Susceptible Gene Screening And Testing Responsible For Cardiopulmonary Bypass Related Lung Injury

Part 1:Differential gene expression in open cardiac surgery patients resulted by cardiopulmonary bypassAcute lung injury (ALI) is a common complication of cardiopulmonary bypass (CPB), 1%～2% of which develop a syndrome of pulmonary dysfunction called the adult respiratory distress syndrome or post-pump syndrome. It is characterized by evidence of pulmonary micro-vascular endothelial damage, increased micro-vascular permeability, increased lung water accumulation, increased intrapulmonary shunting, hypoxia, respiratory failure, and a variable severity of clinical expression. Previous investigations suggested that it is the accumulation and activation of the leukocyte in the lung responsible for ALI, and the neutrophil proteinases play a key role in its development. Thus to study the leukocyte gene transcription profiling would be helpful in revealing the mechanisms of CPB related ALI.Objective To investigate the gene expression profiling during CPB, depict the leukocyte gene differential expression pattern between pre and post CPB.Methods 8 children aged from 3 to 8 years old who scheduled to receive correction of ventricular septal defect under moderate hypothermic CPB were enrolled. Blood samples were taken from each patient immediately before CPB (pre-CPB) and the end of CPB (post-CPB). We had the total RNA extracted from each sample and then transcripted into cDNA through a reverse transcription polymerase chain reaction (RT-PCR) in vitro. And then the cDNA was transcripted into cRNA. Biotin labeled UTP and CTP were used during the course of cRAN synthesis. After purified and fragmented, the biotin labeled cRNA was hybridized with U133A gene chips (Affymetrix). The gene expression level were represented by fluorescent signal of the probe set in genchip, which were came from the scanning to the chip by a laser confocal scanner.Results The number of the expression "present" genes (detected) had a significant increasing after CPB when compared with that of pre-CPB, which reached to 7810 at the end of CPB from 7023 genes. And 2,873 genes showed differential expression at post-CPB in contrast to that of pre-CPB. Furthermore, 96 genes had their expression levels over three times increased. There were 134 proteinase related gene located in the U133A gene chip. And 9 of which detected as differential expression between pre and after cardiopulmonary bypass, 7 up-regulated and 2 down regulated. But the neutrophil elastase gene and the tissue inhibitor of matrix metalloproteinases genes expression showed little change between pre-CPB and post-CPB.Conclusions CPB induces significant alterations in the expression of child neutrophil genes which are associated with a variety of functions. And the gene expression character is coinciding with the generally accepted cognition of systemic inflammatory response syndrome (SIRS) induced by CPB. CPB also induces a lopsided proteinase/anti-proteinase gene expression ratio, and some of the proteinase related gene expression develop an anormal up-regulate. Part 2:The effects of cardiopulmonary bypass on matrix metalloproteinase 9 in human cardiac procedure patientsBackgroud and Objective Activated leukocyte can synthesis and release abundant of proteinase, including matrix metalloproteinase 9 (MMP-9). The latter can degradate type IV, V collagen and gelatin, and was reported responsible for experimental acute lung injury possibly in animal and human. The objective of the present study is to investigate the effects of cardiopulmonary bypass on matrix metalloproteinase 9 in open cardiac procedure patients.Methods Twenty adult patients (12 of male, 8 of female) aged from 24 to 51 yr scheduled to receive open heart surgery under moderate hypothermic cardiopulmonary bypass were enrolled (CPB group). Another 8 patients subject to receive general open chest operation were selected as "control" (control group). With the informed consent of the patients, 2ml of blood was extracted from each patients through a previous placed central venous catheter at the time points of pre-operation, immediately before and be separated from cardiopulmonary bypass in the CPB group, and pre-operation, after open chest and before close chest in the control group as counterpart. Leukocyte gene expression and plasma concentration of total and active pattern of MMP-9 were measured using semi-quantitate reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsorbent (ELISA) assay respectively. Values were presented as mean ± standard deviation, and compared by performing one-way ANOVA between different time points inside group, and independent sample student's t-test between groups. Significance was defined as P<0.05.Results At the time point of pre-operation and before cardiopulmonary bypass, MMP- 9 gene expression level, total and active MMP-9 concentration in plasma all showing a very low level, and little specimen appeared clear DNA fragment band on the gel electrophoregram, but they were increased significantly at the time point of be separated from cardiopulmonary bypass in the CPB group. As for control group, it is still remained low level both of gene expression and plasma concentration of MMP-9 at the time point of before close the chest, which is comparable to that of pre-operation and immediately after open chest.Conclusions Our study demonstrate that the gene expression level, total and active pattern of matrix metalloproteinase 9 plasma concentration elevated significantly induced by cardiopulmonary bypass. These effects are not related with the procedure of open chest. Part 3:The Effects of Dexamethasone on Matrix Metalloproteinase 9 and Pulmonary Function in Open Cardiac Procedure PatientsObjective As one of the most important matrix metalloproteinase family members, it is to be ascribed of matrix metalloproteinase 9 to acute lung injury in some extent. The aim of current study was to investigate the effects of dexamethasone on matrix metalloproteinase 9 and pulmonary function in open cardiac procedure patients.Methods Forty patients subject to cardiac valve replacement operation under moderate hypothermia cardiopulmonary bypass were enrolled and divided into control or dexamethasone group randomly (n=20). With the informed consent of all patients, 0.5mg/kg of dexamethasone or equal volum of normal saline was administered to the patients of corresponding group after general anesthesia was induced. Blood sample were extracted from and platelet-free plasma harvested for each patient at the time points of pre-operation, before and be separated from cardiopulmonary bypass, respectively. Total and active matrix metalloproteinase 9 plasma concentration was measured using enzyme-linked immunoadsorbent or fluorometric enzyme-linked immunoadsorbent assay. Simultaneously, the reverse transcription-polymerase chain reaction was performed to quantify the matrix metalloproteinase 9 gene expression level at the above mentioned time points. And arterial blood-gas analysis was performed to every patient at before and weaning from cardiopulmonary bypass. Values were presented as mean ± standard deviation, and compared by performing one-way ANOVA or independent sample student's t-est. Significance was defined as P < 0.05. Results At the time point of be separated from cardiopulmonary bypass, matrix metalloproteinase 9 gene expression, total and active matrix metalloproteinase 9 plasma concentration are increased significantly for all of the patients than that of the pre-operation and before cardiopulmonary bypass (P<0.05 or 0.01), and still a statistical difference between groups (P<0.05). Furthermore, the alveolar-arterial oxygen difference express obvious increasing when weaning from cardiopulmonary bypass for all of the patients than that of before cardiopulmonary bypass (P<0.05), and still a striking discrepancy between groups (P<0.05).Conclusions Dexamethasone inhibits the increased gene expression, protein synthesis and activation of matrix metalloproteinase 9 and alveolar-arterial oxygen difference caused by cardiopulmonary bypass. This could be partly responsible for its pulmonary protection for cardiac operation patients.