ABSTRACT
Objective To construct a eukaryotic vector of chicken-derived Wnt3a tagged with EGFP (pCAG-MCs-Wnt3a-EGFP) and investigate the influence to the proliferation and axonal formation of neural precursor cells when Wnt3a was overexpressed during the development of chick embryonic spinal cord. Methods Wnt3a gene was amplified from the total RNA obtained from chick embryonic spinal cord using molecular techniques, then connected with pCAG-MCs-EGFP to construct pCAG-MCs-Wnt3a-EGFP, which was identified by digestion and genetic sequencing. At embryonic day (E) 2.5-3.0, pCAG-MCs-Wnt3a-EGFP (experimental group) and pCAG-MCs-EGFP (control group) were transfected into the chick embryonic spinal cord using in vivo electroporation, respectively. Samples were collected at E4 (5 simples of each groups) and then conducted frozen section. The immunofluorescent staining was performed to detect the expression of Wnt3a and proliferating cell nuclear actigen (PCNA) for analyzing the relationship between Wnt3a and cell proliferation, and observe the axonal formation of neural precursor according to the green fluorescence of Wnt3a protein. Results pCAG-MCs-Wnt3a-EGFP was obtained and its gene sequencing was identical with the Gene bank. Green fluorescence was observed at E4 after pCAG-MCs-Wnt3a-EGFP transformed to chick spinal cord. In transversal section of chick embryonic spinal cord, the results of immunofluorescent staining showed Wnt3a was successfully overexpressed. Meanwhile, the amount of neurons projecting axons was dramatically decreased (n=3, P < 0.01), compared to the control group, concomitant with the significant elevation of PCNA level (n =3, P < 0.01). Conclusion pCAG-MCs-Wnt3a-EGFP is successfully constructed and our study confirmed that Wnt3a plays a vital role in the proliferation and axonal formation of neural precursor cells in the developing chick spinal cord.
ABSTRACT
<p><b>BACKGROUND</b>Enhanced apoptosis of cytotrophoblasts in early pregnancy is associated with high risk of intrauterine growth retardation and preeclampsia, which are two common pregnant complications. Its etiological factors remain unclear. Cytotrophoblasts share some traits with innate immune cells and may show response to lipopolysaccharide. This study was conducted to demonstrate whether lipopolysaccharide has apoptosis-inducing effects on cytotrophoblast and the role of innate immune reaction in this process.</p><p><b>METHODS</b>Cytotrophoblasts were isolated from early pregnant villous tissues and cultured with serum-free medium. Subsequently, cytotrophoblasts were treated with lipopolysaccharide at the concentrations of 0 (control), 25, 50, 100 and 200 ng/ml for 24 hours. Apoptosis of cytotrophoblasts was determined by light microscopy, Hoechst 33258 DNA staining with a fluorescent microscope, transmission electron microscope and annexin V-fluorescein isothiocyanate-conjugated/propidium iodide (PI) staining with flow cytometry. Then expression of caspase-3 was detected by Western blot. Confocal immunofluorescence technique was used to detect tumor necrosis factor alpha expression in cytotrophoblasts. The levels of tumor necrosis factor alpha in the culture medium were detected by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Under light, fluorescence microscope and transmission electron microscope, characteristic alternations of apoptosis in cytotrophoblasts were observed after lipopolysaccharide treatment. Flow cytometry results showed that lipopolysaccharide significantly increased apoptosis indexes of cytotrophoblasts. Significant statistical differences were found in the above groups (P = 0.01). The mean relative densities of bands corresponding to caspase-3 were significantly increased in groups treated with lipopolysaccharide, as compared with the normal control (P < 0.001). Tumor necrosis factor a expression was found to increase in cytotrophoblasts by confocal immunofluorescence technique and in culture medium by enzyme-linked immunosorbent assay after lipopolysaccharide treatment. A positive correlation was found between tumor necrosis factor a expression and apoptosis indexes of cytotrophoblasts (r = 0.747, P < 0.001).</p><p><b>CONCLUSION</b>Apoptosis of cytotrophoblasts could be induced by lipopolysaccharide, in which innate immune reaction is the important mechanism.</p>
Subject(s)
Humans , Apoptosis , Blotting, Western , Cells, Cultured , Dose-Response Relationship, Drug , Flow Cytometry , Immunity, Innate , Lipopolysaccharides , Pharmacology , Microscopy, Confocal , Trophoblasts , Cell Biology , Tumor Necrosis Factor-alpha , PhysiologyABSTRACT
Objective To investigate the expression of peroxisome proliferators-activated receptor ? (PPAR?) in trophoblast and relation between PPAR? ligands and trophoblast invasion.Methods We examined the expression of PPAR? by immunohistochemistry,immunocytochemistry and real time quantitative PCR.We next examined,using the cytotrophoblast culture model,the biological role of PPAR? ligands in vitro.Results PPAR? was mainly localized in the nuclei of villous cytotrophoblast and extravillous cytotrophoblast of cell islands and cell columns.In villous tissue and cultured trophoblast from early first trimester,the level of expression of PPAR? mRNA and protein was 36.0?5.1,13.4?3.1 and 1.35?0.08,1.13?0.11;from late first trimester it was 23.3?5.5,6.1?1.3 and 1.17?0.03,0.86 ?0.05,and the expression of PPAR? was obviously decreased (P