ABSTRACT
<p><b>BACKGROUND</b>It has been identified that in patients with pre-eclampsia, several factors were released into the serum, which can regulate the proliferation of vascular smooth muscle cells and stimulate the synthesis of extracellular matrix (ECM). However, the signal transduction pathway of the growth factors and cytokines synthesized by endothelial cells leading to the proliferation and ECM synthesis of human umbilical arterial smooth muscle cell (HUASMC) is unknown. The aim of this study was to research the effects of protein kinase C (PKC) inhibitor, polymyxin B (PMB), on the proliferation, nuclear factor-kappa B (NF-kappaB) activation, and expression of procollagen I and III mRNA in HUASMC cultured with pre-eclamptic umbilical sera.</p><p><b>METHODS</b>Normal HUASMCs were treated with pre-eclamptic umbilical serum (pre-eclamptic group), pre- eclamptic umbilical serum plus PMB (PMB group), or normal umbilical serum (normal group). The expression of I-kappaB, NF-kappaB was detected by Western blotting after the HUASMC was incubated for 2 hours. The proliferation of HUASMC was evaluated by MTT, the cell cycle was analyzed by flow cytomerty, and the expression of procollagen I, III mRNA was measured by RT-PCR assay after HUASMC was incubated for 48 hours. ANOVA was used for statistical analysis.</p><p><b>RESULTS</b>Umbilical sera in pre-eclampsia could stimulate the proliferation, the DNA synthesis, the transition of G(0) + G(1) phase or G(2)/M phase to S and phase, the activation of NF-kappaB, and the expression of procollagen I mRNA of HUASMC as compared with normal umbilical sera (P < 0.01). PMB could inhibit the proliferation, the DNA synthesis, the transition of G(0) + G(1) or G(2)/M phase phase to S phase, the activation of NF-kappaB, and the expressions of procollagen I mRNA of HUASMC stimulated by umbilical sera in pre-eclampsia (P < 0.01).</p><p><b>CONCLUSION</b>PKC-NF-kappaB signal transduction pathway may play a key role in SMC phenotype modulation, which is more important in the pathogenesis of placental blood vessel in pre-eclampsia.</p>
Subject(s)
Female , Humans , Pregnancy , Cell Cycle , Cell Proliferation , Cells, Cultured , Collagen Type I , Genetics , Collagen Type III , Genetics , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Myocytes, Smooth Muscle , Metabolism , NF-kappa B , Physiology , Polymyxin B , Pharmacology , Pre-Eclampsia , Metabolism , Pathology , Protein Kinase C , Physiology , RNA, Messenger , Signal Transduction , Umbilical Arteries , MetabolismABSTRACT
<p><b>BACKGROUND</b>Although great advances in techniques for noninvasive prenatal diagnosis using fetal cells from maternal peripheral blood have achieved, current technology does not meet the demands required for clinical use. In this study, we aimed to establish reliable methods for the gene analysis of fetal cells from maternal peripheral blood.</p><p><b>METHODS</b>Primed extension preamplification (PEP)-polymerase chain reaction (PCR), multiple primed in situ labeling (PRINS), and nested PCR were individually applied to detect the sex determining region Y (SRY) gene in single fetal cells collected from maternal peripheral blood.</p><p><b>RESULTS</b>The sensitivity and specificity of the detection of the SRY gene by PEP-PCR were 97.39% (149/153) and 99.17% (119/120), respectively. The sensitivity and specificity of PRINS were 97.56% (40/41) and 100% (35/35), respectively. The sensitivity and specificity of nested-PCR were 80.00% (24/30) and 87.50% (14/16), respectively.</p><p><b>CONCLUSIONS</b>PEP-PCR and PRINS are reliable techniques for the gene analysis of single fetal cells from maternal peripheral blood because of their high sensitivity and specificity. PEP-PCR and PRINS can be used as standard methods of noninvasive prenatal diagnosis using single fetal cells from maternal peripheral blood.</p>
Subject(s)
Adult , Female , Humans , Pregnancy , Genes, sry , Polymerase Chain Reaction , Methods , Blood , Prenatal Diagnosis , Methods , Sensitivity and SpecificityABSTRACT
Objective To study the effects of nuclear factor-?B(NF-?B)decoy oligodeoxynucleotide(ODN)on the preeclamptic umbilical serum induced expression of precollagen Ⅰ,Ⅲ mRNA and tumor necrosis factor-?(TNF-?)in cultured human umbilical artery smooth muscle cells (HUASMC).Methods Primary cultured HUASMC of normal pregnancy were divided into four groups: group A(HUASMC were incubated with umbilical serum of normal pregnancy);group B(HUASMC were incubated with umbilical serum of preeclampsia);group C(HUASMC were transfected with NF-?B cis decoy ODN 48 h before incubation with umbilical serum of preeclampsia);group D(HUASMC were transfected with NF-?B scramble ODN 24 h before incubation with umbilical serum of preeclampsia).NF-?B cis decoy ODN and NF-?B scramble ODN were transfected with cationic lipofectamine to the latter two groups,respectively.The proliferation of human umbilical artery smooth muscle cells was evaluated by methyl thiazolyl tetrazolium and the apoptosis was analyzed by flow cytometry.The expression levels of precollagen Ⅰ,Ⅲ mRNA were detected by RT-PCR,the expression levels of TNF-? were detected by western blot.Results(1)The proliferation of group B(0.19?0.02)and group D(0.18?0.03)was significantly increased as compared with those of group A(0.11?0.02)and group C(0.14?20.02)(P0.05).(5)The expression of TNF-? of group B(0.74?0.11),group C(0.36?0.09)and group D(0.79?0.12)were significantly higher than that of group A(0.15?0.03)(P0.05).Conclusions NF-?B cis decoy ODN could down-regulate the proliferation,as well as the expression levels of precollagen and TNF-? of HUASMC induced by umbilical serum of preeclampsia.NF-?B may play an important role in the pathogenesis of placental artery abnormalities in preeclampsia.