The Expression Of Nuclear Factor-kappa B P65, Interleukin-8 And Matrix Metalloproteinase-9 In Different Tissue And The Exploration Of Its Clinical Roles In Pregnant With Subclinical Chorioamnionitis

Prematurity is a common complication of pregnancy. The incidence rate is about 5% to 15%.The morbidty and case fatality of premature infant are more than the normal ,which is about 2/3 in perinatal mortality rate . So prematurity is always the focus of Obstetrics. The intrauterine infection is related with premature delivery. About 58 to 88 percent pregnant women of preterm delivery have histological change of chorioamnionitis. But most of pregnant women persent subclinical manifestation,which has not typical clinical symptoms, and great difficulty diagnosis. In the past, the diagnosis of prematurity associated with infection depended on positive outcome of bacterial culture of the amniotic fluid or the histology diagnosis. But both of them could not achieve the purpose of early diagnosis, because the first one took a long time, the later could be later diagnosed after parturition. For the past few years, some researchers predicted preterm delivery by detecting C-reactive pretein(CRP), complement or IgM,IgG,IgA,IgS in maternal blood. But these index had lower sensibility and specificity in clinical application. It is focus for researches begin with the pathogenesy of the preterm delivery that we search the sensitive and specific indexs to reflect amniotic cavity inflection.NF-kappa B is a kind'of nuclear factor, which can start and regulate the transcripion of related gene such as inflammation ,immune reaction ,apoptosis ,cell increase and differentiation.Many inflammatory factor, such as IL-8 and so on who participate the onset course of immature labour and accouchement are regulated by NF-kappa B. It is reported that the up stream regulatory element of MMP-9 gene contain the binding site of NF-kappa B, which can binding NF-kappa B to promote the transcriptionInterleukin-8 is a kind of important inflammatory cytokine, which participate the inflammatory reaction in organism and mediate the amniotic cavity infection and reflect the infection extent objectively. The high level of IL-8 presents the intrauterine infection. A great quantity study presume IL-8 may promote MMPs excrete by stimulating polycyte infiltrating. Matrix metalloproteinases is a family of Zinc dependent proteolytic which can mainly participate the metabolism of extracellular matrix. MMP-9(matrix metalloproteinase, MMP-9) is one of MMPs family which specially degrade typeⅣcollagen. TypeⅣcollagen mainly reside in basal layer of chorion and amnion. The investigation shows that the activity of MMP-9 increased has close relationship with fetal membrane degradation. MMP-9 also participate the development of rupture of membranes in term labor. Further search demonstrate that the level of MMP-9 is obviously higher than normal in premature rupture of membranes(PROM) with amniotic cavity infection. But the relation between MMP-9 and infection is still not clear.NF-κB promote the expression of IL-8or MMP-9. IL-8 enhance the secrete of MMP-9. The abnormal high expression will induce membrane rupture. But if NF-κB, IL-8, MMP-9 are related in development of subclinical chorioamnionitis in preterm delivery? What are they expression in maternal blood ,afterbirth tissue or cord blood? These is no reports about it upto now.So, we select immature labor as the object to research, which is a frequent complication of cyophoria. To observe the expression and changes of histomorphology, and compare the level of NF-κB, IL,8 and MMP-9 in peripheral blood and cord blood of mother.To investigate the contents of NF-κB, IL-8 and MMP-9,whose relationship with subclinical chorioamnionitis and the effect in pathogenesy.It is helpful for offering theory of infective immature labor to estimate and set up therapeutic regimen. Objective: To investigate the change of the p65 submit of the nuclear factor kappa B family in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioamnionitis, and analyze its effect on the pathogenesis of subclinical Chorioamnionitis , then to estimate its clinical value and therapeutic efficacy in predicting subclinical Chorioamnionitis.Methods: To chose 36 cases of preterm birth patients in labor , 14 of cases threatened premature labor , 37 cases of full term gravida with spontaneous inlabor, 38 cases of full term gravida without threatened labor and 18 cases who have the identical gestational age with preterm birth patients. To observe the change of the p65 submit of the nuclear factor kappa B family in maternal blood, umbilical cord blood, placenta and fetal membrane in preterm birth patients with subclinical Chorioamnionitis by five methods,which were HE staining ,immunohistochemical staining , immunocytochemical staining,Western Blotting and RT-PCR.Results:1. The pathematology results of placenta and fetal membrane in each groups:There were 24 cases who were subclinical chorioamnionitis patients in 36 cases ofpreterm birth patients in labor (66.67%) . 12 cases are mild,10 cases are midrange,2 cases are severe among them. There were 7 cases who were subclinical chorioamnionitis patient in 37 cases of the full term gravida with spontaneous inlabor group (18.92%). 5 cases are mild,2cases are midrange,none is severe among them. There were 3 cases who were subclinical chorioamnionitis patient in 38 cases of the full term gravida without threatened labor group (7.89%). 3 cases are mild, none is midrange and severe..2. The expression of NF-κB P65 in placenta and fetal membrane:2.1 Compared the expression of NF-κB P65 in placenta and fetal membrane by IHC in each groups:The expression of NF-κB P65 were detected in placenta and fetal membrane by im_munohistochemistry staining method in 2 groups,which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group.The median and range of the average staining intensity of NF-κB P65 was [8.00 (2.00～8.00) ] in pregnant with subclinical chorioamnionitis group,and [4.00(2.00～6.00)] in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).2.2 Compared the expression of NF-κB P65 in placenta and fetal membrane by Western Blotting in each groups:The median and range of the average staining intensity of NF-κB P65 was 48.43 (7.75～97.27) in pregnant with subclinical chorioamnionitis group, and 27.98 (7.75～61.83)in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).2.3 Compared the expression of NF-κB P65 mRNA in placenta and fetal membrane by RT-PCR in each groups:The average staining intensity of NF-κB P65 was 47.51±17.21 in pregnant with subclinical chorioamnionitis group, and 31.30±13.60 in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).3. The expression of NF-κB P65 in umbilical cord blood in each groups:3.1 The expression of NF-κB P65 in umbilical cord blood by ICC in each groups:The expression of NF-κB P65 were detected in umbilical cord blood by immunocytochemical staining method in 2 groups, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of NF-κB P65 was 4.00 (2.00～8.00) in pregnant with subclinical chorioamnionitis group, and 2.00 (2.00～6.00) in pregnant without subclinical chorioamnionitis group. There was significant difference between the two groups(P＜0.001).3.2 The expression of NF-κB P65 in umbilical cord blood by Wstern Blotting in each groups:The median and range of the expression of NF-κB P65 was 27.38 (17.89～69.40) in pregnant with subclinical chorioamnionitis groupthan, and 25.18 (13.92～32.46) in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).3.3 The expression of NF-κB P65 mRNA in umbilical cord blood by RT-PCR in each groups:The median and range of the expression of NF-κB P65 was30.76 (17.79～74.72) in pregnant with subclinical chorioamnionitis group, and 22.67 (13.57～30.12) in pregnant without subclinical chorioamnionitis group. There was significant difference between the two groups(P＜0.001).4. The expression of NF-κB P65 in maternal blood in each groups:4.1 The expression of NF-κB P65 in maternal blood by ICC in each groups:The expression of NF-κB P65 were detected in maternal blood by immunocytochemical staining method in 2 groups, which included pregnant with subclinical chofioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of NF-κB P65 in pregnant with subclinical chofioamnionitis group is 8.00 (3.00～8.00), and 4.00 (2.00～6.00) in pregnant without subclinical chofioamnionitis group. There was significant difference between the two groups(P＜0.001).4.2 The expression of NF-κB P65 in maternal blood by Wstern Blotting in each groups:The median and range of the expression of NF-κB P65 was 34.22 (19.24～88.49) in pregnant with subclinical chorioammionitis group, and 31.45 (11.33～48.44) in pregnant without subclinical chorioamnionitis .group.There was significant difference between the two groups(P＜0.001).4.3 The expression of NF-κB P65 mRNA in maternal blood by RT-PCR in each groups:The expression of NF-κB P65 mRNA were detected in maternal blood by RT-PCR method in pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of NF-κB P65 was the higher in pregnant with subclinical chorioamnionitis group[42.58 (28.57～81.11) ] than those in Pregnant without subclinical chorioamnionitis group[23.56 (14.92～42.87) ].There was significant difference between the two groups(P＜0.001).5. The correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood:5.1 The correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by IHC/ICC:To compare the correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood ,we found that the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.498,p＜0.01); the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.581 ,p＜0.01); the concentration of NF-κB P65 in umbilical cord blood was positively correlated with that of maternal blood (r=0.769,p＜0.01). 5.2 The correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by Western Blotting:To compare the correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by Western Blotting, we found that the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.583,p＜0.01); the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.714,p＜0.01); the concentration of NF-κB P65 in umbilical cord blood was positively correlated with that of maternal blood (r=0.803,p＜0.01).5.3 The correlation of NF-KB P65 mRNA in placenta and fetal membrane, umbilical cord blood, maternal blood by RT-PCR:To compare the correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by RT-PCR,, we found that the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.522,p＜0.01); the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.571,p＜0.01); the concentration of NF-κB P65 in umbilical cord blood was positively correlated with that of maternal blood (r=0.892,p＜0.01).6. The ROC curve of NF-κB P656.1 The ROC curve of NF-κB P65 in umbilical cord blood:The critical point of NF-κB P65 in umbilical cord blood were 3.50, 25.64 and 22.83 respectively by ICC,Western Blotting,RT-PCR, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve were 0.728, 0.653 and 0.749 respectively. There was significant difference between any groups of the three (P＜0.05). It was NF-KB P65 tested by ICC that had better sensitivity(79.4%) and specificity(51.9% )for diagnosing chorioamnionitis.6.2 The ROC curve of NF-κB P65 in maternal blood:The critical point of NF-κB P65 in umbilical cord blood were 5.00, 31.61 and 31.68 respectively by ICC,Western Blotting,RT-PCR three methods, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve were 0.899, 0.676 and0.920 respectively. There was significant difference between any groups of the three (P＜0.05). It was NF-KB P65 tested by RT-PCR that had better sensitivity (87.9 %)and specificity (89.3 %)for diagnosing chorioamnionitis.Conclusion:1. The high expression of the p65 submit of the nuclear factor kappa B family in maternal blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical Chorioamnionitis patients and in evaluating the therapeutic effect on patients with subclinical Chorioamnionitis.2. The high expression of the p65 submit of the nuclear factor kappa B family in umbilical cord blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical chorioamnionitis patients and in evaluating the therapeutic effect on patients with subclinical chorioamnionitis.3. It was high expression of the p65 submit of the nuclear factor kappa B family in placenta and fetal membrane in subclinical Chorioamnionitis. which indicates that the p65 submit of the nuclear factor kappa B plays an important part in the pathogenetic of subclinical Chorioamnlonitis patients. Objective: To investigate the change of the interleukin-8 in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioamnionitis patients,, to analyze its effect on the pathogenesis of subclinical Chorioamnionitis , to estimate clinical value in predicting subclinical ChorioamnionitisMethods: To chose 36 cases of preterm birth patients in labor, 14 cases of threatened premature labor, and 37 cases full term gravida with spontaneous in labor, 38 cases full term gravida without threatened labor and 18 cases who have the identical gestational age with preterm birth patients. It were enzyme-linked immunosorbentassay, HE staining, immunohistochemical staining that used to observe the change of the interleukin-8 in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioanmionitis.Results:1. The pathematology results of placenta and fetal membrane in each groups:The pathematology results of placenta and fetal membrane were the same that of the ChapterⅠ.2. Compared the expression of IL-8 in placenta and fetal membrane by IHC in each groups:The expression of IL-8 were detected in 2 groups by immunohistochemistry staining method, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the average staining intensity of IL-8 was the higher in pregnant with subclinical chorioamnionitis group[4.00 (2.00～8.00)] than those in pregnant without subclinical chorioamnionifis group[2.00 (2.00～6.00) ]. There was significant difference between the two groups(P＜0.001)3. The expression of IL-8 in unbilical cord blood in each groups:The expression of IL-8 were detected in umbilical cord blood by enzyme-linked immunosorbent assay method in 2 groups, including pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The expression, of IL-8 was the higher in pregnant with subclinical chorioamnionitis group (33.43±12.98ng/ml) than those in pregnant without subclinical chorioamnionitis group (20.21±11.47ng/ml) .There was significant difference between the two groups(P＜0.001).4. The expression of IL-8 in maternal blood in each groups:The expression of IL-8 were detected in maternal blood by enzyme-linked immunosorbent assay method in 2 groups, which included the pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of IL-8 was the higheer in pregnant with subclinical chorioamnionitis group[45.14 (25.15～63.21) ng/ml]than those in pregnant without subclinical chorioamnionitis group[24.18 (0.57～65.13) ng/ml].There was significant difference among the two groups(P＜0.001).5. The dependable comparison of IL-8 in placenta and fetal membrane, umbilical cord blood, maternal blood:To analyze the correlation of IL-8 in afterbirth, umbilical cord blood and maternal blood ,we found that the positive correlation was exist in placenta and fetal membrane and umbilical cord blood in expression of IL-8(r=0.296,p＜0.01); The concentration of IL-8 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.304,p＜0.01); The concentration oflL-8 in umbilical cord blood was positively correlated with that of maternal blood (r=0.532,p＜0.01); 6. The ROC curve of IL-86.1 The ROC curve of IL-8 in umbilical cord blood:The critical point of IL-8 in umbilical cord blood is 25.40ng/ml, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve is 0.773. The sensitivity and specificity were 85.3% and 70.1% respectively.6.2 The ROC curve of IL-8 in maternal blood:The critical point of IL-8 in maternal blood is 34.76ng/ml, which is the optimization value to estimate chorioamnionitis. The integral area under the curve is 0.798. The sensitivity and specificity were 79.4% and 79.2% respectively.Conclusion:1. The high expression of the interleukin-8 in maternal blood in subclinical Chorioamnionitis, could be one of an biochemical marker in predicting subclinical Chorioamnionitis and in evaluating the therapeutic effect on subclinical Chorioamnionitis patients.2. The high expression of the interleukin-8 in umbilical cord blood in subclinical Chorioamnionitis, could be one of an biochemical marker in predicting subclinical chorioamnionitis and in evaluating the therapeutic effect on subclinical chorioamnionitis patients.3. It was high expression of the interleukin-8 in placenta and fetal membrane in subclinical Chorioamnionitis that indicates the participation of the morbility of subclinical Chorioamnionitis.4. The high expression of the interleukin-8 in core blood in fetals with subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting and assisting to the early diagnostic of infectious of newborn, and also could be one of an biochemical marker in evaluating the therapeutic effect on infectious of newborn. Objective: To investigate the change of the matrix metalloproteinase-9 in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioamnionitis, to analyze its effect on the pathog.enesis of subclinical Chorioamnionitis, to estimate the clinical value of the matrix metalloproteinase-9 anticipation in predicting subclinical Chorioamnionitis.Methods: To Chose 36 cases ofpreterm birth patients in labor , 14 cases of threatened premature labor, and 37 cases full term gravida with spontaneous in labor, 38 cases full term gravida without threatened labor and 18 cases who have the identical gestational age with preterm birth patients . It were enzyme-linked immunosorbentassay, HE staining, immunohistochemical staining that used to observe the change of the matrix metalloproteinase-9 in maternal blood , umbilical cord blood, placenta and fetal membrane in preterm birth patients with subclinical Chorioamnionitis.Results:1. The pathematology results of placenta and fetal membrane in each groups:The pathematology results of placenta and fetal membrane were the same as that of the Chapter I.2. Compared the expression of MMP-9 in placenta and fetal membrane by IHC in each groups:The expression of MMP-9 were detected in 2 groups by immunohistochemistry staining method in 2 groups, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group.The median and range of the average staining intensity of MMP-9 was the higher in pregnant with subclinical chorioamnionitis group[6.00 (2.00～8.00) ] than those in pregnant without subclinical chorioamnionitis group[2.00 (2.00～6.00)].There was significant difference between the two groups(P＜0.001).3. The expression of MMP-9 in umbilical cord blood in each groups:The expression of MMP-9 were detected in umbilical cord blood by enzyme-linked immunosorbent assay method in 2 groups, including pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The expression of MMP-9 was the higher in pregnant with subclinical chorioamnionitis group (368.41±110.55ng/ml) than those in pregnant without subclinical chorioamnionitis group (281.67±96.73ng/ml) .There was significant difference between the two groups(P＜0.001).4. The expression of MMP-9 in maternal blood in each groups:The expression of MMP-9 were detected in maternal blood by enzyme-linked immunosorbent assay method in 2 groups, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of MMP-9 was the higheer in pregnant with subclinical chorioamnionitis group[458.19 (315.52～522.15) ng/ml]than those in pregnant without subclinical chorioamnionitis group[307.15 (181.29～533.30) ng/ml].There was significant difference among the two groups(P＜0.001).5. The correlation of MMP-9 in placenta and fetal membrane , umbilical cord blood, maternal blood:To compare the correlation of MMP-9 in placenta and fetal membrane, umbilical cord blood, maternal blood ,we found that the concentration of MMP-9 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.420,p＜0.01); the concentration of MMP-9 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.412,p＜0.01); the concentration of MMP-9 in umbilical cord blood was positively correlated with that of maternal blood (r=0.561,p＜0.01).6. The ROC curve of MMP-96.1 The ROC curve of MMP-9 in umbilical cord blood:The critical point of MMP-9 in umbilical cord blood is 317.50ng/ml, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve is 0.720, the sensitivity and specificity were 73.5% and 71.4%,respectively.6.2 The ROC curve of MMP-9 in maternal blood:The critical point of MMP-9 in maternal blood is 360.81ng/ml, which is the optimization value to estimate chorioamnionitis.The area under the curve is 0.823. The sensitivity and specificity were 87.4% and 72.0% respectively.Conclusion:1. The high expression of the matrix metalloproteinase-9 in maternal blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical Chorioamnionitis and in evaluating the therapeutic effect on subclinical Chorioamnionitis patients.2. The high expression of the matrix metalloproteinase-9 in umbilical cord blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical chorioamnionitis and in evaluating the therapeutic effect on subclinical chorioamnionitis patients.3. It was high expression of the matrix metatloproteinase-9 in placenta and fetal membrane in subclinical Chorioamnionitis. which indicates that it is the important pathogenetic reason for subclinical Chorioamnionitis.