子宫内膜体外三维重建的形态学研究

目的 :探索建立子宫内膜体外三维模型的方法。方法 :将分离的子宫内膜基质细胞和上皮细胞依次种植于生物可降解胶原 -聚乳酸羟基乙酸共聚物杂交聚合网上下两面 ,光镜及扫描电镜观察细胞的生长状况。结果 :接种后第 5天子宫内膜细胞能在聚合网上融合生长 ,第 1 4天细胞融合成片 ,呈复层生长 ,形成三维结构。结论 :生物可降解的杂交聚合网作为支架可建立体外子宫内膜三维模型。     

子宫内膜三维模型上小鼠囊胚着床的扫描电镜观察

目的　观察胚胎与体外子宫内膜三维模型共培养时 ,子宫内膜上皮细胞与胚胎的超微结构变化。　方法　将妊娠第 4天的小鼠囊胚在已建立的体外子宫内膜三维模型上进行培养 ,扫描电镜 (SEM )观察胚胎着床时子宫内膜上皮细胞及胚胎的超微结构。　结果　小鼠囊胚能正常脱透明带、粘附和扩展。电镜扫描显示体外培养的子宫内膜可形成胞饮突 ,但数量比体内子宫内膜少 ,胚胎粘附于胞饮突上。　结论　体外子宫内膜三维模型可形成胞饮突 ,且胞饮突可能直接参与了囊胚与子宫内膜的粘附。     

人子宫内膜体外构建的实验研究

目的探讨体外构建人子宫内膜的新方法。方法将采用筛网分离法获得的原代人子宫内膜基质细胞和腺上皮细胞依次接种到自制的液态胶原材料上共培养,形成子宫内膜组织,并与二维条件下正常培养的子宫内膜基质细胞和腺上皮细胞进行比较。采用组织学和免疫组织化学方法观察所构建的子宫内膜的组织学特征,以及两种细胞在不同培养条件下的生长和分布情况。结果分离获得的子宫内膜基质细胞和腺上皮细胞的纯度分别达到95%和90%。在Ⅰ型液态胶原形成的凝胶内,子宫内膜基质细胞排列紧密,腺上皮细胞呈腺体样结构。结论两种细胞与Ⅰ型液态胶原凝胶复合后构建的子宫内膜具有正常子宫内膜组织的结构。     

表皮生长因子对子宫内膜细胞体外增殖的影响

本研究观察了表皮生长因子（ＥＧＦ）在体外对子宫内膜上皮细胞及基质细胞增殖的影响。采用酶消化加物理方法分离人子宫内膜上皮细胞及基质细胞,分别在体外培养,细胞汇合后消化,一部分用盖片法培养,用特异性抗体鉴定细胞纯度,另一部分做细胞增殖实验。在细胞中加入不同浓度的ＥＧＦ,培养２４、４８、７２小时,用ＭＴＴ法及流式细胞仪测定ＥＧＦ对子宫内膜上皮细胞及基质细胞增殖的作用。结果：ＥＧＦ浓度为５．０及１０．０ｎｇ／ｍｌ时,明显刺激子宫内膜上皮细胞及基质细胞的增殖,ＭＴＴ与流式细胞仪两法一致。ＥＧＦ不刺激细胞凋亡。结论：采用酶消化结合物理方法分离的人子宫内膜基质细胞及上皮细胞,方法简便、快速,细胞纯度高,在体外生长良好,可用于体外研究着床及异位子宫内膜生长的机制。当ＥＧＦ浓度为５．０及１０．０ｎｇ／ｍｌ时,确实刺激子宫内膜细胞的增殖。     

着床期小鼠子宫内膜有丝分裂原活化蛋白激酶的表达及LeY寡糖对其表达的调控

目的观察围着床期小鼠子宫内膜有丝分裂原活化蛋白激酶(MAPK)的定位及表达变化,探讨LeY寡糖对其表达的调控。方法应用免疫组织化学、Western-blot方法,检测小鼠着床期(D1~D8)子宫内膜MAPK的两种亚型ERK1(p44 MAPK)和ERK2(p42 MAPK)的表达部位及水平;并通过点杂交方法,分析细胞表面的LeY寡糖被抗体阻断后,对培养的D4子宫内膜上皮细胞ERK1/2表达的影响。结果免疫组织化学分析显示D2、3、4磷酸化活性ERK1/2分布于子宫内膜腔上皮和腺上皮,D4主要集中在腔上皮,而D5、6则以蜕膜细胞表达最强;Western-blot结果显示ERK1亚型表达水平高于ERK2,后者在围着床期的变化趋势较前者明显;LeY寡糖被抗体阻断后D4内膜上皮细胞ERKs的表达下降。结论MAPK在围着床期小鼠子宫内膜中有特异的定位和表达规律,其表达强度与子宫内膜的接受态及蜕膜化有关。LeY寡糖可能作为一种信息分子,对MAPK信号转导通路起调控作用。     

子宫内膜异位症患者在位内膜分泌RANTES及对单核细胞的趋化活性

目的了解子宫内膜异位症(Em)患者的在位内膜在分泌RANTES及对单核细胞的趋化活性是否有别于非Em患者子宫内膜。方法白细胞介素(IL)-1β刺激培养Em与非Em患者子宫内膜基质细胞,分别在刺激培养后12、24、36、48、60、72、84和96 h取上清酶联免疫吸附试验(ELISA)检测RANTES水平;Boyden小室趋化实验分析刺激培养上清液对单核细胞的趋化活性及RANTES趋化单核细胞的能力。结果Em在位内膜从刺激培养60 h始,RANTES分泌量及趋化单核细胞的能力显著高于非Em在位内膜;Em在位内膜基质细胞刺激培养60 h上清所趋化的单核细胞中有55%是由RAN-TES趋化而来。结论在相似的培养条件下,Em患者的在位内膜基质细胞RANTES的表达模式和趋化单核细胞的活性不同于非Em患者子宫内膜。     

Luminex液相蛋白芯片用于研究着床窗期子宫内膜和早孕蜕膜基质细胞的细胞因子的表达谱

目的探索着床窗期子宫内膜和早孕期蜕膜基质细胞的细胞因子谱表达及其对胚胎着床和早期妊娠的可能影响。方法获取着床窗期子宫内膜基质细胞(ESC)和早孕期蜕膜基质细胞(DSC)进行体外培养,采用Luminex液相蛋白芯片技术检测细胞培养上清液中15种细胞因子:肿瘤坏死因子(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素(IL)-1β、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12、颗粒-巨噬细胞集落刺激因子(GM-CSF)、颗粒细胞集落刺激因子(G-CSF)、巨噬细胞炎症蛋白-1α(MIP-1α)、单核细胞趋化蛋白因子-1(MCP-1)、受调节激活的正常T细胞排泌的因子(RANTES)和γ干扰素诱导蛋白10(IP-10)的含量变化,比较两者表达的差异。结果DSC分泌多种细胞因子,与ESC相比,其中TNF-α、IFN-γ、IL-1β、IL-6、IL-8、IL-10、IP-10、GM-CSF、G-CSF、MIP-1α、RANTES表达下调,有极显著差异(P<0.001);IL-4表达上调,有显著差异(P<0.01);MCP-1、IL-2和IL-12表达无明显变化。不同时期表达的细胞因子之间存在复杂的相关关系。结论从着床窗期子宫内膜到早孕期蜕膜基质细胞表达的细胞因子谱的水平发生变化,可能对调控胚胎着床和维持早期妊娠起着重要作用。     

子宫内膜异位症子宫内膜及输卵管细胞对鼠胚胎发育的影响

目的探讨子宫内膜异位症(内异症)子宫内膜及输卵管上皮细胞对小鼠胚胎体外发育的影响。方法小鼠2细胞期胚胎202个与卵巢巧克力囊肿患者术中切除的子宫内膜及输卵管上皮细胞共培养(实验组),314个与非卵巢巧克力囊肿患者子宫内膜及输卵管上皮细胞共培养作为对照。结果体外培养72~96h后,与对照组输卵管上皮细胞及子宫内膜共培养时有25.7%和26.2%的2细胞期胚胎发育到扩张期囊胚,与实验组输卵管上皮细胞共培养有25.9%发育到扩张期囊胚,二者无显著差异(P>0.05),而子宫内膜仅为11.1%,二者有显著差异(P<0.05)。结论内异症患者的输卵管上皮细胞对鼠早期胚胎体外发育无影响;子宫内膜对胚胎发育有明显的抑制作用,这可能与内异症的生育率降低有关。     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的:研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架.方法:应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液,接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物.应用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性,并用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态.结果:网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性.尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞;长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架紧密贴附,适度伸展并有基质分泌.结论:网状尿道支架适合尿道移行上皮细胞黏附生长,可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道.     

子宫内膜体外构建及其应用研究进展

体外构建子宫内膜是利用组织工程学原理,在体外构建结构、形态与功能上与体内子宫内膜相似的三维模拟体。种子细胞分离培养、生物支架材料的合理选择及体外构建方法的优化是体外构建子宫内膜的重要因素。构建的子宫内膜在病理研究、三维培养体系、胚胎发育学及移植等方面具有重要的研究意义。只有更进一步模拟自然子宫的结构,优化构建技术路线和培养条件,才可以再造出真正意义上的组织工程化子宫。     

聚乙烯醇/胶原共聚物在组织工程前交叉韧带支架材料中的实验研究

目的 :探索体外构建组织工程前交叉韧带 (anteriorcruciateligament,ACL)的支架材料。方法 :聚乙烯醇 (polyvinylalcohol,PVA)与胶原共聚后 ,深低温冷冻干燥成形、制孔。用酶消化法体外培养ACL细胞 ,种植到该支架上 ,立体培养。结果 :支架的表面和孔内均有细胞生长 ,状态良好。结论 :聚乙烯醇 /胶原共聚物支架具有良好的组织相容性和力学相容性 ,适合体外培养ACL细胞 ,通过进一步改善力学性能 ,有可能成为体外构建组织工程前交叉韧带的理想支架。     

人子宫内膜植入兔眼前房模型的研究

本实验成功地将人子宫内膜植入到１５只去卵巢新西兰雌兔的眼前房，并给每只兔外源补充雌二醇（Ｅ２）１００μｇ／ｄ和孕酮（Ｐ）８ｍｇ／ｄ。在内膜植入期间每只兔皮下注射总量为９ｍｇ的霉酚酸（ｍｙ－ｃｏｐｈｅｎｏｌｉｃａｃｉｄ）以防免疫排斥反应。在眼前房的人子宫内膜存活了２８．４４±８．６５天，兔血清Ｅ２和Ｐ的平均值分别为１６１．７１±１５．１３和３８．８８±７．４５ｐｍｏｌ／Ｌ。前房液中Ｅ２和Ｐ浓度在植入人子宫内膜前后分别为２４．８０±１６．５３和１７０９．２５±４７５．１０ｐｍｏｌ／Ｌ，２．００±０．５３和１２．２２±３．８１ｎｍｏｌ／Ｌ。树脂切片验证人子宫内膜植入兔眼前房第５天就能与虹膜附贴并有血管发生。来自巩膜的血管分布在子宫内膜周围。扫描电镜观察人子宫内膜分化成分泌细胞和纤毛细胞。植入人子宫内膜后，前房液的蛋白浓度高于植入前。可以证明该模型可用来研究人子宫内膜对药物的反应和通过对前房液的测量研究子宫内膜的分泌功能。     

组织工程心脏瓣叶的体外培养

目的 探讨瓣叶支架种植细胞后,不同体外培养时间瓣叶的生成情况. 方法杂种猪9只,取主动脉,分离内皮细胞、成纤维细胞和平滑肌细胞,培养扩增.将成纤维细胞、平滑肌细胞和内皮细胞按顺序种植在预湿处理的支架材料上,以首次种植成纤维细胞、平滑肌细胞为起点,取7天、14天和28天样本使用戊二醛固定,用扫描电子显微镜(SEM)检测,对培养28天的样本进行组织学检查. 结果 SEM检测显示:随着培养时间的延长,细胞数量增加,细胞间连接出现,并有基质生成;至28天,瓣膜支架上可见大量细胞黏附,细胞融合成片,表面可见基质形成.支架的细胞覆盖率也随时间的延长明显增加.组织学检测见大量细胞黏附材料,细胞已长入材料中部. 结论体外培养的瓣叶存在生长活性,使用组织工程方法有可能生成组织工程心脏瓣膜.     

ECV304细胞用于三维血管新生的研究

目的:采用ECV304细胞(人脐静脉内皮细胞株)构建体外血管新生的三维模型,为ECV304细胞应用于体外血管新生的研究提供依据.方法:制作鼠尾胶原三维凝胶,在凝胶表面种植ECV304细胞,培养至接近汇合状态,换用含碱性成纤维细胞生长因子(bFGF)的培养液继续培养3～12d,用倒置相差显微镜观察内皮细胞株向凝胶内迁移、生长状况.结果:bFGF组ECV304细胞迁移到凝胶内不同层次,形成细胞条索并吻合成网;垂直切面上可见ECV304细胞从凝胶表面的单细胞层向凝胶内发芽生长,形成树枝状毛细血管样结构.结论:ECV304细胞在三维胶原凝胶基质培养模型中,在适当的因子诱导下,能够表现出血管新生中的增殖、迁移、发芽等步骤,可以作为内皮工具来研究体外血管新生.     

Tissue-engineered urinary bladder wall using PLGA mesh-collagen hybrid scaffolds: a comparison study of collagen sponge and gel as a scaffold

Purpose: Tissue engineering of the urinary bladder using autologous cells and biodegradable scaffold is a promising method for augmentation. The authors developed 2 hybrid scaffolds by combining poly (DL-lactic-co-glycolic acid; PLGA) mesh for mechanical strength with collagen sponge or gel suitable for cell seeding. The aim of this study was to compare collagen as a scaffold between collagen sponge and gel and to construct a tissue-engineered urinary bladder wall utilizing these hybrid scaffolds.Methods: The PLGA mesh-collagen hybrid scaffolds were prepared by introducing collagen sponge or gel into the PLGA knitted mesh. Urothelial and smooth muscle cells were obtained from porcine urinary bladder wall and were cultured in their respective media. The cells were seeded on these hybrid scaffolds. These constructs were analyzed morphologically and immunohistochemically.Results: The urothelial layer was generated 3 dimensionally by culturing urothelial cells with PLGA mesh and collagen sponge. The smooth muscle layer was constructed by culturing smooth muscle cells with PLGA mesh and collagen gel. And a novel tissue-engineered urinary bladder wall was constructed laminating the urothelial and smooth muscle layers.Conclusions: Ex vivo construction of urinary bladder wall using hybrid scaffolds prepared by combining PLGA mesh with collagen sponge or gel was successful. This tissue-engineered urinary bladder wall allows easy handling and may become a promising tool for bladder augmentation.     

Comparison of mechanical properties and host tissue response to OviTex™ and Strattice™ surgical meshes

Hernia - This study compared the in vitro/benchtop and in vivo mechanical properties and host biologic response to ovine rumen-derived/polymer mesh hybrid OviTex™ with porcine-derived...     

In-vitro study of the cellular response of human fibroblasts cultured on alloplastic hernia meshes. Influence of mesh material and structure]

BACKGROUND: The biocompatibility of meshes in hernia surgery seems to be influenced markedly by the amount of the selected material and its structure. Fibroblasts play a major key role during the process of mesh incorporation. This study was performed to investigate differences in cell morphology and proliferation of human fibroblasts cultured on different polypropylene meshes. METHODS: In the present in vitro study the cellular response of human fibroblasts was investigated by scanning electron microscopy (SEM), comparing three different polypropylene meshes: a newly constructed low-weight and microporous mesh (NK1), a low-weight and macroporous mesh with absorbable polyglactin filaments (Vypro), and a heavy-weight and microporous mesh (BiomeshP1). Human fibroblasts (1,5.10(5) cells) were incubated with the meshes (each 12 mm(2)) for 6 hours, 5 days, 2, 4, 6, and 12 weeks. Computer-assisted morphometry of the fibroblast/mesh surface ratio served to reflect the biological cell response. RESULTS: The Vypro mesh showed the significantly highest fibroblast density during the first 6 weeks, but cell growth was nearly exclusively limited to the polyglactin filaments. At 3 months, after reabsorption of the polyglactin, the fibroblast-coated polypropylene mesh surface was only 50% compared to NK1 and BiomeshP1. The morphologic aspect of the fibroblasts on the BiomeshP1 mesh was much more degenerative and unphysiological, compared to NK1 and Vypro, with isolated, single cells instead of a broad, connective growth. The BiomeshP1 showed a significantly higher fibroblast proliferation around the nodes of the mesh compared to the straight filaments. On the NK1 mesh fibroblasts exclusively proliferated on the filaments but not on the pressed mesh surface. CONCLUSIONS: The polymer surface and structure appears to be of major importance for the biocompatibility of meshes: human fibroblasts preferably grow on low-weight meshes, thin filaments, and mesh nodes. Heavy-weight meshes induce degenerative cell reactions. Polyglactin seems to further improve cell proliferation whereas a pressed mesh surface without pores hinders fibroblast growth.     

In vitro three-dimensional reconstruction of human bladder mucosa

ObjetiveThe purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to human urothelium, to reconstruct an in vitro three-dimensional human bladder mucosa, suitable for graftingMaterial and MethodsBiopsy specimens of human bladder mucosa were obtained from patients undergoing suprapubic prostatectomy, in vitro cultured and finally, an immunohistochemical study was madeResultsA three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding. We used a biodegradable polyglycolic acid mesh to facilitate the tissue manipulation and implantation. An immature epithelium was obtained with a weak immunostaining to cytokeratins. The immunohistochemical study could not demonstrate the development of basement membraneConclusionsIn vitro keratinocyte culture techniques could be applied to other epithelial tissues like the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time, which needs the matrix interactions in order to mature     

人早期胚胎体外培养中自体子宫内膜细胞共培养与序贯培养联合系统的建立

目的:建立早期胚胎与自体子宫内膜细胞共培养与序贯培养联合系统的培养方法。方法:黄体中期获取子宫内膜,细胞培养后程序化冻存。取卵前解冻自体内膜细胞,受精后将双原核胚转移至已更换分裂期胚胎培养液的自体子宫内膜细胞行共培养,取卵后72 h更换囊胚培养液行共培养。结果:人黄体中期子宫内膜有良好的体外培养活力,程序化冷冻与快速复温后细胞存活高,自体胚胎共培养的应用比率为74.04%。种植前各时期早期胚胎联合应用序贯培养液,在自体内膜细胞上生长良好。建立联合培养系统的临床妊娠率68.83%,种植率44.23%。结论:人早期胚胎与自体子宫内膜细胞共培养联合序贯培养系统的建立,为早期胚胎体外培养的模式提供了一种新的思路,力求提高种植前胚胎的质量与发育潜能。