The Mechanism Of HLA-G In Pathogenesis Of Preeclampsia & Development And Function Of Brain In Preeclampsia Like Rat Offspring

Preeclampsia is a multisystem and multiorgan disorder of unclear cause that is unique to human pregnancy. It occurs in 3-5% of pregnant women and higher percentage in development country. It clsssically manifests as new-onset hypertension and proteinuria after 20 weeks of gestation. Preeclampsia is a major cause of maternal and neonatal morbidity and mortality. Recent studies indicate that insufficient invasion of placenta is the major characteristic in preeclampsia. And the insufficient invasion of extraembryonic trophoblasts might contribute to the lower placenta perfusion. Extraembryonic trophoblasts are major cell types including cytotrophoblasts, syncytiotrophoblasts and extrovillous trophoblasts in the placenta. Normal development of the placenta and adequate trophoblast functioning are prerequisites for a successful pregnancy. Reduced human leucocyte antigen-G (HLA-G) expression in trophoblasts contributes to adverse obstetric outcomes including preeclampsia. HLA-G is a non-classical human leucyte antigens (HLAs) which are the most complex polymorphism system involved in immune tolerance. Previous studies have documented roles for the HLA-G proteins in the interactions between immune cells, such as natural killer (NK.) cells and trophoblasts, whereby HLA-G isoforms inhibit NK cytotoxicity and cytokine production. However, it remains unclear whether HLA-G might exert an independent regulatory role on the biological property of trophoblasts. It would provide insight to understand the pathogenesis of preeclampsia and be helpful for preventing and curing preeclampsia.Therefore, in the first part of this thesis, we revealed the function of HLA-G in JEG-3 cells through regulating HLA-G expressionUnfortunately, there is yet no cure for the preeclampsia. As unique disease in human pregnancy, preeclampsia not only is adverse for the heath of mother, but also the development of fetus. Furthermore, the epidemiologic evidences have shown that infants from preeclamptic mothers are susceptible to develop respiratory distress syndrome, hypertension and stroke in adult life.Thus, it is necessary for establishing the preeclamptic animal model to understand pathophysiology of preeclampsia and the long-term adverse effects on the offspring.In previous studies, it has been reported the rats treated by Nω-Nitro-L-Arginine Methy Ester (L-NAME), a nitric oxide synthase (NOS) inhibition drug, during from last third of gestation would be produced preeclampsia-like conditions.There are many studies which focus on lung,kidney and vascular endothelium development and function of infants from preeclampsia mothers or offspring from preeclampsia animal models.However, the later pregnancy stage (embryonic 14.5-20.5days in rodent;25 week-40week in human) which is after preeclampsia onset time (approximately 20weeks) is critical for the fetus brain development in utero.Thus, it is necessary to study the development and function of brain in the offspring from preeclamptic rats.Section 1 The mechanism of HLA-G regulating invasive property of JEG-3 choriocarcinoma cellsObjectiveTo find the function of HLA-G in JEG-3 cells without immunity cells co-culture.Methods Wesynthesized HLA-G specific siRNA to scilence the expression of HLA-G in JEG-3 cells. We increased HLA-G expression in JEG-3 cells via adding human galectin 1 in culture medium. We observed the invasion of JEG-3 cells after down-regulation or up-regulation of HLA-G. We detected the proliferation and apoptosis of JEG-3 cells after down-regulation of HLA-G by immunostaining with Brdu, PH3 and activated cleavagecaspase 3. We detected the phosphorylation status of STAT3 in JEG-3 cells after down-regulaiton or up-regulation of HLA-G. We studied whether IL-6 can rescue the phosphorylation status of STAT3 and invasion of JEG-3 cells after down-regulation of HLA-G.Results 1) We found down-regulation of HLA-G resulting in a notably attenuated invasion capacity of the cells in a Transwell assay without alterations in cell proliferation and apoptosis.2) The down-regulation of HLA-G dampened the activation of signal transducer and activator of transcription 3 (STAT3).3) The up-regulation of HLA-G promoted STAT3 activation and invasion in JEG-3 cells treated with human galectin-1.4) We found interleukin-6 (IL-6), but not galectin-1, was shown to rescue invasion deficiency in a dose-dependent manner.DisscusionThus, we demonstrate that HLA-G is able to regulate JEG-3 cell invasion directly by influencing STAT3 activation without interating on immunity cells, which may underlie the implantation defects accompanying HLA-G hypo-expression in pre-eclampsia.Section 2 Development and Function of Brain in preeclampsia like rat offspringObjectives We wanted to established the preeclampasi like rat model. We wanted to document the development and function of brain in preeclampsia like rat offspring.Methodsweused pregnant Sprague-Dawley rats in whole experiments. The pregnant dams were randomly divided into two groups to be given 50 mg·kg-1·d-1 L-NAME or pure water by daily gavage from day 14.5 to day 21 of gestation. A portion of P0 day rats pups were sacrificed for early brain development assays by immunostaining. The other postnatal P0 pups mice were kept with their mothers in the cages and done for water maze test and adult brain development assays by immunostaning.We detected the expression and promoter methylation of neurogenensis associated genes by realtime PCR and BSP. We also detected the expression and phosphorylation status of LTP relative genes such as ERK and JNK by Western blot.Results 1)We established the preeclampsia-like rats model.2) We found the weight of P0 offspring and their brains in the L-NAME treated group were less than those in the water treated group, but the brain body weight ratio was similar between these groups.3) We found the cell proliferation but not apoptosis process,radial glia morphology and differentiation was impaired in P0 rat brains.4) The low body weight and brain development delay could be resolved soon froml week onward. In P56 rat brains, we found the radial dimension (thickness) and laminar structures were unchanged which may due to the overgrowth of astrocytes.5) We found the learning& memory in drug treated group were impaired.6) We found gliosis and deficiency of adult hippocampus neurogenesis in the drug treated group which may explain away the deficiency in learning& memory ability. To further analysis, we found the adult hippocampus neurogenesis associated genes were hypo-expression.7) However, we did not find any difference in promoter methylation of Fgf2, Creb and Ep300 between L-NAME and control group.8) we also did not find any difference in expression and phosphorylation status of LTP relative genes such as ERK and JNK.Discussion Our results have shown the severe effect on development and function of brain in preeclamspsi like rats. Major discoveries are the delay of neurogenesis in eary brain, gliosis and defects in adult hippocampus neurogenesis which might result in deficiency in learning & memory ability. These discoveries advance our understanding of the effects on preeclampsia offspring and provide an in vivo model for studying and changing long-term effects on them.