【作者】 刘惠萍；

【导师】 王若光；

【作者基本信息】 湖南中医药大学 ， 中医妇科学， 2006， 硕士

【摘要】 目的改善体外分离、培养人早孕绒毛细胞滋养层细胞（cytotrophoblast,CTB）的方法,并对其进行鉴定,探讨CTB的一些生物学特性。研究TNF-α和黄芩苷对CTB细胞活力的影响及黄芩苷对体外TNF-α诱导培养CTB凋亡的影响。方法取人工流产6～8周妊娠绒毛,机械法分离,复合酶消化,差速贴壁法纯化细胞后继续培养。倒置显微镜观察其大体形态及体外转化,HE染色观察其胞核情况,免疫细胞化学观察细胞的细胞角蛋白18（cytokeratin,CK18）、波形蛋白（vimentin,Vim）和人胎盘催乳素（human placental lactogen,hPL）的表达。流式细胞仪测定其细胞周期。选取不同浓度的TNF-α作用于CTB,四甲基偶氮唑盐（3-（4,5-dimethylthiazo-2-yl）-2,5-diphenyl-tetrazolium bromide,MTT）比色实验分析TNF-α对CTB细胞活力的影响。选取不同浓度的黄芩苷稀释液作用于CTB,MTT比色实验分析黄芩苷对CTB细胞活力的影响。选取TNF-α作用浓度10ng/mL造模及10000倍稀释液的黄芩苷浓度作用于CTB,MTT比色实验和流式细胞术分析黄芩苷对CTB凋亡的影响。荧光显微镜CTB凋亡的形态学观察。结果分离培养的CTB呈不规则多角形片状,单层铺展生长。所有的细胞都表达CK18,而Vim只在部分细胞表达,hPL免疫反应阳性物质在胞浆表达。细胞周期显示约有60%的细胞处于G₀/G₁期。TNF-α作用后的CTB活性（A值）均低于于空白对照组,并且随着TNF-α浓度越大,A值越小。5、10、20ng/ml浓度TNF-α作用后CTB与空白对照组比较差异有显著性,P＜0.05。黄芩苷作用后的CTB活性（A值）高于空白对照组。黄芩苷作用后的CTB活性（A值）均高于凋亡模型组,差异具有显著性,P＜0.05。流式细胞术示黄芩苷作用后的CTB凋亡率低于凋亡模型组及黄体酮组。在荧光显微镜下,凋亡的CTB细胞核变小皱缩,可见致密强荧光。结论成功分离培养了人早孕绒毛CTB,方法简便易行,获得的细胞形态单一、生长稳定,为进一步研究CTB在妊娠生理和妊娠免疫耐受中的作用提供了实验基础。TNF-α明显抑制细胞增殖分裂的过程。黄芩苷对滋养细胞的生长增值有促进作用。黄芩苷对TNF-α诱导培养的CTB凋亡有抑制作用。更多还原

【Abstract】 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.更多还原