Initiative Research On Effects Of Baicalin On CTB Apoptosis Induced By TNF-α

ObjectiveTo improve the methods for isolation and culture of human cytotrophoblasts(CTBs) from early pregnancy villi,to identify it and to investigate its partial biological properties.To study the effects of TNF-αand baicalin on the activity of CTB.To reseach the effects of baicalin on CTB apoptosis induced by tumornecrosis factorα(TNF-α).MethodsHuman villi from 6～8 weeks legal voluntary interruption pregnancy were mechanically isolated,complex enzyme-digested,and purified by different speeding adherence.The cellular morphology was observed by inverted phase contrast microscope and the nucleus and cytoplasm were observed by staining of haematoxylin and eosin(HE).The immunocytochemistry was used to examine the expression of cytokeratin 18(CK18),vimentin(Vim) and human placental lactogen(hPL).Flow cytometer was used to observe the cell cycle.CTB were cultured with different concentrations of TNF-αand baicalin.After culturing for 24h, the activity of CTB was examined by MTT assay.Flowcytometric analysis were used to determine the apoptotic cells.And fluorescent microscope were used to determine nuclear shape of CTB.ResultsThe cultured cells appeared as patchy spreading and irregular polygonal with big oviform nucleus and transparent cytoplasm.CK18 expressed in all the cells cultured and Vim expressed only in some of the cells.hPL immunoreactive positive substances were distributed in cytoplasm.Cell cycle revealed that 58%cells were at G₀/G₁.The activety of the hepatocyte(A) in the groups of 10 ng·ml^-1 and 20 ng·ml^-1 of TNF-αwere significantly lower than black control group(P＜0.05).The activety of the hepatocyte(A) in the different concentrations of baicalin groups were all higher than black control group.The activety of the hepatocyte(A) in the groups of baicalin were significantly higher than those in apoptotic model group(P＜0.05).The flowcytometry showed that the apoptosis rates in the baicalin group were lower than that of the apoptotic model group(P＜0.05).Fluorescent microscope showed nuclear contraction, and dense and strong fluorescent can be observed.ConclusionWe have successfully cultured human cytotrophoblasts isolated from early pregnancy villi and obtained monomorphic cells with stable growth,which provides a useful experimental technique for the studies of the roles of CTBs in gravidity physiology and immune tolerance.TNF-αcan restrain CTB's proliferation.Baicaline can advance CTB's growth. Baicaline can restrain CTB apotosis induced by TNF-α.Hepatocytes were cultared with different concentrations of TNF-αand baicalin.