Expression Of SPOCK2 Gene In Neonatal Rat Lung With Hyperoxia Exposure And Plasmid Construction Of Its Highly Expressed Lentiviral Vector

Background and Objective Bronchopulmonary dysplasia(BPD)may be one of the most destructive disorders in preterm infants,including the detrimental effects on lung,neurodevelopment,and multiple systems.Its pathogenesis is complex and there is no effective prevention and control measure.Based on the evidences that SPOCK2gene is highly related to BPD,we designed this study to explore the role of SPOCK2gene in lung development by construct a neonatal rat model of BPD.In this study,SPOCK2 plasmid was constructed,then packaged with lentiviral vector and transfected into A549 and UCB-MSCs to provide experimental basis for related functional studies of SPOCK2 gene.Methods 1.BPD models were induced by 85%O2 exposure.Newborn SD rats were randomly divided into the air group and the hyperoxia(85%oxygen)group.The pathological changes of pulmonary tissues were observed by hematoxylin-eosin(HE)staining.The expressions of SPOCK2 mRNA were detected by real-time quantitative PCR.The expression of SPOCK2 protein was detected by immunohistochemistry.2.Through real-time quantitative polymerase chain reaction(RT-PCR),the SPOCK2CDS(Coding sequence)region was captured using a primer and cloned into the lentiviral expression plasmid pHAGE-CMV-MCS-IzsGreen(expressing green fluorescent protein).3.Recombinant expression plasmids were identified by sequencing and transfected into 293T cells to detect SPOCK2 mRNA expression levels.4.Recombinant expression plasmid and packaging plasmid were co-transfected into 293T cells to construct a lentiviral expression vector,which was purified and concentrated to determine its titer.5.The recombinant lentiviral expression vector infected A549 and UCB-MSCs to establish stable transfected cell lines.Flow cytometry was used to detect the efficiency of virus infection.Western Blot and qPCR were used to detect protein and SPOCK2 mRNA expression.Results 1.(1)HE staining:The pathological changes of BPD were found in the pulmonary tissue of the model group.The value of pulmonary radical alveolar counts(RAC)in the model group was significantly lower than that of the control group at 10days and 14 days(P<0.05).(2)As shown with immunohistochemistry,the expression of SPOCK2 protein in control group was higher than that in model group,especially at 10 days and 14 days.(3)The expression of SPOCK2 mRNA in the control group was higher than that in model group,which reached its peak at 18 days and decreased in adulthood.(4)Compared with the control group,the expression of SPOCK2 mRNA in model group decreased.The difference was statistically significan at 10 days and 14 days(P<0.05).2.(1)A recombinant lentivirus plasmid pHAGE-SPOCK2 was successfully constructed with a virus titer of approximately6*10~7 Tu/ml.The expression of SPOCK2 in 293T could be detected by the expression of IzsGreen and sequencing.(2)Recombinant lentivirus vector was successfully transduced into A549 and UCB-MSCs as evidenced by significantly increased expression levels of SPOCK2 mRNA and protein(P<0.0001),indicating successful construction of lentivirus-mediated stable transfection cell lines.Conclusion 1.Successfully constructed a neonatal rat model of BPD.The SPOCK2 gene may be involved in the development of lung in neonatal rat,especially during the development of alveoli.2.Successful construction of SPOCK2-IzsGreen fusion gene lentivirus expression vector.Lentivirus-mediated A549 and UCB-MSCs stably transfected cell lines carrying the SPOCK2 gene were successfully constructed.For further study of the mechanism of action of the SPOCK2 gene provides cell lines.