The Study of Cytokine Profiles of Whole Blood in Chronic Lung Disease Infants
|School||Southern Medical University,|
|Keywords||Cytokines Chronic lung disease Whole blood Liquid suspension array technology Premature children|
The background chronic lung disease (chronic lung disease, CLD) is a common complication of premature children, the final outcome of pulmonary fibrosis, preterm children maimed, an important cause of death. About 10% -15% of patients die from respiratory failure within 1 year after birth, the survivors need long-term dependence on oxygen or ventilator therapy, a direct impact on the future quality of life of the children, to the family and the community was a tremendous financial and mental burden. With the continuous development of perinatal medicine, the survival rate of very low birth weight infants significantly improve the overall incidence of chronic lung disease showed an increasing trend, foreign reports was 1.5% in the incidence of chronic lung disease in all newborns has become one of the most intractable problems in the neonatal intensive care unit (NICU). Pathogenesis of CLD is very complex, but the essence is the basis of the genetic susceptibility, iatrogenic factors (such as oxygen toxicity, barotrauma or capacity injury), and infection and inflammation caused by immature lung injury ultimately lead to lung tissue abnormalities repair. Currently considered inflammatory responses play a crucial role in the pathogenesis of chronic lung disease, and cytokines involved in the inflammatory response in the lungs media to promote the occurrence and development of CLD, but the exact mechanism is still not clear today. There are a large number of studies to assess the high risk of CLD or CLD amniotic fluid, cord blood, plasma cytokines in airway secretions, indicating lung tissue or bronchoalveolar lavage fluid (BALF) IL-1β, IL- 6, IL-8, TNF-α and other proinflammatory cytokines early showed a high level of sustained expression of CLD caused by a variety of causes, but the duration is still controversial. Chemokine monocyte chemoattractant protein (MCP) -1, MCP-2, MCP-3, macrophage inflammatory protein (MIP)-1α, MIP-1β, and chronic lung disease. MCP-1 concentrations in airway secretions in the airway colonization with Ureaplasma urealyticum (CLD of one of the risk factors). Bronchoalveolar lavage fluid of IL-4, IL-10, IL-12, IL-13 and other anti-inflammatory cytokine secretion or irregular secretion may lead to pulmonary inflammation out of control and the persistence of the final result fibroplasia CLD occur. But there are some studies failed to confirm these results: studies have shown that in the bronchoalveolar lavage fluid and blood IL-6, was no correlation between the occurrence of TFN-α, IL-1β levels CLD; CLD children also found that the BALF, IL-10, lowered inflammatory cytokines compared with other children did not change. Which cytokines does participate in the occurrence of the CLD, each of the CLD acute phase and a non-acute phase function or play a key role factor which, the relationship between each factor how whether the duration before and after the active factor are the same, or only acute phase cytokines, these cytokines in the peripheral blood of children with similar change specific duration, still need to be further explored. The CLD cytokines at home and abroad is mostly focused on the lungs local less for CLD children with systemic immune status. Study the majority of animal experiments, few clinical studies, and clinical research samples are usually less. Few studies of whole blood in children with cytokines. Systemic immunity is involved in the pathogenesis of CLD CLD different stages of children with systemic immune state, the local immune and pulmonary whether a correlation These problems are uncommon reported in the literature. Due to sample processing and detection of the different methods, the detection result of the cytokine differences, can be used to compare the fixed reference value. Does not reflect the complexity of the interactions of cytokines network by one or the detection of individual cytokines, the key to solve this problem is to have uniform sample processing method, the use of high sensitivity, and can detect a variety of factors detection equipment. Liquid suspension system-on-a-chip is the only chip detection system approved by FDA, to meet fast trace the simultaneous detection of multiple cytokines need to reduce the detection error. In this study, the cytokine detection in liquid suspension array technology applied to CLD solve blood sampling in preterm children less contradiction detection indicators, can simultaneously detect 17 cytokines in whole blood. Objective To study the preterm children with chronic lung disease (CLD) 17 whole blood cytokine expression, to explore the possible role of systemic immune factors in chronic lung disease, to seek new treatment of CLD guide targeted immune adjust the treatment in the CLD, provide a reliable theoretical basis for improving the prognosis of children. Method 1, the choice of the immune stimulant (Zhujiang Hospital, immune Transplant Institute) Liu Zhanguo previous studies, the PHA concentration 10ug/ml, stimulation of whole blood 12 hours, for the best whole blood cell cytokine secretion stimulants. We therefore chose to PHA as a stimulating agent, concentration of 10ug/ml, stimulation time was 12 hours. 2, the choice of the object of study from October 2007 to March 2009, the collection Nanfang Hospital, Southern Medical University, Southern Medical University, Zhujiang Hospital, the Third Affiliated Hospital of Guangzhou Medical College, Guangzhou Children's Hospital, Guangdong Provincial People's Hospital, five The top three hospital neonatal Lee NICU children as an object of study. Inclusion criteria: 1), premature children; 2), gestational age, weight difference: 3), hospitalized for 28 days; 4), under 28 days whether out of oxygen divided into two groups: group of chronic lung disease (can not deoxy ) and control group (without oxygen). Exclusion criteria: congenital lung hypoplasia causes oxygen according to resistance to be excluded. Peripheral blood specimens collected in 28d. Children undiluted heparin whole blood after obtaining the consent of the families of children with 2ml as compared to the study sample (with the separation of mononuclear cells and diluted whole blood samples, to better simulate the environment within which the whole blood cells, better reflect the situation in the body). We collected a total of 27 specimens, 1 case of congenital pulmonary hypoplasia be removed, so the specimens of 26 cases for analysis. Chronic lung disease group of 14 patients in the control group of 12 patients. For 28-33 weeks gestational age, weighing 700-2500g, no significant difference between the two groups of gestational age and weight comparison (gestational age Z = -1.794, P = 0.0729; weight Z = -0.464, P = 0.643). Cytokine detection methods select cytokine detection, detection of single cytokines does not reflect the complexity of the interactions of cytokines network, so the traditional ELISA technology, proteomics technologies are unable to meet our demand, liquid suspension protein chip technology combined flow detection chip technology can be detected in a sample of up to 100 cytokines levels at once, and the short detection time (about 2 hours), meet our rapid and comprehensive analysis of whole blood the cytokines requirements, so we choose by the U.S. FDA certification suspended liquid protein chip technology as cytokines. 4 Statistical Methods SPSS13.0 statistical software for statistical analysis, the skewed distribution of inter-group comparison of two independent samples was used to compare the Wilcoxon rank sum test, the measured data to the median, the average rank, P lt ; 0.05 was considered statistically significant; t test was used to compare the homogeneity of variance in the normal distribution of the data between the two groups, the measured data X ± S; gender distribution between the two groups compared using Fisher's Exact Test at α = 0.05 for Inspection standards. 1, the general situation of chronic lung disease and control groups of 26 specimens CLD group of 14 male and 11 cases, 3 females, 28-32-week and 13 cases, gt; 32 weeks, lt; 1000g the two cases, 1000g-1500g seven cases, 1500g-2500g, 5 cases; control group of 12 patients, including 9 males and 3 females ,28-32 weeks, 11 cases gt; 32 weeks, lt; 1000g 1 cases, 1000g-1500g seven cases, 1500g-2500g 4 cases. Gender distribution differences of the two groups was not statistically significant (P = 1.000), the men and women of the CLD group than the difference was statistically significant (P = 0.000). The two groups have similar basic treatment: prevention and treatment of infections, improve microcirculation, micronutrient supplementation to maintain electrolyte and acid-base balance, intravenous nutrition support. The difference: CLD group after birth 28d blood collection still need oxygen therapy, the control group does not. 2, chronic lung disease and control groups of 17 cytokine levels in chronic lung disease and control groups of 17 cytokine IL-1β, IL-2, IL-4, IL-5, IL-6 and of IL- 7, IL-8, IL-10, IL-12, IL-13, IL-17, GM-CSF, IFN-γ, TNF-α, G-CSF, MCP-1, MIP-1β, respectively between the two groups . Groups of GM-CSF, IL-7, IL-4, IL-13, IL-17, the G-CSF, IFN-γ, IL-5, IL-12 to stimulate agent almost no reaction or a poor response. Groups 17 cytokine levels: IL-1β: Z = -1.234, P = 0.217; IL-2: Z = -0.522, P = 0.602; IL-4: Z = -0.267, P = 0.789; of IL- 5: Z = -0.874, P = 0.382; IL-6: Z = -0.206, P = 0.837; IL.7: Z = -1.668, P = 0.095; IL-8: t = -0.764, P = 0.452; IL-10: t = -0.947, P = 0.353; IL-12: Z = -0.440, P = 0.660; IL-13: Z = -0.749, P = 0.454; IL.17: Z = -0.515, P = 0.606: GM-CSF: Z = -1.251, P = 0.211; G-CSF: Z = -0.945, P = 0.344; MCP-1: Z = -0.900, P = 0.368: IFN-γ: Z = -0.313, P = 0.754; TNF-α: Z = -0.721, P = 0.471, above the 16 cytokines P gt; 0.05, no statistically significant difference between the two groups. MIP-1β: Z = -2.623, P = 0.009, P lt; 0.05. 16 cytokines no statistically significant difference, but we analyzed the data still can be found: the CLD group, IL-6, IL-13, G-CSF, MCP-1, TNF-α in the median, and IL-8, IL-10 in number than the control group, IL-2, IL-4, and of IL-17, IFN-γ, IL-1β, IL-5, IL-12 in the digits lower than the control group. Conclusion 1, MIP-1β, CLD children than in the control group was significantly reduced, we speculate that it may be involved in the pathogenesis of CLD's, but the exact mechanism is not clear. 2, systemic immune factors in the acute phase of chronic lung disease may not critical to study the significance of non-acute phase of chronic lung disease of systemic immune mechanisms may need further discussion. 3, GM-CSF, IL-7, IL-4, IL-13, IL-17, G-CSF, IFN-γ, IL-5, IL-12 may be provided in whole blood is not secreted or secreted rarely, this may be immature and cellular immunity and humoral immunity in preterm children, the ability to secrete these factors is relatively insufficient. 4, the the CLD IL-6, IL-8, IL-10, IL-13, the G-CSF, MCP-1, TNF-α concentration of the trend of higher than the control group, IL-2, IL-4 of IL -17, IFN-γ, IL-1β, IL-5, IL-12 concentrations lower than the control group, but the difference between the two groups was not statistically significant, this result is the number of samples related to insufficient and needs further study. Specimens difficult to obtain sex, our sample size is small, the accuracy of the conclusions that may require further research.