ABSTRACT
Objective To observe the transport of hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC)through placental barrier set up by choriocarcinoma trophoblast cells (Bewo cells),and to explore the biological role of PBMC as a carrier for HBV transport.Methods Bewo cells and PBMC were cultured and their proliferation and activity were detected by cell counting kit (CCK)-8.One hundred μL serum containing 5 ×10 6 copy/mL HBV DNA was used to infect PBMC,and cells infected with HBV were labeled by fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE).A co-culture model of Bewo cells and HBV-infected PBMC was set up by transwell chamber. The migration of HBV-infected PBMC was detected by flow cytometry.Realtime fluorescence quantitative polymerase chain reaction method was used to detect HBV DNA contents of PBMC under transwell chamber.Results PBMC and Bewo cells proliferated at around 24 h and entered into growth stagnation at around 120 h.The contents of PBMC labeled by green fluorescent at 0,12,24 and 48 h during co-culture under chamber were (0.445 ±0.021)%,(21 .180 ± 4.653 )%,(34.830 ± 7.156 )% and (64.185 ± 3.161)%,respectively.The amount of PBMC marked green fluorescence increased over prolonged incubation time (F =68.983,P =0.001 ).PBMC HBV DNA contents at 24 and 48 h of co-culture under chamber were (1.925±0.431)×103 copy/mL and (2.565 ±0.361)×103 copy/mL,respectively,indicating that PBMC under chamber were infected with HBV.Conclusions PBMC may be a target for HBV infection in extrahepatic tissues.Placental trophoblastic barrier built by transwell chambers may provide new ideas to investigate HBV transmission across the placenta in vitro .
ABSTRACT
AIM: To investigate the potential role of 14-3-3 tau in trophoblast cells on invasiveness. METHODS: 14-3-3 tau expression was detected in first-trimester villi, deciduas and human trophoblastic cell line (BeWo) by immunohistochemistry. Small interference RNA (siRNA) targeting 14-3-3 tau was transfected into BeWo cells. The effects of down-regulated 14-3-3 tau on invasion of human trophoblasts cell line BeWo were examined by matrigel invasion assay, and the transcription, translation of E-cadherin and snail were estimated by RT-PCR or Western blotting. U0126 was used to detect the extracellular-signal related kinase 1/2 (ERK1/2) function on down-regulation of 14-3-3 tau induced cell invasion. RESULTS: 14-3-3 tau was detected in the invasive trophoblastic cells in the first trimester villi and that invaded to the deciduas. BeWo cells also expressed 14-3-3 tau. Down-regulation of 14-3-3 tau increased the invasive cell-number of BeWo, as well as the expression of snail, and inhibited E-cadherin. U0126 inhibited the enhanced invasiveness in these cells induced by the down-regulation of 14-3-3 tau. CONCLUSION: 14-3-3 tau may regulate the invasiveness of human trophoblastic cells through ERK1/2 signaling pathway.
ABSTRACT
Las células estromales son las células más abundantes presentes en la decidua y juegan un papelmuy importante durante la implantación, la nutrición fetal y el mantenimiento del embarazo. Losprocedimientos que se han descrito para el aislamiento de células estromales requieren el uso de muchosanticuerpos monoclonales ya que hay contaminación con otros tipos celulares en la decidua y ademásalgunos marcadores características de células estromales, muestran variabilidad en los diferentes díasde la gestación. En este estudio se estandarizó un procedimiento de aislamiento por digestión enzimática,gradiente de densidad y adherencia al plástico y se caracterizaron las células estromales murinas porexclusión de marcadores que se expresan en macrófagos (F4-80), células epiteliales y trofoblasto(citoqueratina-7), obteniéndose un 98% de células negativas para estos marcadores que correspondería alas células estromales. Esta técnica de aislamiento permite obtener células estromales con métodos menoscostosos y altamente eficientes que facilita el acceso a un modelo celular de gran utilidad en el estudio dela fisiología de la gestación en diferentes especies.
Stromal cells are the most abundant cell population present in decidual tissue; they are involved inkey processes during embryo implantation, fetal nutrition and the pregnancy maintenance. Described procedures for stromal cells isolation require the use of many monoclonal antibodies due to contaminationwith another cell types in the decidua; besides, some markers of stromal cells show variability during thedays of gestation. In this study, we standardized a procedure for isolation by enzymatic digestion, densitygradient and adherence to plastic. Murine stromal cells were characterized by exclusion of markers thatare expressed in macrophages (F4-80), epithelial cells and trophoblast (cytokeratin-7), yielding a 98% ofnegative cells for these markers that correspond to stromal cells. This isolation procedure permits to obtainstromal cells with less expensive and high efficiency methods that provide a useful cellular model to studythe physiology of gestation in different species.
As células estromales são as células mais abundantes presentes na decídua e tem um papel muitoimportante durante a implementação, a nutrição fetal e a manutenção da gravidez. Os procedimentosdescritos para o isolamento das células estromales necessitam o uso de muitos anticorpos monoclonais jáque há contaminação com outros tipos celulares na decídua e demais alguns marcadores característicosdas células estromales que tem variabilidade nos diferentes dias de gestação. No presente trabalho foiestandardizado um procedimento de isolamento por digestão enzimática, gradiente de densidade eaderência ao plástico e foram caracterizados as células estromales murinas por exclusão de marcadoresque expressão em macrófagos (F4-80), células epiteliais e trofoblasto (citoqueratina-7), obtendo-se um98% de células negativas para estes marcadores que corresponderia às células estromales. Esta técnicade isolamento permite obter células estromales com métodos menos custosos e altamente eficientes quefacilita o aceso a um modelo celular de grande utilidade no estudo da fisiologia da gestação em diferentesespécies.