| Background Hypertension Disorder Complicating Pregnancy (HDCP) as a pregnancy-dependant disease is a leading cause of fetal and maternal mortality and morbidity. In the past decades, achievements have been made in the treatment of HDCP and better understandings have achieved in the pathology of the disease, however, the etiology and pathogenesis remain obscure and there are no specific diagnostic test, Which restrains the prevention and further improvement of the treatment. Thus, Until now HDCP is still one of the most important and long-time subject in perinatology.In recent year, with the development of medical basic theory, especially, with the molecular biology developing, reproductive immunology has become an active area of research. Numerous findings indicate that widespread endothelial cell dysfunction is central to the pathophysiology of HDCP, of which immune factor play an important role in the process. Appropriate balance of immunity between Maternal and fetal is thought to be crucial for maintaining the normal pregnancy, non-specific immunity is one important part of immunity. Various representation of activated non-specific immunity during pregnancy gradually attract more attention. Recent foreign reports have shown that the function of non-specific immunity is significantly enhanced during pregnancy, suggest that non-specific immunity response may be one supplementary for the depressed specific immunity during pregnancy. Which play a key role in regulating the relationship between Maternal and fetal. Macrophage is a major part of non-specific immunity, it not only can directly participate in immune function , but also can produce high levels of pro-inflammatory cytokines in case of activated , such as tumor necrosis factor( TNF- α ) and interleukin-6(IL-6), which play a key role in pathogenesis of many diseases . In recent year, some investigators consider that excessive maternal inflammatory response to pregnancy is responsible for the clinical syndrome of HDCP. Lots of studies have demonstrated that macrophage is one important kind of antigen presenting cell (APC) during immune response. Greater numbers of macrophages are found in many organs of woman with HDCP. In case some factor active monocyte or macrophages in vivo of HDCP, activated macrophages will secrete lots of pro-inflammatory cytokines, damage endothelial cell, down-regulate the function of trophoblast cell invasion. Here, we hypothesize that Macrophages may play a key role in the pathogenesis of HDCP.In this study, Peritoneal macrophages were obtained from patients with HDCP and normal pregnant, after isolated, refined and cultured in vitro, macrophages are detected by biological and immunological methods to study the expression of TNF-α,IL-6,TGF β1and CD54 on peritoneal macrophages of normal pregnant and HDCP, to discuss the role of macrophages and cytokines in the pathogenesis of HDCP. In addition, peritoneal macrophages are intervened by Anisodamine In vitro, to investigate the etiology and pathogenesis of HDCP , which provide the theoretic basis for working out prevention and treatment measures.This study is divided into three parts: Part IExpression of TNF- α and TGF- β1 mRNA in peritonealmacrophages during normal pregnancy.Objective To study the expression of TNF-α mRNA and transforming growth factor- betal(TGF-β1) mRNA in peritoneal macrophages of normal pregnancy. Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect TNF-α mRNA and TGF-β1mRNA in the peritoneal macrophages of normal pregnancy and myoma of uterus. Results (1) The level of TNF-α mRNA in peritoneal macrophages of normal pregnancy was significantly higher than that in myoma of uterus (P<0.01) . (2) The level of TGF-β1 mRNA in peritoneal macrophages of normal pregnancy was significantly higher than that in myoma of uterus (P<0.001). Conclusions macrophages might be activated by pregnancy, the function of non-specific immune response are enhanced. TNF-α and TGF-β1 might play an important role in the balance of immunity. Part IIThe changes in function of releasing cytokine and expression of CD54 on peritoneal macrophages in patients with hypertensiondisorder complicating pregnancy.objective To study the changes in cell viability and capacity of releasing TNF-α , TGF-β1 or IL-6 and the expression of CD54 on peritoneal macrophages in the patients with HDCP, and to discuss the potential role of these cytokines were explored. Methods Peritoneal macrophages were obtained from patients with HDCP and normal pregnant. The concentration of TNF-a, TGF β 1 and IL-6 in the supernatant of cultured macrophages was measured by enzyme linked Immunosorbent assay (ELISA), The expression of TNF- α mRNA, TGF β 1 mRNA and IL-6mRNA in peritoneal macrophages was detected by reverse transcription-polymerase chain reaction (RT-PCR) , The expression of CD54 on peritoneal macrophages was measured by flow cytometry. Results (1) The concentration of TNF- α , TGF β 1 and IL-6 in the supernatant of cultured macrophages of HDCP : The level of TNF-a in the supernatant of cultured macrophages was significantly elevated in HDCP group compared with that in control group. Of which The level of TNF-a was significantly different between GH, MP or SP and control group (P<0.05, P<0.01, P<0.01) , The level of TNF-a was significantly different between MP or SP and GH (P<0.01, P<0.01 ) ;The level of IL-6 in the supernatant of cultured macrophages was significantly elevated in HDCP group compared with that in control group. Of which The level of IL-6 was significantly different between MP or SP and control group (P<0.05, P<0.01) , The level of IL-6 was significantly different between SP and GH (P<0.01) ; The level of TGF-β1 in the supernatant of cultured macrophages was elevated according with the state of HDCP , The level of TGF- β1 was significantly different between SP and MP, GH or control group (P<0.05 ,P<0.05, P<0.05) .(2) The mRNA expression of TNF- α , TGF β 1 and IL-6 in peritoneal macrophages of HDCP. The mRNA expression of TNF-α in peritoneal macrophages of HDCP group was significantly higher than that in control group. Of which The level of TNF-a mRNA was significantly different between GH, MP or SP and control group (P<0.05, P<0.01, P<0.01) , The level of TNF-a mRNA was significantly different between MP or SP and GH (P<0.01, P<0.01) ;The mRNA expression of TGF-β1 in peritoneal macrophages of SP was significantly higher than that in MP, GH or control group (P<0.01, P<0.01, P<0.01) ;The level of IL-6 mRNA in peritoneal macrophages of GH ,MP and SP was significantly higher than that in control group (P<0.05, P<0.01, P<0.01) , The level of IL-6mRNA was significantly different between SP and GH (P<0.01) .(3) The expression of CD54 on peritoneal macrophages of HDCP: the percentage of CD54 positive cells in MP and SP were significantly higher than those in control group (P< 0.05, P< 0.05), The level of CD54 positive cells was no significantly different between GH and control group(P>0.05). Conclusions (1) The expression of TNF-α , TGF β 1 and IL-6 of in vitro cultured macrophages of HDCP are elevated at both transcription and translation levels , on the other hand, which prove that the viability and capacity of cytokine secretion in peritoneal macrophages of HDCP are reinforced. It shows that macrophages are in a state of activation in vivo, Macrophages play an important role in the Immunomechanism of HDCP.(2) The Production of TNF- α in peritoneal macrophages of HDCP are significantly elevated at both transcription and translation levels, it shows that in vivo the level of TNF- α is elevated, overproduced TNF- α may play an important role in the pathogenesis of HDCP.(3) The level of TGF- β 1 of cultured macrophages is elevated according with the state of HDCP , The level of TGF- β 1 is significantly different between SP and control group.(4) The Production of IL-6 in peritoneal macrophages of HDCP are significantly elevated at both transcription and translation levels, IL-6 may be concerned with the< damage process of vascular endothelial cell. The high level of IL-6 play an important role in the pathogenesis of HDCP. (5) the percentage of CD54 positive cells in preeclampsia are significantly higher than those in normal pregnancy , The level of CD54 positive cells of cultured macrophages is elevated according with the state of HDCP. The anomaly expression of intercellular adhesion molecule-1 may play an important role in the pathogenesis of HDCP. Part IIIThe effect of Anisodamine on cytokine secretion in peritoneal macrophagesof Hypertension Disorder Complicating PregnancyObjective To explore the immunomechanism of anisodamine (654-2) in the treatment of HDCP, the effect of 654-2 on TNF-α and IL-6 levels released by peritoneal macrophages were investigated. Methods The isolated peritoneal macrophages from pregnant women with HDCP were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum or in RPMI 1640 with 5mg/mL 654-2 and 10% fetal bovine serum, by cultured 24 hour ,both the TNF- α , EL-6 levels and their mRNA expression were detected by ELISA and RT-PCR respectively. Results (1) 654-2 significantly reduced TNF- α level at GH ( P<0.05 ), MP ( P<0.01 ) and SP ( P<0.01 ). (2) 654-2 significantly reduced IL-6 level at MP ( P<0.05 ) and SP ( P <0.01 ). (3) 654-2 significantly reduced TNF- α mRNA expression at GH ( P< 0.05 ), MP ( P<0.01 ) and SP ( P<0.01 ). (4) 654-2 significantly reduced IL-6 mRNA expression at MP ( P<0.05 ) and SP ( P<0.05 ). Conclusions Anisodamine not only have spasmolysis function for HDCP, but also can inhibit the excessive activation of peritoneal macrophages and inhibit TNF- α , IL-6 production at both transcription and translation levels, prevent the occurrence progression of HDCP. |
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