人子宫内膜基质细胞和腺体细胞的分离培养及鉴定

目的：人子宫内膜分离培工得到纯度较高的内膜基质细胞和腺体细胞。方法：采用二次滤网过滤法进行分离并通过光镜观察和免疫细胞化学染色对其进行鉴别。结果：基质细胞在接种后0．5h开始贴壁,可见梭形和多角形两种形态的细胞。免疫细胞化学染色显示这两种细胞均具有波形蛋白免疫反应,反应率可达95％以上,而细胞角蛋白无免疫反应性,提示它们为来源于内膜基质的基质细胞,大部分腺体细胞在接种24h后贴壁,培养后4d左右腺体细胞呈旋涡状排列,单个细胞为多角形,核大而圆,腺细胞呈细胞角蛋白反应性,反应率在90％以上,结论：采用二次滤网过滤法可成功地分离入人子宫内膜基质细胞和腺体细胞。     

丹那唑对体外培养的原位与异位子宫内膜细胞生长的影响

应用胶原凝胶细胞培养和传代培养的方法培养原位与异位子宫内膜细胞，并经丹那唑处理在无丹那唑对照培养中，细胞生长良好，形态呈星形，以胞浆突起相互连接。而经两种浓度丹那唑处理后，细胞生长受抑，传代后２４ｈ细胞贴壁力受损，部分细胞呈致死性病理改变，丹那唑作用的超微结构变化包括胞浆内大量空泡与脂滴，溶酶体增多，线粒体肿胀与细胞核不规则，核膜内陷，提示胶凝胶培养系统为较佳的培养子宫内膜细胞的方法，丹那唑可直接     

种植窗期子宫内膜细胞体外培养的形态学特点与妊娠结局的关系

目的对种植窗期人子宫内膜进行体外培养,探讨不同妊娠结局的子宫内膜细胞体外培养的细胞形态学特点。方法收集拟行IVF-ET助孕的患者种植窗口期（排卵后第5～7天）子宫内膜组织进行体外培养,于倒置显微镜下观察子宫内膜细胞形态学特性,并通过免疫荧光法进行鉴定,采用培养板单层贴壁细胞的原位计数方法,计算子宫内膜间质细胞和腺上皮细胞数量及比例。按助孕后妊娠结局,分为妊娠组和未妊娠组,比较两组患者子宫内膜细胞体外培养的细胞形态学特点。结果39例子宫内膜组织,培养成功36例。其中妊娠组19例,培养成功18例,培养成功率94.7％;未妊娠组20例,培养成功18例,培养成功率90.0％,两者的培养成功率差异无统计学意义（P〉0.05）。子宫内膜间质细胞接种后即开始贴壁,细胞呈扁平状,胞浆透明,核圆居中,波形蛋白表达阳性。子宫内膜腺细胞于接种2h后开始贴壁,细胞呈多角形或蝌蚪形,旋涡状排列,核圆而大,角蛋白表达阳性。妊娠组腺细胞贴壁率为64.8％±4.7,贴壁培养后腺上皮细胞的比例为17.3％±9.5,间质细胞比例为82.7％±9.5;未妊娠组子宫内膜组织腺细胞贴壁率为57.6％±3.4,贴壁培养后腺上皮细胞的比例为14.7％±4.8,间质细胞比例为85.3％±4.8。妊娠组比未妊娠组腺上皮细胞贴壁率及贴壁后腺细胞比例高,但差异无统计学意义（P〉0.05）。结论种植窗期人子宫内膜细胞进行体外培养后,妊娠组与未妊娠组细胞的外观特征相似,虽妊娠组腺上皮细胞贴壁率及贴壁后腺细胞比例较高,但两组差异无统计学意义。单以种植窗期子宫内膜体外培养的细胞形态特点,无法判断内膜的容受性,也无法预测妊娠结局。     

子宫内膜异位症在位内膜腺上皮细胞及基质细胞的分离培养和鉴定

目的分离培养子宫内膜异位症在位内膜组织中子宫内膜腺上皮细胞及基质细胞,建立研究子宫内膜异位症的细胞模型。方法对分泌期子宫内膜异位症在位内膜组织用混合酶消化,滤网过滤和差速梯度离心的方法分离,体外培养后通过光镜观察及免疫细胞化学、免疫荧光化学方法对分离细胞鉴定。结果分离的细胞角质蛋白阳性子宫内膜腺细胞百分率约90～95%;分离的骨架蛋白形成蛋白阳性的基质细胞百分率达90%以上。结论本研究获得较高纯度的子宫内膜腺细胞和基质细胞,成功建立了研究子宫内膜异位症的细胞模型。     

子宫内膜基质细胞作为饲养层培养人胚原始生殖细胞

目的探讨子宫内膜基质细胞作为饲养细胞培养人胚原始生殖细胞的可能。方法从5—9周人胚胎生殖嵴、中肾嵴、肠系膜中消化分离原始生殖细胞(PGCs),种植于经丝裂霉素c处理的人子宫内膜基质细胞饲养层上培养。结果PGCs可以在体外培养3个多月,传14代。这样培养的PGCs表达胚胎干细胞的多种标志,如碱性磷酸酶染色、SSEA和TRA抗原等。另外细胞保持正常的染色体核型。结论人子宫内膜基质细胞可以作为饲养细胞在体外培养人胚原始生殖细胞,保持未分化状态。     

PGE2促进培养的人子宫内膜细胞VEGF的表达

目的探讨炎性介质前列腺素E2（PGE2）对体外培养的人子宫内膜细胞VEGF的分泌、VEGF mRNA影响和调控的信号通路的作用。方法收集子宫内膜异位症（EMs）患者的子宫内膜组织进行体外培养、纯化，采用免疫组化方法对细胞进行鉴定。子宫内膜细胞培养24h后，用酶免法测定细胞培养液上清VEGF蛋白的含量，RT-PCR法测定不同浓度PGE2对体外培养的子宫内膜细胞VEGF mRNA表达的影响。酶免法测定不同浓度的Forskolin、SQ 22536及PGE2＋SQ 22536等对子宫内膜细胞培养液上清VEGF分泌的影响。结果加入不同浓度的PGE2 24hA，VEGF mRNA及其蛋白的表达呈剂量依赖性增加。不同浓度的Forskolin可剂量依赖性地增加VEGF的表达，而SQ 22536明显抑制PGB所诱导的VEGF浓度升高。PGE2可促进子宫内膜细胞VEGF的表达，促进效应呈剂量依赖性，该效应可能部分通过cAMP介导。结论PGE2作为一种炎性介质可能通过增加子宫内膜细胞VEGF的表达参与内异症血管形成的发生。     

子宮内膜异位症在位内膜腺上皮细胞及基质细胞的分离培养和鉴定

目的 分离培养子宫内膜异位症在位内膜组织中子宫内膜腺上皮细胞及基质细胞,建立研究子宫内膜异位症的细胞模型.方法 对分泌期子宫内膜异位症在位内膜组织用混合酶消化,滤网过滤和差速梯度离心的方法分离,体外培养后通过光镜观察及免疫细胞化学、免疫荧光化学方法对分离细胞鉴定.结果 分离的细胞角质蛋白阳性子宫内膜腺细胞百分率约90～95%;分离的骨架蛋白形成蛋白阳性的基质细胞百分率达90%以上.结论 本研究获得较高纯度的子宫内膜腺细胞和基质细胞,成功建立了研究子宫内膜异位症的细胞模型.     

人子宫内膜干细胞的分离、培养及鉴定

目的 分离、培养子宫内膜干细胞并进行鉴定.方法 采用酶消化和机械方法相结合分离人子宫内膜细胞,两次滤网过滤分离基质细胞和上皮细胞.采用有限稀释法培养基质细胞和上皮细胞,观察细胞的形态及生长特性,免疫荧光检测波形蛋白和细胞角蛋白表达情况.原代培养15 d后,计算细胞克隆形成率,流式细胞术检测抗原CD133、CD34、CD45、CD90、CD73及CD29表达.结果 基质细胞呈纤维样细胞形态,排列呈辐射状,免疫荧光显示其波形蛋白表达阳性;原代培养15 d后,细胞克隆形成率达(1.34±0.44)％.上皮细胞呈多角形或椭圆形,免疫荧光显示其细胞角蛋白表达阳性;原代培养15d后,未发现克隆形成.流式细胞术检测克隆形成的基质细胞CD29、CD90、CD73表达阳性,CD34、CD45、CD133表达阴性.结论 人体存在少量具有克隆形成能力和高度增生潜能的子宫内膜干细胞,这些干细胞也可能来源于骨髓间充质干细胞.     

兔子宫内膜片层体外构建的实验研究

探索以扩增培养的子宫内膜细胞为种子细胞、以液态胶原为支架材料,构建组织工程化子宫内膜片层的可行性。体外分离并规模化扩增子宫内膜上皮细胞与基质细胞,以Ⅰ型液态鼠尾胶原为支架,按自然子宫内膜的结构在体外重建具有两层结构的组织工程化子宫内膜,体外培养14天时进行H.E.染色与免疫组化鉴定。H.E.染色与免疫组化染色结果表明：再造子宫内膜片层在胶原材料中能够较好地维持双层结构,上皮层呈CK18阳性,在某些部位还能够形成极化的柱状上皮。利用体外扩增培养的子宫内膜上皮细胞与基质细胞构建的组织工程化子宫内膜片层具有子宫内膜组织的双层结构与极化上皮特征。     

改良差时贴壁法分离培养鉴定小鼠骨髓间充质干细胞和内皮前体细胞

目的 探索同时从小鼠骨髓分离培养间充质干细胞(MSCs)与内皮前体细胞(EPCs)及对其鉴定的方法。方法 小鼠骨髓细胞经改良差时贴壁法分离,以48h为时间点,48h内贴壁细胞传至3代后行成骨、成软骨、成脂分化诱导实验,流式细胞术（FCM）检测其表面标记;48h后收集未贴壁细胞,传至3代后行血管形成实验,传至5代后行CD31免疫荧光细胞染色实验,FCM检测其表面标记。 结果 第3代48h内贴壁细胞可诱导分化为骨、软骨和脂肪细胞,FCM 检测Sca-1、CD29、CD45、CD11b 阳性率分别为（98.30±0.75）%,（97.47±1.32）%,（1.87±0.15）%,（1.03±0.71）%;第3代48h后贴壁细胞在基质胶上可形成血管样结构,第5代48h后贴壁细胞特异性表面抗原CD31呈阳性表达,FCM检测CD34、CD133、血管内皮生长因子受体（VEGFR2） 阳性率分别为（88.90±1.18）%,（92.73±2.90）%,（87.63±1.79）%。 结论 采用改良差时贴壁法可同时分离培养扩增小鼠骨髓 MSCs和 EPCs,且简便高效稳定可重复。     

人外周血内皮祖细胞的分离、培养和扩增

目的:改进从人外周血中分离、培养和体外扩增血管内皮祖细胞(EPC)的方法。方法:采用不同淋巴细胞分离液,用密度梯度离心法从外周血分离EPC;用CD34免疫磁性活化细胞分选系统(MACS)分离CD34 细胞;分别培养在包被和不包被有人纤维连接蛋白(HFN)的培养板内;采用细胞免疫化学法检测内皮细胞表面标志CD31、CD34和vWF的表达。结果:从成人外周血可分离获得EPC;不同的分离条件可影响获得EPC的数量和质量,HFN对EPC的生长有促进作用。结论:进一步改进从人外周血分离获取EPC并行体外扩增的方法,为EPC的研究奠定了基础。     

The correlation between human adipose-derived stem cells differentiation and cell adhesion mechanism

In recent years, research in the areas of stem cells has dramatically increased, including studies of cellular adhesion to a substrate. We sought to determine the adhesive properties of human adipose-derived stem cells (hASCs) for extracellular matrix proteins. The adhesion of hASCs to collagens and laminin was completely inhibited by a monoclonal antibody, Mab 2253, which binds to the β1 integrin subunit. These data indicate that hASC adhesion to collagens and laminin was exclusively mediated by an integrin. Cell adhesion on fibronectin (Fn) was inhibited by the heparin-binding peptide (HBP) in the presence of Mab 2253, but not by either Mab 2253 or HBP alone. These results indicate that both the β1 subunit and the heparan sulfate proteoglycan participated in the cell adhesion to Fn. Microscopic views showed extensive spreading of hASCs cultured on Fn, whereas the cells maintained a round shape when cultured on a heparin-binding domain (HBD) substrate. hASCs differentiated into adipocytes, which stained positive for lipid vacuoles by Oil Red-O analysis, more readily on HBD substrate than on FN substrate. These results suggest that hASCs have an adhesion mechanism for the HBD of Fn and hASC morphology is controlled by the adhesion mechanism and strongly correlated with adipogenic differentiation.     

Expression of focal adhesion kinase in endometrial stromal cells of women with endometriosis was adjusted by ovarian steroid hormones

The aim of our study is to investigate the effects of ovarian steroid hormones on focal adhesion kinase (FAK) expression in ESCs and whether there is alteration in women with endometriosis. FAK expression was assessed by western blotting analysis. Elevated expression of FAK was seen in the cultured ESCs treated with estrogen (P < 0.05). Expression of FAK protein was not changed in ESCs after treated by progesterone or treated by estrogen and progesterone. The level of up-regulation by estrogen in endometriosis is significantly higher than that from women without endometriosis (P < 0.05). FAK expression in endometrial stromal cells from endometriosis was more sensitive to estrogen, which might contribute to the pathogenesis and progress of endometriosis.     

Expression of leukocyte-endothelial cell adhesion molecules on monocyte adhesion to human endothelial cells on plasma treated PET and PTFE in vitro

We used a coculture model to evaluate the inflammatory potential of ammonia gas plasma modified PET and PTFE by flow cytometry and immunohistochemistry. In these studies, human endothelial cells from umbilical cord (HUVEC) and promonocytic U937 cells were used. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-) were used as controls. U937 adhesion to endothelium on each surface was evaluated at day 1 and day 7. To further investigate the role of leukocyte–endothelial cell adhesion molecules (CAMs) in cell-to-cell interaction on material surfaces, the expression of the leukocyte–endothelial CAMs: ICAM-1, VCAM-1, PECAM-1, and E-selectin on HUVECs were evaluated after U937 cell adhesion. The results demonstrated that plasma treated PET (T-PET) and treated PTFE (T-PTFE) did not increase U937 cell adhesion compared to the negative control. Maximal adhesion of U937 cells to HUVEC was observed on TNF- stimulated endothelium with significant differences between day 1 and day 7, which is consistent with our prior observation that T-PET and T-PTFE did not cause HUVECs to increase the expression of adhesion molecules. After U937 cell adhesion, the expression of ICAM-1 and VCAM-1 of HUVECs were not different on T-PET and T-PTFE compared with the negative control. However, the expression of E-selectin was reduced on day 1, but not on day 7. The effects of plasma treated PET and PTFE on HUVEC adhesion and proliferation were also studied. On day 1 there were slight increases in the growth of HUVECs on both of T-PET and T-PTFE but this was not statistically significant. On day 7, the cell number increased significantly on the surfaces compared to the negative control. The results demonstrate that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and these surfaces do not exhibit a direct inflammatory effect in terms of monocyte adhesion and expression of leukocyte–endothelial CAMs. The monocyte adhesion to endothelial cells on surfaces can be used as a tool for the evaluation of material surface modification and further to study the mechanisms of cell-to-cell interactions in response to surfaces.     

小鼠骨髓间充质干细胞分离培养方法的建立

目的建立培养小鼠骨髓间充质干细胞(none mesenchymal stem cells,BMSCs)的分离培养方法。方法采用全骨髓体外分离并采用差速贴壁法纯化扩增C57BL/6小鼠BMSCs,形态学观察细胞生长情况,流式细胞仪检测其表面抗原情况并观察其多项分化潜能。结果使用该方法纯化扩增的BMSCs形态均一,生长良好,表达CD29、CD44和Sca-1,不表达CD11b、CD45和CD34,并具有成骨成脂潜能。结论通过全骨髓贴壁法可以成功的分离培养出小鼠BMSCs。     

Antibody ligation of human leukocyte antigen class I molecules stimulates migration and proliferation of smooth muscle cells in a focal adhesion kinase-dependent manner

Chronic rejection manifests as transplant vasculopathy, which is characterized by intimal thickening of the vessels of the allograft. Intimal thickening is thought to result from the migration and proliferation of vascular smooth muscle cells (SMC) in the vessel media, followed by deposition of extracellular matrix proteins. The development of post-transplantation anti-human leukocyte antigen (HLA) antibodies (Ab) is strongly correlated with the development of transplant vasculopathy and graft loss. Here we demonstrate that cross-linking of HLA class I molecules on the surface of human SMC with anti-HLA class I Ab induced cell proliferation and migration. Class I ligation also increased phosphorylation of focal adhesion kinase (FAK), Akt, and ERK1/2 in SMC. Knockdown of FAK by siRNA attenuated class I-induced phosphorylation of Akt and ERK1/2, as well as cell proliferation and migration. These results indicate that ligation of HLA class I molecules induces SMC migration and proliferation in a FAK-dependent manner, which may be important in promoting transplant vasculopathy.     

粘着斑激酶在血管内皮细胞黏附和迁移中的作用

目的 采用粘着斑激酶（focal adhesion kinases,FAK)抑制剂抑制FAK在Y397位点的酪氨酸磷酸化,测定不同浓度的FAK抑制剂的对内皮细胞黏附、迁移及下游信号Rac1蛋白表达的影响,探索粘着斑激酶在内皮细胞黏附和迁移中的作用。方法 运用内皮损伤模型（划痕法）测定FAK抑制剂在2、4、8、24 h各时间点对EA.hy 926细胞迁移的影响。Western blot结合免疫荧光测定不同浓度的0~250 nmol/mL的FAK抑制剂的加入对Rac1蛋白分布和表达的影响。结果 随着FAK抑制剂浓度的增加,细胞迁移距离减少,Rac1蛋白表达逐渐减弱。结论 抑制FAK的磷酸化将抑制内皮细胞的黏附和迁移的生物学行为,下游Rac1蛋白表达降低。内皮细胞的黏附和迁移与FAKRho GTPases 信号轴相关。     

Expression of a secretory product by microvillous and ciliated cells of the human endometrial epithelium in vivo and in vitro

A monoclonal antibody which identifies a component of post-ovulatoryendometrial secretions is now shown to be expressed within thecytoplasm and on the cell surface of both microvillous and ciliatedepithelial cells. A glandular explantatlon model was developedin order to study the ‘carry over’ of this secretionto the regenerative phase endometriwn. A loss of cytoplasmicantigen was observed in vitro. However, it was retained on thecell surface in a fashion consistent with its expression atthe time of explantation. Mosaicism of expression of this secretorycomponent occurs throughout the secretory-phase and is particularlypronounced at the time of transition from proliferative to secretoryphase. It is conduded that both ciliated and microvillous epithelialcells produce a post-ovulatory secretory component which maybe retained on the cell surface in the absence of hormonal stimulation.