| Objective The AEC? model of primary premature rats in experimental experiments was established,and the separation,purification and culture methods were further explored,which laid a foundation for subsequent high-oxygen-induced AEC? injury and calcitonin gene-related peptide(CGRP)intervention experiments.To investigate the effect and significance of CGRP on AEC? in premature rats exposed to oxygen volume fraction greater than 95%.Whether the protective effect of CGRP on hyperoxia-induced AEC? injury involves Notch signaling pathway,and changes in Notch1,Hes and HERP signaling molecules in this signaling pathway were observed.To explore the effects of exogenous neuropeptide-like transmitters on Notch signaling pathway during lung injury,providing a drug target for possible treatment.Methods The SPF-class SD premature rats were used as the experimental cell source for 19 days,and the lung tissues were taken out and the lung tissues were digested with trypsin combined with DNase and collagenase I to separate the lung cells.AEC? was purified by differential centrifugation and differential adherence,and then cultured in DMEM/F12 complete medium(10%fetal bovine serum concentration).Trypan blue staining was used to detect AEC ? activity and modified Pap stain to detect AEC ? purity.After isolation and purification of primary AEC? in premature rats,they were randomly divided into normoxic group,hyperoxia group,hyperoxia+CGRP group,hyperoxia+Gamma secretase inhibitor MW167 group,hyperoxia+CGRP+CGRP8-37group,hyperoxia+Gamma secretase inhibitor MW167+CGRP group.The normoxic group was cultured in an indoor air condition,and the hyperoxia group was administered with oxygen having an oxygen volume fraction greater than 95%.After 24 hours of culture,the morphological changes of AEC? were observed.The cell viability was detected by CCK8 assay.The apoptosis of AECI and AEC? cells was detected by flow cytometry.Malondialdehyde(MDA)and superoxide dismutase(SOD)were respectively observed.The oxidative damage and anti-oxidative damage were detected.The expression levels of Notch1,Hes and HERP protein were detected by Western blot.The expression of Notch1,Hes and HERP mRNA was detected by QPCR.Results 1.The primary cultured AEC? yield is better.The number of AEC? available in each premature rat is(8.2±1.2)×10~6,the viability of the cells is(95.5±1.6)%;the purity of the cells is identified as(95.7±2.2)%.2.Compared with the normoxic group,the survival rate and SOD content of AEC? in the hyperoxia group decreased significantly,and the apoptotic rate and MDA content increased(P<0.05).Compared with the hyperoxia group,the apoptotic rate of AEC? decreased and survived in the hyperoxia+CGRP group.Increased rate,down-regulated MDA,up-regulated SOD,CGRP8-37 can block the biological effects of CGRP.3.Compared with normoxic group,the expression of Notch1,Hes and HERP protein and mRNA in hyperoxia group decreased significantly(P<0.05).Compared with hyperoxia group,the expression of Notch1,Hes and HERP protein and mRNA in alveolar cells of hyperoxia+CGRP group(P<0.05).The expression of Notch1,Hes and HERP protein and mRNA in hyperoxia+CGRP and its antagonist group decreased to the level of hyperoxia group(P<0.05).The expression of Notch1,Hes and HERP protein and mRNA in the hyperoxia+CGRP+MW167 group was significantly higher than that in the hyperoxia group(P<0.05).Conclusions1.Improved by cell culture experiments,using differential adherence and centrifugation to separate and purify,cultured AEC?,high purity,good vigor compared with the previous method,low mortality,can be used in AEC? and other related experimental research.2.By successfully producing AEC? injury model under high volume fractional oxygen exposure,it was found that CGRP has partial anti-oxidative stress ability,which can promote the proliferation of AEC? in the case of high volume fractional oxygen,and has a certain protective effect on damaged AEC?.3.The study found that Notch signaling pathway may be involved in the repair process of AEC? injury under high volume fractional oxygen exposure.The protective effect of CGRP on AEC ? after injury may be related to the activation of Notch signaling pathway. |
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