Changes and significance of plasma fibrinogen gamma-chain concentration in preeclampsia patients.

OBJECTIVE: To investigate the plasma fibrinogen gamma-chain concentration in preeclampsia patients and explore its value in preeclampsia prediction and auxiliary diagnosis. METHODS: Follow-up of pregnant women who regularly attended perinatal care at two hospitals in China was performed, and clinical data and plasma samples were collected at each examination until delivery. The gamma-chain concentration was detected by Western blotting, and Quantity One Software was used for gamma-chain grayscale value measurements. RESULTS: Forty-two patients with preeclampsia and 42 control patients completed the follow-up. In the control group, the gamma-chain concentration at 32 weeks of gestation was higher than that at 20 weeks of gestation, but the difference was not statistically significant (p > 0.05). In the experimental group, the gamma-chain concentration at preeclampsia diagnosis was significantly higher than that at 20 weeks of gestation (p < 0.05). Compared with the control group, the gamma-chain concentration was higher at 20 weeks of gestation in the experimental group, but the difference was not statistically significant. However, at 32 weeks of gestation and at the time of diagnosis, the gamma-chain concentration in the experimental group was significantly higher than that in the control group (p < 0.05). At 32 weeks of gestation and at the time of diagnosis, the AUCs from ROC curve analysis of plasma fibrinogen gamma-chain concentrations were 0.64 and 0.71, respectively. CONCLUSION: Plasma fibrinogen synthesis and degradation were disrupted in preeclampsia patients before and after diagnosis, and gamma-chain concentration was significantly increased. Plasma fibrinogen gamma chain may be of some value in preeclampsia prediction and auxiliary diagnosis.

Elevated microRNA-520g in pre-eclampsia inhibits migration and invasion of trophoblasts.

INTRODUCTION: Pre-eclampsia (PE) is a common and severe obstetric complication. MicroRNAs (miRs) have emerged as molecules that are associated with the disease. METHODS: Quantitative reverse transcription PCR (RT-qPCR) was used for serum miR-520g characterization from 19 severe pre-eclamptic and 19 normal pregnancies. In situ hybridation was adopted to localize microRNA-520g (miR-520g). Migration and invasion of HTR-8/SVneo cells were evaluated after miR-520g mimic treatment with transwell system. MiR-520g target gene was verified in luciferase reporter system. RESULTS: The expression of serum miR-520g displayed an upward trend as pregnancies progress. At first-trimester, miR-520g in pre-eclampsia was significantly higher than that in the control, but no significant differences were found in the second and last trimesters. MiR-520g localized in cytoplasm of early trimester placental trophoblasts. The migration and invasion of HTR8/SVneo were inhibited by miR-520g mimic treatment. Matrix metalloproteinase 2 (MMP2) was verified as a direct target of miR-520g. CONCLUSIONS: Elevated maternal serum level of miR-520g level in first trimester was detected in patients with severe PE. By suppressing the migration and invasion of trophoblast via at least partial inhibition of MMP2 translation inhibition, miR-520g might play a role in the defective spiral artery remodeling, and thus contribute to pre-eclampsia pathophysiology.

Association between the maternal serum levels of 19 eicosanoids and pre-eclampsia.

OBJECTIVE: To investigate whether serum levels of 19 eicosanoids are associated with pre-eclampsia. METHODS: A case-control study was performed using data for pregnant women with pre-eclampsia, normotensive pregnant women, and nonpregnant women, for all of whom serum samples had been collected at a hospital in Shanghai, China, between December 2012 and December 2013. Ultra-performance liquid chromatography-tandem mass spectrometry was used to measure the serum levels of 19 eicosanoids. RESULTS: Overall, 49 pregnant women with pre-eclampsia, 26 normotensive pregnant women, and 14 nonpregnant women were included. Women with pre-eclampsia had significantly higher serum levels of 11,12-epoxyeicosatrienoic acid (11,12-EET), the hydroxyeicosatetraenoic acids 5-HETE, 8-HETE, 12-HETE, and 15-HETE, and leukotriene B4 than did women with a normal pregnancy and nonpregnant women, both before and after the onset of pre-eclampsia (P<0.01 for all comparisons). Women with severe pre-eclampsia had significantly higher serum levels of 5-HETE, 15-HETE, and leukotriene B4 than did women with mild pre-eclampsia, women with a normal pregnancy, and nonpregnant women (P<0.01 for all comparisons). CONCLUSION: The eicosanoids 11,12-EET, 5-HETE, 8-HETE, 12-HETE, 15-HETE, and leukotriene B4 might play important parts in the occurrence and development of pre-eclampsia.

Quantification of preeclampsia-related microRNAs in maternal serum.

To identify the specific serum preeclampsia (PE)-related biomarkers, 10 microRNAs (miRNAs) were selected based on their reported aberrant (4 upregulated and 6 downregulated) expression in PE placenta. A total of 1,035 pregnant patients were enrolled. Finally, 32 pregnancies with PE and 32 healthy pregnancies were incorporated in the study. The expression of these 10 miRNAs in the different trimesters was determined by SYBR-Green reverse transcription-quantitative polymerase chain reaction. Compared with that in the healthy controls, the expression levels of miR-152, miR-183 and miR-210 in PE serum were higher in the second and third trimester, whereas the expression of miR-182 was only higher in the third trimester. The expression levels of 6 miRNAs (miR-1, miR-328, miR-363, miR-377, miR-500 and miR-584) that were downregulated in PE placenta showed no significant differences between pregnancies complicated by PE and healthy pregnancies throughout the 3 trimesters. Areas under the receiver operating characteristic [standard error (SE)] during the 20-24th gestational week for predicting PE were miR-152: 0.94 (SE, 0.026), miR-183: 0.97 (SE, 0.031) and miR-210: 0.93 (SE, 0.018). In conclusion, the expression levels of serum miR-152, miR-183 and miR-210 were elevated since the second trimester in pregnancies complicated with PE, indicating their potentials as serum biomarkers for forecasting PE.

Detection of 19 types of para-arachidonic acids in five types of plasma/serum by ultra performance liquid chromatography-tandem mass spectrometry.

The aim of this study was to examine the consistency of ultra performance liquid chromatography-tandem mass spectrometry (UPLC-TMS) in detecting the levels of para-arachidonic acids (PAAs) among differently processed plasma/serum samples. Ethylenediaminetetraacetic acid (EDTA)-K2, sodium citrate, heparin lithium, coagulant/separation gel, and coagulant-free vacuum blood-sampling tubes were used to collect the fasting blood samples from 15 volunteers. All blood samples were subjected to solid-phase extraction using an Oasis HLB µElution 96-well plate, and UPLC-TMS was used to detect 19 types of PAAs in the blood samples. Within the plasma samples, the concentrations of 5, 6-DHET; 11, 12-epoxyeicosatrienoic acid (EET); 5-hydroxyeicosatetraenoic acid (HETE); leukotriene B4 (LTB4); plasma thromboxane B2 (TXB2); and 12-HETE were significantly higher in the heparin lithium group than in the EDTA-K2 and sodium citrate groups. Within the serum samples, the concentration of LTB4 was significantly higher in the coagulant/separation gel group than in the coagulant-free group, while that of TXB2 was significantly higher in the coagulant-free group than in the coagulant/separation gel group. The levels of some types of PAAs in differently processed plasma/serum samples were inconsistent, and the concentrations of 5, 6-DHET; 5-HETE; 12-HETE; TXB2; and LTB4 were significantly higher in the two serum samples and the heparin lithium group than in the EDTA-K2 and sodium citrate groups.

Effect of 20-hydroxyeicosatetraenoic acid on biological behavior of human villous trophoblasts and uterine vascular smooth muscle cells.

During pregnancy, disorders in uterine spiral artery remodeling (USAR) cause preeclampsia and other diseases. The aim of this study was to investigate the effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on the biological behavior of human villous trophoblasts (HVTs) and human uterine vascular smooth muscle cells (HUVSMCs), and explore the role of 20-HETE in USAR. 20-HETE and its inhibitor HET0016 were used to study migration, invasion and apoptosis in the HVT and HVT-HUVSMC models. 20-HETE inhibited the migration and invasion of HVTs, and inhibited apoptosis in HUVSMCs and HUVSMCs co-cultured with HVTs. 20-HETE had thus obvious effects on the biological behavior of HVTs and HUVSMCs. These effects may cause USAR disorders and vascular dysfunction, leading to preeclampsia.

[Correlaion between serum Golph2 protein and hepatocellular carcinoma].

OBJECTIVE: To study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein. METHODS: Golph2 gene was cloned by RT-PCR using RNA from WBF44 cell line as template, the point mutations of the cloned sequence were corrected by PCR, then the gene (1206 bp) was cloned into pET-21 plasmid, and the resulted plasmid was transformed into E.coli DH5a. The expression of 6xHis and Golph2 fusion protein was induced by isopropylthio-beta-D-galactoside (IPTG). The expression of fusion protein was detected by SDS-PAGE and Western blot, and was purified by Ni NTA chelating agarose. The rabbit antibody against Golph2 protein was obtained by immunizing 2 rabbits with the purified Golph2 protein. The specificity and titer of the antibody was determined by Western-blot and ELISA respectively. Sandwich ELISA was used to detect the level of serum Golph2 protein. RESULTS: There were two replacement mutation and 1 deletion mutation in the cloned sequence contrasted to NM177937 in Genbank, including 644(T-->C, L-->P) , 970 (G-->A, V-->I) and 802 G deletion. The sequence was completely reversed by PCR. The sequence of Golph2 gene cloned into expression vector was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that Golph2 protein was expressed in E.coli DH5a. The antiserum could bind to the 52 kD recombinant protein and serum 73 kD protein specifically. The mean A450 value of ELISA for serum Golph2 protein were significantly higher in HCC and other liver diseases than that in control groups. The sensitivity and specificity for HCC were 44.5% and 82.0%, respectively, at the cut off value is more than or equal to 0.40. CONCLUSION: The polyclonal antibody against Golph2 protein is specific. The level of serum Golph2 is significantly higher in patients with HCC and other liver diseases than that in healthy controls.

[A study on the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein].

OBJECTIVE: To study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein. METHODS: Four specific siRNAs were obtained by transcription in vitro. After transfection with the siRNAs into HepG2.2.15 cells for 3 days, the inhibitive effects of La protein were analyzed by Western blot; the content changes of HBsAg, HBeAg and HBV-DNA were detected by ECL and RT-PCR. RESULTS: In comparison to normal cells, La protein was less in the cells. There was less La protein in the cells trans-infected with siRNAs. HBsAg, the HBeAg and HBV-DNA secreted by the cells transfected with siRNA were also less than that in the normal cells. CONCLUSION: There is a correlation between La protein and HBV mRNA and the expression of HBV protein.

[Real-time quantitative PCR detecting minimal residual disease in multiple myeloma patients after autologous peripheral blood stem cell transplantation].

In order to explore the role of real-time PCR in detecting minimal residual disease in multiple myeloma and Waldenstrom's macroglobulinemia after autologous peripheral blood stem cell transplantation (APBSCT), real-time PCR was used to quantitate the IgH rearrangement in 8 patients with multiple myeloma (MM) and 1 case of Waldenstrom's macroglobulinemia before and after APBSCT. The results showed that the copies of IgH rearrangement pre- or post-APBSCT were 3108 +/- 1043 and 549 +/- 660 (P < 0.05) respectively. The number of IgH copies was positively correlated with the amount of plasmocytes in patient 's bone marrow and the M-protein in peripheral blood (r = 0.86, P < 0.05). Similar result was obtained in a case of relapsed Waldenstrom's macroglobulinemia. In conclusion, the quantitative analysis of IgH rearrangement by real-time PCR is a novel way to evaluate the therapeutic efficaciousness and predict the prognoses in MM patients.