The Function Of NFAT-1、GAS/ISRE And Plasma In The Pathogenesis Of Pre-eclampsia

PartⅠ：Immuno-beads Isolation and Validation of Monocytes and T cellsfrom Human Peripheral Blood Mononuclear CellObjectiveTo isolate and validate the mnocytes and Tcells from hman pripheral boodmononuclear cells. To test its purity and extract the cytoplasmic and nuclear protein fromthe samples.Methods50ml of blood was centrifuged and plasma was isolated. Dilute the rest of the bloodcells with PBS-E and PBMC were isolated from Non-pregnant, Normal-Pregnant,Pre-eclamptic samples using Ficoll-Paque. Monocytes and T cells were isolated fromdifferent PBMCs using negative immuno-isolation. Purity of the cells were tested focusingon the surface marker CD14and CD3by flow-cytometer. Proteins were extracted and theconcentration of the proteins was tested. Western blotting was used to test the purity ofboth cytoplasmic and nuclear proteins. Result1. Generally1.24±0.36×108of PBMCs per50ml of blood was isolated byFicoll-paque. The mean of Non-pregnant, Normal-Pregnant and Pre-eclamptic sample was1.24±0.36×108/50ml，1.14±0.41×108/50ml，1.03±0.28×108/50ml，respectively.（P>0.05）。2. The ratio of isolated monocytes to isolated PBMCs of pre-eclampsia was higher(8.93±3.19)%than that of Normal-Pregnant（6.74±2.54%, P<0.05）or Non-pregnancy（6.15±2.35%,P<0.01）.3. The ratio of isolated T cells to isolated PBMCs of pre-eclampsia was similar (34.00±7.197%)of that of Normal-Pregnant（32.64±9.447%, P>0.05）or Non-pregnancy（33.83±15.61, P>0.05）.4. The purity of the monocytes we got were over95%, which is qualified for futherexperiments.5. The purity of the T cells we got were over99%, which is qualified for futherexperiments.6. Stain cytoplasmic and nucler proteins with both Histone-H1and Tubulin. Therewas no Tubulin expression in nuclear proteins and no Histone-H1expression incytoplasmic proteins, which means the both parts of the proteins were pure withoutcontamination.ConclusionAll the cells (PBMCs, Monocytes, T cells) and proteins were qualified for the furtherexperiments. Part II：Transcription Factor Array for Monocytes and T cells inpre-eclampsiaObjectiveTo test the expression of transcription factors in both monocytes nucleus and T cellnucleus in different groups and different status. Try to find out which transcription factorwas up-regulated in monocytes while down-regulated in T cells.MethodsUsing the monocytes and T cells isolated from the part I, transcription factor array(Protein/DNA Array I, Panomics) was performed to test the expressions in different groupof samples.Result1．In unstimulated monocytes, the expression of NFAT-1was64560±3685，89160±2338and103900±5204for non-pregnancy, normal-pregnancy and pre-eclampsia,respectively.（P<0.05）.2．In stimulated monocytes, the expression of NFAT-1was22640±4582，63380±11690and86020±7218for non-pregnancy, normal-pregnancy and pre-eclampsia,respectively.（P<0.01）.3．In unstimulated T cells, the expression of NFAT-1was92400±4473，24050±1217and11550±3399for non-pregnancy, normal-pregnancy and pre-eclampsia,respectively.（P<0.01）.4．In stimulated monocytes, the expression of NFAT-1was82170±885.6，45770±3097and31980±8096for non-pregnancy, normal-pregnancy and pre-eclampsia,respectively.（P<0.01）5．In unstimulated monocytes, the expression of GAS/ISRE was2244±446.5，43250±4087and83107±4702for non-pregnancy, normal-pregnancy and pre-eclampsia, respectively.（P<0.01）.6．In stimulated monocytes, the expression of GAS/ISRE was21220±614，37180±837and42660±1754for non-pregnancy, normal-pregnancy and pre-eclampsia,respectively.（P<0.01）.7．In unstimulated T cells, the expression of GAS/ISRE was14350±2198，6012±167and5352±183for non-pregnancy, normal-pregnancy and pre-eclampsia, respectively.（P<0.01）.8．In stimulated monocytes, the expression of GAS/ISRE was40470±17596，15680±3259and2348±194for non-pregnancy, normal-pregnancy and pre-eclampsia,respectively.（P<0.01）Conclusion1. The expression of transcription factor NFAT-1in monocytes was up-regulated inpregnancy than that in non-pregnancy.And this is further amplified in pre-eclampsia.2. The expression of transcription factor NFAT-1in T cells was down-regulated inpregnancy than that in non-pregnancy.And this is further amplified in pre-eclampsia.3. The expression of transcription factor GAS/ISRE in monocytes was up-regulatedin pregnancy than that in non-pregnancy.And this is further amplified in pre-eclampsia.4. The expression of transcription factor GAS/ISRE in T cells was down-regulated inpregnancy than that in non-pregnancy.And this is further amplified in pre-eclampsia. Part III：The Function of the peripheral plasma in the Pathogenesis ofPre-eclampsiaObjectiveTo find out the function of peripheral plasma in the pathogenesis of pre-eclampsia.MethodsIsolate PBMCs from6non-pregnant women using Ficoll-Paque. Mix10samples ofplasma from each group from the first part of the experiment. Then co-culture the normalPBMCs with different type of mixed plasma for72hours. Isolate the monocytes and Tcells from these PBMCs using immuno-beads. Then stimulate the cells by12groups aswas shown:1）Monocytes+Non-pregnant plasma+LPS2）Monocytes+Non-pregnantplasma plasma+DMSO3）Monocytes+Normal-pregnant plasma+LPS4）Monocytes+Normal-pregnant plasma+DMSO5）Monocytes+pre-eclamptic plasma+LPS6）Monocytes+pre-eclamptic plasma+DMSO7） T cell+Non-pregnant plasma+PMA/ionomycin8） T cell+Non-pregnant plasma+Ethanol9） T cell+Normal-pregnant plasma+PMA/ionomycin10）T cell+Normal-pregnant plasma+Ethanol11）T cell+pre-eclamptic plasma+PMA/ionomycin12）T cell+pre-eclampticplasma+Ethanol.Extract the mRNA and test the volum of the TNF-α mRNA production of monocytesand INF-γ mRNA production of T cells using PCR.Result1. The TNF-αmRNA production of unstimulated non-pregnant monocytes co-culturedwith pre-eclamptic plasma was4.16±1.18，and the production of the monocytes co-culturedwith non-pregnant and normal pregnant plasma were0.712±0.132and3.278±1.037,respectively.(P<0.01）。The TNF-αmRNA production of stimulated non-pregnant monocytes co-cultured with pre-eclamptic plasma was3.84±0.74，and the production of the monocytes co-culturedwith non-pregnant and normal pregnant plasma were0.97±0.17and2.07±0.61,respectively.(P<0.01）。2. The INF-γmRNA production of unstimulated non-pregnant monocytes co-culturedwith pre-eclamptic plasma was0.53±0.08，and the production of the monocytes co-culturedwith non-pregnant and normal pregnant plasma were2.66±0.245and1.77±0.21,respectively.(P<0.01）。ConclutionThe pre-eclamptic plasma changed the function of normal immune cells.1. The pre-eclamptic plasma made the normal monocytes express less TNF-αmRNA,so to up-regulate the innate immunity. And this change was further than that happend innormal pregnant plasma.2．The pre-eclamptic plasma made the normal T cells express more INF-γmRNA, soto down-regulate the addaptive immunity. And this change was further than that happend innormal pregnant plasma. Part IV：Prognostic value of ambulatory blood pressure measurements forthe diagnosis of gestational hypertension and pre-eclampsiaObjectiveTo estimate the value of Ambulatory Blood Pressure Monitoring for early diagnosispre-eclampsia. The study included12gestational hypertension，15severepre-eclampsia)，33mild pre-eclampsia. Ambulatory blood pressure monitoring indexsand clinical outcomes were obtained. Using ROC curve to analyse the indexs and to get thecutoff value which can differ preeclampsia and hypertension disorder complicatedpregnancy.MethodUsing the ambulatory blood pressure monitoring impliment, we can get the bloodpressure of the50cases with preeclampsia and that of the20cases with gestationalhypertension and20cases of normal pregnancy. Also the clinical performances arerecorded. Using the Receiver Operating Characteristic curve, we found cutoff values ofthat can divide gestational hypertension and preeclampsia.ResultsThe best6index to distinguish gestational hypertension from normal pregnancy wasas follow ordered by their value: Mean24h Systolic> Mean Daytime Systolic> Mean24hMap> Mean Night Systolic> Max Night Systolic> Mean24h DiastolicThe best6index to distinguish pre-eclampsia from gestational hypertension was asfollow ordered by their value: Mean Night Systolic> Mean24h Systolic> Mean24hSystolic> Mean24h Map> Mean24h Diastolic> Max24h DiastolicConclusionThe value of ambulatory blood pressure mornitoring to prognosis gestationalhypertension and pre-eclampsia has been studied in this study.