tissues were digested by trypsin and collagenase, then purified for AECⅡwith different centrifugal force and repeated attachment. AECⅡwere cultured in dulbecco's modified eagle's medium(DMEM/F12) complemented with 10% fetal calf serum. Cells viability was assessed by trypan blue exclusion. Modified papanicolaou staining was employed to evaluate cells purity. The cells were identified by transmission electron microscopy. Meanwhile, cellular growth status and morphologic change were observed with inverted phase contrast microscope.ResultsThe number of preterm rats we obtained from one pregnant rat was 8~11 (95% CI), and (7.7±0.9)×106 AEC II could be achieved from one preterm rats. The viability of AEC II was (95.5±1.6)%, and the purity was (95.0±1.5)%. The lamellar bodies in cytoplasm and abundant microvilli on cell surface were observed under electron microscope. AECⅡattached 12~18h after culture. At 24~48h of culture, the AECⅡwas in exponential growth phase. Cells became flat after 72h and attached cells decreased.ConclusionTrypsin combined with collagenase digestion, and different centrifugal force and repeated attachment were the efficient methods to obtain AECⅡwith highly yield, purity and activity. It could be useful in research. Cells grew well at 24~48h after the primary culture, thus AECⅡcould be obtained for study in vitro in this period. Part two The effects of endothelin-1 on preterm rat typeⅡalveolar epithelial cells exposed to hyperoxiaObjective To establish oxidative damage pattern of primary AECⅡfrom preterm rats, and investigate the proliferation, death and surfactant protein C (SP-C) expression of hyperoxia-injured AECⅡtreated with ET-1 and ET receptor antagonists.Methods1. Primary AECⅡwere cultured in plates for 15 to 18 hours at first, then randomly divided into 3 groups: air group, hyperoxia group, hyperoxia plus ET-1 (10-10 mol/L～10-6 mol/L) group. Cells of hyperoxia group and hyperoxia plus ET-1 groups were placed in the sealed oxygen container with 95% O2 and 5% CO2, then all of the three groups were placed in the incubator for 24h. The methyl thiazolyl tetrazolium (MTT) assay was taken to determine the proliferation of AECⅡin each group.2. AECⅡwere randomly divided into 4 groups: (1)air group; (2)hyperoxia group; (3) hyperoxia plus ET-1 group: 10~-8 mol/L ET-1 was added into the cell culture medium before hyperoxia were treated; (4) hyperoxia plus ET-1 and ET receptor antagonist (PD145065) group: 10~-8 mol/L ET-1 and 10~-6 mol/L PD145065 were added. Then the cells were exposed to air or hyperoxia for 24h respectively. The morphologic changes of AECⅡwere observed under inverted phase contrast microscope. Cell proliferation was determined by MTT assay and cell death detected by flow cytometry. The mRNA levels and protein contents of SP-C in each group were measured by RT-PCR and immunofluorescence respectively.ResultsThe proliferation of hyperoxia-exposed AECⅡwas obviously inhibited by ET-1 when the concentration of ET-1 was from 10~-8 mol/L～10~-6 mol/L. These data suggested that 10-8 mol/L ET-1 had effect on AECⅡcells exposed to the hyperoxia and could be used for further research. The result of MTT assay showed that the proliferation of AECⅡwas obviously inhibited by hyperoxia and ET-1 enhanced the inhibition; Annexin V/PI staining assay showed that AECⅡexposed to the hyperoxia environment underwent death, and ET-1 can further aggravate this process; PD145065 attenuated the ET-1-induced inhibition of cell proliferation and increase of cell death. Hyperoxia significantly inhibited SP-C mRNA expression, and the inhibition was further enhanced by ET-1, PD145065 reversed ET-1 mediated SP-C expression inhibition. We likewise found the same change of SP-C protein expression by Immunofluorescence assay.ConclusionHyperoxia induced AECⅡdeath, and inhibited the cell proliferation and SP-C expression of AECⅡin vitro; ET-1 aggravated hyperoxia-induced injure of fetal rat AECⅡ, increased cell death rate and inhibited SP-C expression were further enhanced. The non-selective endothelin receptor antagonist, PD145065, inhibited these effects of ET-1; it may play a protective role in ET-1 exacerbated cell proliferation inhibition and increased death rate of hyperoxia-injured AECⅡ.Part three The signal mechanism for ET-1 aggravated hyperoxia-induced injury of preterm rat typeⅡalveolar epithelial cellsObjectiveTo investigate the proliferation, death and surfactant protein C (SP-C) expression of hyperoxia-injured AECⅡtreated with ET-1 and ET receptor antagonists, and the effect of ROS-MAPKs signal pathway on ET-1 aggravated hyperoxia-induced injury of AECⅡ.MethodsPrimary AECⅡwere cultured in plates for 15 to 18 hours at first, then randomly divided into 5 groups: (1)air group; (2)hyperoxia group: cells were placed in the sealed oxygen container with 95% O2 and 5% CO2; (3) hyperoxia plus ET-1 group: 10-8 mol/L ET-1 was added into the cell culture medium before hyperoxia were treated; (4) hyperoxia plus ET-1 and ET receptor A antagonist (BQ123) group: 10~-8 mol/L ET-1 and 10~-6 mol/L BQ123 were added; (5) hyperoxia plus ET-1 and ET receptor B antagonist (BQ788) group: 10~-8 mol/L ET-1 and 10~-6 mol/L BQ788 were added. Then the cells were exposed to air or hyperoxia for 24h respectively. Cell proliferation was determined by MTT assay; the death rate and intracellular reactive oxygen species (ROS) levels of AEC II were analyzed by flow cytometry. Western blot analyses for SP-C, phosphorylated and total nonphosphorylated ERK, JNK or p38.ResultsET-1 accentuated the inhibition of AECⅡproliferation induced by hyperoxia and increased the death rate of hyperoxia-injured AECⅡ. In addition, ET-1 reduced SP-C expression and increased ROS production in AECⅡ. The phosphorylated ERK, JNK and p38 increased in AECⅡexposed to the hyperoxia, ET-1 reduced phosphorylated ERK and further up-regulated phosphorylated JNK and p38. The BQ123, not BQ788, inhibited these effects of ET-1. While the total nonphosphorylated ERK, JNK or p38 were no difference in each group.ConclusionET-1 markedly aggravated hyperoxia-induced AECⅡinjury by increasing ROS generation and regulation of MAPKs signaling pathways mediated by the ET receptor A. The ET receptor A antagonist, BQ123, had a potential protective effect on ET-1 potentiating hyperoxia-induced injury of AECⅡ.
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