Study On Peripheral Blood Related Immune Cells In Premature Infants With Bronchopulmonary Dysplasia

BackgroundBronchopulmonary dysplasia（BPD）is the most common and severe chronic lung disease in infancy,which mainly occurs in premature infants,especially very premature infants（VPIs）and extremely premature infants（EPIs）.It is one of the leading causes of death in premature infants,seriously jeopardizing their health.The main risk factors of BPD include prematurity（immature lung development）,oxygen inhalation,mechanical ventilation,perinatal infection,and genetic susceptibility etc.Despite the rapid development of medicine,the pathogenesis of BPD has not been fully elucidated,effective prevention and treatment methods are still lacking,and BPD has not been effectively curbed.Previous studies have shown that inflammation plays an important role in the occurrence and development of BPD,but there is no study about the impact of changes in peripheral blood related immune cells on the prognosis of BPD.For instance,children with BPD have a high incidence of secondary respiratory tract infection,but few related mechanisms have been studied.Neutrophils are at the forefront of resistance to pathogen invasion and play an important role in non-specific immunity.As the constitutive proteins of neutrophils,S100A8 and S100A9 are also involved.If they are insufficient,the chance of infection may increase.So,how do S100A8,S100A9 and peripheral blood related immune cells,including neutrophils,change in BPD?What are the possible mechanism leading to these changes and what are the clinical implications of these changes?All of these prompted us to conduct the following research.ObjectivesTo investigate the changes of S100A8 and S100A9 expression in lung tissues and the number of peripheral blood related immune cells（including neutrophils,lymphocytes and monocytes）of BPD through animal experiments and clinical retrospective analyses,and to explore the possible mechanism,potential significance of these changes,so that it can provide a theoretical basis for better clinical management of children with BPD.MethodsPart I:Animal experiment:Firstly,20 neonatal SD rats with less than one hour difference in birth time were randomly divided into 21%O₂group（control group,air group or non-BPD group,n=10,the same below）and 85%O₂group（experimental group,hyperoxia group or BPD group,n=10,the same below）within 6 hours after birth.The hyperoxia（85%O₂）-induced BPD model of newborn SD rats was established.The general state of neonatal SD rats was observed,and their body weight was aslo measured and recorded every day.Secondly,the lung tissue samples were extracted at 14 days after birth for HE and CD34 immunohistochemical staining,and the modeling effects were evaluated by observing the morphological changes of the lungs.Thirdly,the changes of gene expression in the lung tissues of 14-day-old newborn BPD rats were detected by RNA sequencing,then the target genes were screened out,and their changes and pathological significance were discussed.Fourthly,the results of RNA sequencing were further validated by RT-PCR for the target genes.Finally,the peripheral blood of 14-day-old newborn SD rats was subjected to a fully automated blood analyzer for complete blood count test to find out the changes of their peripheral blood neutrophils.Part II:Clinical Research:At first,the inclusion criteria of cases was established and the cases meeting the criteria were included in the research;then,retrospective analysis was made on the gestational age,birth weight and peripheral blood immune cells counts（including neutrophils,lymphocytes and monocytes）of full-term infants(gestational age 37⁺⁰-41⁺⁶weeks,n=88),preterm infants without BPD（gestational age<32 weeks,n=41）,preterm infants with BPD（gestational age<32 weeks,n=35）discharged from neonatal intensive care unit of Shiyan Taihe Hospital（affiliated hospital of Hubei University of Medicine）from January 1,2014 to December 31,2017.Results1.Compared to the newborn SD rats in the 21%O₂group,with the prolongation of hyperoxia exposure time,those in the 85%O₂group showed weight loss,dry fur,insufficient vitality,oxygen dependence,and at 14 days after birth,their lungs were pale and dull with alveolar structure simplification,uneven alveolar,partial alveolar fusion,alveolar cavity enlargement（MLI↑）,alveolar number reduction（RAC↓）and decreased pulmonary microvessel density（MVD↓）.2.RNA sequencing results showed that the gene expression in lung tissue of85%O₂-BPD model rat changed at 14 days after birth.Among them,S100A8m RNA and S100A9 m RNA decreased significantly,which was further confirmed by RT-PCR.3.Complete blood count（CBC）showed that the number of peripheral blood neutrophils in 85%O₂-BPD rats was lower than that in the 21%O₂group at 14days after birth.4.The number of peripheral blood neutrophils in preterm infants with gestational age less than 32 weeks（divided into preterm infants without BPD group and preterm infants with BPD group）was significantly lower than that in full-term infants.There was no significant difference in the number of peripheral blood neutrophils at birth between preterm infants with or without BPD.After birth,the number of peripheral blood neutrophils in preterm infants with BPD decreased gradually,and at 36-37 weeks of postmenstrual age（PMA）,the number of peripheral blood neutrophils in preterm infants with BPD were significantly lower than those in preterm infants without BPD.However,regardless of the difference of peripheral blood lymphocytes or monocytes number at birth between preterm infants and full-term infants,it was not as obvious as that of peripheral blood neutrophils.Furthermore,the levels of peripheral blood lymphocyte and monocyte in preterm infants with or without BPD were relatively stable from birth to 36-37 weeks of postmenstrual age.Conclusions1.Continuous inhalation of 85%O₂for 14 days resulted in oxygen dependence,obstruction of alveolarization and pulmonary microvascular dysplasia in neonatal SD rats,showing typical clinical and pathological features of BPD.85%O₂inhalation can be used to construct BPD model.2.The gene expression in lung tissue of 85%O₂-BPD model newborn SD rats changed,and the down-regulation of S100A8m RNA and S100A9m RNA was observed.3.Long-term oxygen inhalation can lead to the decrease of peripheral blood neutrophils in children with BPD.4.Unlike peripheral blood neutrophils,peripheral blood lymphocytes and monocytes are less affected by gestational age and long-term oxygen therapy after birth.It indicates that peripheral blood lymphocytes and monocytes are not sensitive to long-term high oxygen exposure.5.With the prolongation of oxygen therapy after birth,the number of peripheral blood neutrophils in children with BPD decreased,which may be one of the reasons for their susceptibility to respiratory tract infection.