Objective To determine the expression of aquaporin-1,3,8,9 mRNA (AQP-1,3,8, 9) in amniotic membranes in pregnant women with polyhydramnios. Methods Amniotic membranes were collected from women who presented with either polyhydramnios (n= 5)or normal amniotic fluid volume (control, n= 5) underwent elective cesarean sections at term. The AQP-1,3,8,9 mRNA expression were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Results The expression of AQP-9 mRNA on fetal membranes were significantly higher in polyhydramnios groups (1. 1403±0. 0831) than that of control (0. 5903±0. 1909) (P = 0. 002), although the expression of AQP-1, 3 and 8 showed no significant difference between the two groups (P= 0. 972, 0. 242,0. 608, respectively). Conclusions AQP-9 may play an important role in maintaining the volume and the balance of different components of amniotic fluid in polyhydramnios cases.

BACKGROUND: Placenta and fetal membrane play an important role In maternal-fetal homeostasis. However, the molecular and cellular mechanisms underlying water transfer across placenta and amniotic membrane remain unknown. It is hypothesized that maternal-fetal fluid exchanges via aquaporin (AQP) water channels in the placenta and fetal membrane.OBJECTIVE: To investigate AQP8 protein expression in normal human placenta and fetal membrane.DESIGN, TIME AND SETTING: A control observation was performed at the Central Laboratory of Guangzhou Medical College from July to December 2005.MATERIALS: Human placenta and fetal membrane tissues from 5 elective cesarean section deliveries of normal term pregnancies (range 37-42 weeks) were studied. Maternal age averaged (27?) years old. Experimental protocol was approved by the Hospital's Ethics Committee.METHODS: Thirty minutes after delivery, fetal membrane and placenta were dissected and washed with sterile physiological saline. Some were frozen at -80?, and the remaining tissues were fixed for 24-48 hours with 10% neutral formalin and paraffin embedded for immunohistochemical staining.MAIN OUTCOME MEASURES: AQP8 expression and distribution in human placenta and fetal membrane were detected by the reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blotting analysis.RESULTS: RT-PCR results showed that AQP8 mRNA was expressed in both placenta and fetal membrane tissues. Western blotting analysis also yielded positive results in placenta and fetal membrane with a specific band site at approximately 45 000.Immunohistochemistry results revealed that AQP8 protein was expressed in placental syncytiotrophoblasts, amniotic epithelial cells, and chorion cytotrophoblasts.CONCLUSION: At protein level, AQP8 is expressed in placental syncytiotrophoblasts, amniotic epithelial cells, and chorion cytotrophoblasts.

Objective To investigate the correlation between the CD40 gene( - 1 C/T)single nucleotide polymorphism(SNP) and acute coronary syndromes(ACS), and the expression of CD40 on platelets. Method A total of 562 patients with ACS canfirmed by coronary angiography were divided into 3 groups according to the clinical characteristics, namely ACS patients( n = 210), stable angina(SA) patients( n = 189) and control group( n = 163).ACS was defined as ischemic chest pain at rest resulting in admission to hospital and > 50% stenosis in a major coronary artery with or without a rise in troponin Ⅰ. SA was defined as stable effort-related angina without change in angina pattern in 3 months. Patients with infection, tumor, or liver or kidney disease were excluded The gene polymorphism was measured by the polymerase chain reaction and restriction fragment length polymorphism(PCR-RFIP) and identiffed by sequencing. The expression of CD40 on platelets was detected by flow cytometry. The frequency, distribution of genotypes was compared using cross-tabulation and standard X~2 test. Result The CC genotype(31% ) and C allele of frequency(57.9%)of CD40 gene in ACS patients were significantly higher than those in SA(15.9%, 43.1% ) and control groups( 16.1%, 42.6% ). No significant difference of the genotypes or allele frequencies was found between SA and control group(X~2 = 0.053, P = 0.974;X~2 = 0.017, P = 0.897). 1he expression of CD40 on platelets in patients with C alleles carries was significandy higher than that of T allele carries in each group( P <0.0001). Conclusions CD40- 1C/T polymorphism was associated with ACS in Chinese Han nationallity.

OBJECTIVETo explore the clinical significance of vasohibin-1 expression in colorectal cancer tissues and its correlation with vascular endothelial growth factor A(VEGF-A) and microvessel density (MVD).

METHODSTumor tissues and paired adjacent normal tissue (distance to cancer >5 cm) from 60 colorectal cancer patients undergoing resection in the Second Hospital of Tianjin Medical University from June 2013 to November 2013 were included in this study. The protein expressions of vasohibin-1, VEGF-A and MVD were detected by immunohistochemical staining. The mRNA expressions of vasohibin-1 and VEGF-A were detected by RT-PCR. The protein expressions of vasohibin-1 and VEGF-A were observed by Western blot. Correlation among parameters was examined.

RESULTSVasohibin-1 expression was mainly localized in the cytoplasm of tumor cells and endothelial cells. VEGF-A expression was mainly localized in the cytoplasm and membrane of tumor cells. The expressions of vasohibin-1, VEGF-A and MVD in colorectal tumor tissues were significantly higher than those in corresponding adjacent tissues [43.3% (26/60) vs. 16.7% (10/60), 51.7%(31/60) vs. 18.3% (11/60), (39.67 ± 16.80)/mm² vs. (17.85 ± 6.43)/mm², all P<0.05]. Higher vasohibin-1 expression was significantly associated with TNM stage and metastasis (P<0.05). Vasohibin-1 expression was positively correlated with VEGF-A and MVD (r=0.378, 0.628, all P<0.05). Vasohibin-1 and VEGF-A mRNA expressions and protein expressions in colorectal cancer tissues were significantly higher than those in corresponding adjacent tissues (all P<0.05).

CONCLUSIONVasohibin-1 expression in colorectal cancer tissues is significantly higher as compared to corresponding adjacent tissues. Vasohibin-1 expression is positively correlated to VEGF-A and MVD, and associated to TNM stage and metastasis. Positive vasohibin-1 expression indicates a poor prognosis of patients with colorectal cancer.

Cell Cycle Proteins ; Colorectal Neoplasms ; Humans ; Microvessels ; Neovascularization, Pathologic ; Prognosis ; Vascular Endothelial Growth Factor A

To develop Senecavirus A (SVA) virus-like particles (VLPs), a recombinant prokaryotic expression plasmid pET28a-SVA-VP031 was constructed to co-express SVA structural proteins VP0, VP3 and VP1, according to the genomic sequence of the field isolate CH-FJ-2017 after the recombinant proteins were expressed in E .coli system, and purified by Ni+ ion chromatographic method. The SVA VLPs self-assemble with a high yield in vitro buffer. A typical VLPs with an average diameter of 25-30 nm which is similar to native virions by using TEM detection. Animals immunized by SVA VLPs shown that the VLPs induced high titers neutralizing antibodies in Guinea pigs. This study indicated that the VLPs produced with co-expressing SVA structural proteins VP0, VP3 and VP1 in prokaryotic system is a promising candidate and laid an important foundation for the development of a novel SVA VLPs vaccine.
Animals ; Antibodies, Neutralizing ; Escherichia coli/genetics* ; Genomics ; Guinea Pigs ; Picornaviridae/genetics*

The aim of this study was to produce E^rns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified E^rns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E^rns was constructed based on the gene sequence of BVDV-1 NADL strain. The E^rns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-E^rns into CHO cells. The expression and purification of the E^rns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the E^rns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified E^rns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the E^rns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the E^rns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log₁₀ was induced in rabbits. In this study, purified BVDV E^rns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.
Rabbits ; Animals ; Cricetinae ; Cricetulus ; CHO Cells ; Antibodies, Viral ; Diarrhea Viruses, Bovine Viral/genetics* ; Antibodies, Monoclonal/genetics* ; Diarrhea ; Viral Vaccines/genetics*

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×10⁷ U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.
Swine ; Animals ; Cricetinae ; Interferon-gamma/pharmacology* ; Cricetulus ; CHO Cells ; Sincalide ; Recombinant Proteins/pharmacology* ; Antiviral Agents/pharmacology*