| BACKGROUNDChronic lung disease(CLD) is one of the most common and significant medical complications associated with preterm birth, and is characterized as a fibrotic pulmonary endpoint. CLD is an important source of morbidity and mortality in premature neonates. About 10 percentage to 15 percentage of the patients died of respiratory failure within 1 year after birth, and the survivors need to rely on oxygen or ventilator therapy that affect the future quality of life,also increase financial and spiritual burden to family and society. Advances in perinatal medicine have resulted in increasing numbers of very-low-birth-weight infants who are at risk of CLD,and the incidence of BPD appears to be growing in conjunction with the increased survival.Infants develop CLD in about 1.5 percentage of all newborn births.And it has become one of the most intractable problems in the neonatal intensive care unit.The pathogenesis of chronic lung disease is very complex, but its essence is the injury (such as oxygen toxicity, barotrauma or volume injury, infection or inflammation)on the basis of genetic susceptibility disturbed normal lung development and caused disorder of the repair.Pulmonary inflammation has a central role in the complex pathogenesis of CLD. Cytokines are the main immune mediators involved in this process and to promote the occurrence and development of CLD. However,our understanding of the underlying processes which lead to the development of CLD remains rudimentary. Many studies have been conducted to evaluate the presence of cytokines in the amniotic fluid, cord blood, plasma and tracheal secretions of preterm neonates at risk or who have CLD,confirmed that "pro-inflammatory"cytokines (include interleukin(IL)-1β, IL-6, IL-8, TNF-αetc) which showed early and sustained high levels of expression in lung tissue or bronchoalveolar lavage fluid (BALF) occurred in CLD caused by species of causes. However,the duration remains controversial.Chemokines, including monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, macrophage inflammatory protein (MIP)-1a, MIP-1βhave a relationship with chronic lung disease. Detectable high levels of MCP-1 in tracheal aspirates could correlate with airway colonization of ureaplasma urealyticum(a risk factor of CLD). Cytokines include IL-4,IL-10,IL-12 and IL-13 usually act in an anti-inflammatory capacity.The decreased levels of those cytokines would predispose to the development of CLD given the role that inflammation likely has in this disorder. However, there are discrepant findings to above results:studies have shown that the levels of cytokines (IL-6,TFN-α,IL-1β) in blood and BALF have no correlation with CLD;some experiments also found that the levels of "anti-inflammatory" cytokines in the tracheal fluid of preterm infants at risk for CLD,particularly IL-10,eemingly no difference compared with other infants.So what cytokines exactly participate the occurrence of CLD, which cytokines play a role or play a key role in the stages of acute and non-acute of CLD, how the relationship among various cytokines, whether the work factors are the same, and is it just cytokines involved in the acute phase, whether corresponding changes occurred in peripheral blood, Specific duration etc, still need further study. Most studies of CLD focus on cytokines locally in the lungs, no matter at home or abroad. There's little studies about systemic immunological condition of CLD. The majority of research methods are animal experiments, very little clinical,and samples are usually small. The studies of cytokines in whole blood is rare. Whether systemic immunity is involved in the pathogenesis of CLD, how are the systemic immune status in different stages of CLD, and whether relating to local immunity in the lungs. These problems are still rare reported in the literature.Because sample processing and detection methods are different, the results of cytokines vary greatly, and there is no fixed reference values can be used to compare. Single or several cytokines test can hardly reflects the whole situation in vivo because of the complexity of immune responses.The key to solving this problem is to have a unified method of sample processing, using detection equipment with high sensitivity and can simultaneously detect a variety of cytokines each time. The system of Luminex is the only FDA-certified by the U.S It could meet our need of rapiding, micro-volume and mufti-cytokine test to reduce the testing error.Thus, in order to detect multiple cytokines in tiny volume of whole blood,we selected the Luminex technique,it can detect 17-cytokine simultaneously.OBJECTIVETo explore the possible role of general immunity of chronic lung disease (CLD) in premature infants by investigating the concentration of 17 cytokines of whole blood. In order to guide the application of targeted immune therapy, search for new treatments, improve prognosis and provide some theoretical and experimental evidence in CLD.METHOD1,The selection of immunologic stimulantsPrevious studies of LIU zhanguo etal(Immune Transplant Institute of Zhujiang Hospital) showed that PHA (concentration of 10ug/ml,stimulation of whole blood for 12 hours)has been proved to be the most effective stimulant for cytokine production in whole blood.Therefore,we choose PHA as a stimulant, the concentration of lOug/ml, stimulation for 12 hours.2,The choice of subj ectsOur study objects were consisted of infants who were admitted to neonatal intensive care unit (NICU) at Nanfang Hospital-the first affiliated hospital of Southern Medical University,Zhujiang Hospital-the second affiliated hospital of Southern Medical University,the Third affiliated hospital of Guangzhou Medical University,Guangzhou Children's Hospital and Guangdong General Hospital between October 2007 and March 2009 and who met the following criteria:1) preterm infants and 2) gestational age-matched and birth weight-matched and 3) length of stay to 28d and 4) according to whether on oxygen at 28 d postnatally can be divided into two groups:who were on oxygen at 28 d postnatally were divided into chronic lung disease group,otherwise were divided into control group. Exclusion criteria:on oxygen at 28 d postnatally caused by congenital pulmonary hypoplasia and other diseases. Peripheral blood samples were collected at the 28d postnatally We selected 2ml undiluted and heparinized whole blood samples for research after obtained the consent of their parents (It is better to simulating the environment and reflecting the in vivo situation compared to the separated mononuclear cells and diluted whole blood sample).During the study period, a total of 27 preterm infants were enrolled. Among these preterm infants,1 infant with congenital pulmonary hypoplasia was excluded from the analysis. Finally 26 preterm infants were analyzed. Of these,14 preterm infants who were on oxygen at 28 d postnatally were divided into chronic lung disease group,12 preterm infants who were not need oxygen at 28 d postnatally were divided into control group, gestational age=28～33 weeks, birth weight= 700～2500g.The two groups were gestational age-matched and birth weight-matched (gestational age Z=-1.794,P=0.0729,weight Z=-0.464,P=0.643).3,The selection of methods to detect cytokines.Single cytokine test can hardly reflects the whole situation in vivo because of the complexity of immune responses. Therefore, the traditional ELISA technology and proteomics can not meet our needs.In order to detect multiple cytokines in tiny volume of whole blood, we selected the Luminex technique which combines flow cytometry and flow chip techniques together. Theoretically, Luminex can detecte up to 100 cytokines in only 15ul samples within 2 hours. So we choose the Luminez technique which is the only FDA-certified by the U.S.to detect cytokines4,StatisticsData analyzed with SPSS 13.0 software and comparisons between groups were made with two independent samples of Wilcoxon rank sum test when the data was skewed distribution,and data presented by median and mean rank,P<0.05 was considered statistically significant. Inter-group comparison group using t-test when the data was normal distribution and data presented by X±S.The comparison of gender distribution between the two groups was made with Fisher's Exact Test. a=0.05 for the test.Results1,The general conditions of preterm infants with and without CLD26 infants were enrolled in this study. The CLD infants were 14 in number: male11 cases, female 3 cases.GA=28～32 weeks 13cases, GA>32 weeks 1 case.WT <1000g 2 cases,1000-1500g 7 cases,1500g～2500g 5 cases.The control infants were 12 in number:male 9 cases,female 3 cases. GA=28～32 weeks 11 cases,> 32 weeks 1 case.WT<1000g 2 cases, 1000g～1500g 7 cases,1500g～2500g 4 cases.Gender distribution between the two groups was statistically insignificant (P=1.000).The comparison of incidence in CLD group between male and female was significantly(P=0.000). The two groups had similar basic treatment:prevent infection, improve microcirculation, to add trace elements, to maintain electrolyte and acid-base balance, parenteral nutrition support. The difference:the need for supplemental oxygen at the postnatal age of 28 days.2,Cytokine levels in the two groupsThe levels of IL-1p(Z=-1.234,P=0.217,P>0.05),IL-2(Z=-0.522,P=0.602, P>0.05),IL-4(Z=-0.267,P=0.789,P>0.05),IL-5(Z=-0.874,P=0.382,P>0.05),IL-6(Z=-0 .206,P=0.837,P>0.05),IL-7(Z=-1.668,P=0.095,P>0.05),IL-8(t=-0.764,P=0.452,P>0.0 5),IL-10(t=-0.947,P=0.353,P>0.05),IL-12(Z=-0.440,P=0.660,P>0.05),IL-13(Z=-0.74 9,P=0.454,P>0.05),IL-17(Z=-0.515,P=0.606,P>0.05),GM-CSF(Z=-1.251,P=0.211,P >0.05),G-CSF(Z=-0.945,P=0.344,P>0.05),MCP-1(Z=-0.900,P=0.368,P>0.05),IFN-y (Z=-0.313,P= 0.754,P>0.05),TNF-α(Z=-0.721, P=0.471, P>0.05) detected in 26 premature infants were no significant differences in two groups. The levels of MIP-1β(Z=-2.623,P=0.009, P<0.05) detected in 26 premature infants was significant differences in two groups.GM-CSF,IL-7,IL-4,IL-13,IL-17,G-CSF, IFN-γ, IL-5, IL-12 were no response or poor response to stimulator. Although 16 cytokines were statistically insignificant,we analyzed the data also found that:The concentrations of IL-2,IL-4,IL-17,IFN-γ,IL-1β,IL-5 and IL-12 were lower in preterm infants with CLD, and IL-6,IL-8,IL-10,IL-13,G-CSF,MCP-1,TNF-αwere higher in preterm infants with CLD.Conclusion1,We speculate that MIP-1βmay be involved in the pathogenesis of CLD, but the exact mechanism is not clear.2,General immune factors may not play a key role in the late period of chronic lung disease.The meaning of study general immune mechanism may require further discussion.3,GM-CSF,IL-7,IL-4,IL-13,IL-17,G-CSF,IFN-γ,IL-5 and IL-12 of whole blood may be not secreted or secreted rare.This may be related to immature cellular immunity and humoral immunity in premature infants and the relative lack of capacity in secretion these cytokines.4,The concentrations of IL-6,IL-8,IL-10,IL-13,G-CSF,MCP-land TNF-αhave a rising trend in preterm infants with CLD,but the concentrations of IL-2,IL-4,IL-17,IFN-γ,IL-1β,IL-5 and IL-12 have a decreasing trend in preterm infants with CLD. However,there are statistically insignificant.Whether it is related of the insufficient number of samples needs further study.Because it is difficult to obtain specimens, we have small sample size. The accuracy of the conclusion may need further research. |
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