改良大鼠肺微血管内皮细胞原代培养技术及细胞鉴定

目的 改进大鼠肺微血管内皮细胞的原代培养方法,并对所培养的原代细胞进行鉴定.方法 应用SD大鼠的外周肺组织,进行大鼠肺微血管内皮细胞的原代培养.从动物选取、取材、肺灌注、植块贴壁、培养基及添加物的选择等方面对肺微血管内皮细胞原代培养技术方法进行改进.从细胞形态学特征,免疫组织化学检测血管内皮细胞表面标志物（CD31抗原和Ⅷ因子相关抗原的表达）,荧光显微镜观察异硫氰酸荧光标记植物凝集素与肺微血管内皮细胞特异性结合情况等方面鉴定所培养的肺微血管内皮细胞.流式细胞仪检测细胞纯度,噻唑蓝测量细胞生长曲线.结果 原代培养肺微血管内皮细胞成多角形,融合为单层后呈典型的鹅卵石或铺路石样生长,传代培养后可变为梭形呈漩涡样生长或聚集生长.免疫组织化学显示细胞CD31和Ⅷ因子相关抗原的表达阳性;异硫氰酸荧光标记植物凝集素结合试验阳性.细胞生长状态良好、细胞纯度达93.2％.结论 改进后的原代培养方法简便、可靠,获得的肺微血管内皮细胞污染少、纯度高,状态良好,保持了内皮细胞的结构和特性.     

肺微血管内皮细胞原代培养方法的建立

目的建立大鼠肺微血管内皮细胞原代培养方法,观察细胞生长状态并鉴定细胞性质。方法 4~5周龄大鼠随机分成3组,无菌状态下剥除胸膜,将肺组织边缘剪成1 mm3的大小,贴入一次性培养瓶,分别用含肝素钠的DMEM完全培养液、RPMI1640完全培养液以及含原代内皮细胞培养特制添加剂的完全培养液培养,在倒置显微镜下观察细胞生长和形态,免疫荧光组织化学染色观察细胞CD31的表达。结果与2组对照组相比,采用含原代内皮细胞培养特制添加剂的完全培养液培养,获得的肺微血管内皮细胞数量多,纯度高。内皮细胞于24 h迁移出肺组织块,14 d左右汇合,呈多角形,以铺路石样镶嵌状排列生长。传代细胞呈长梭形,生长迅速,具有自发形成血管的倾向。细胞为CD31免疫反应阳性,证实其为内皮细胞。结论采用组织块贴壁法,选择原代内皮细胞培养特制添加剂以及优化分离过程,可以获得高纯度原代大鼠肺微血管内皮细胞。     

改良贴壁法结合内皮细胞培养基培养小鼠肺微血管内皮细胞*☆

背景：肺微血管内皮细胞是研究微循环的重要内皮细胞模型之一,众多培养方法中单纯贴壁法操作相对简便,但耗时长,杂质细胞多,是批量培养细胞的最大障碍。 目的：建立优化的小鼠肺微血管内皮细胞体外培养方案,观察细胞生长状态并鉴定细胞性质。 方法：无菌状态下快速剪碎5日龄C57BL/6J小鼠的肺叶外周组织,肺组织颗粒贴壁法获得肺微血管内皮细胞,并用内皮细胞培养基培养。倒置显微镜观察培养细胞生长和行为状态,Ⅷ因子相关抗原免疫组化和免疫荧光进行细胞鉴定。 结果与结论：肺组织块培养24 h内可见梭形细胞爬出,传代后细胞生长迅速,形态规则呈鹅卵石状,纯度高达98%以上,结合Ⅷ因子相关抗原检测证实其为内皮细胞。结果可见联合运用肺组织颗粒贴壁和内皮细胞培养基可高效获得原代小鼠肺微血管内皮细胞。关键词：肺微血管内皮细胞;细胞培养;优化;鉴定;小鼠;血管组织工程 缩略语注释：PMVECs: pulmonary microvascular endothelial cells,肺微血管内皮细胞 doi:10.3969/j.issn.1673-8225.2012.15.007     

小鼠肺微血管内皮细胞磁珠分选法分离和原代培养

背景:肺组织的功能依赖于肺微血管内皮细胞的活性,因此肺微血管内皮细胞是相关研究的重要细胞模型,但目前国内多采用的组织块贴壁法培养所得的肺微血管内皮细胞常有其他细胞混杂。目的:建立一种有效分离、培养、扩增小鼠肺微血管内皮细胞的方法。方法:采用酶消化、免疫磁珠二次分选法分离纯化小鼠肺微血管内皮细胞,贴壁培养法体外扩增,CCK-8法测定细胞的生长情况,相差显微镜观察培养细胞的形态,透射电子显微镜观察细胞的超微结构,流式细胞仪对其表型进行鉴定。结果与结论:培养所得小鼠肺微血管内皮细胞具有典型的铺路石样形态学特征,含有大量内皮细胞特有的杆状细胞器Weibel-Palade小体,较稳定地表达内皮细胞特异性表面标记CD105,不表达淋巴管内皮细胞特异性表面标记血管内皮生长因子受体3。说明免疫磁珠二次分选法可成功分离纯化小鼠肺微血管内皮细胞,体外培养所获细胞纯度高、自我更新能力强,并保留了包括构成、表面抗原表达等特性。     

脂肪源干细胞向血管内皮细胞的分化

背景：脂肪来源干细胞在体内储备丰富,体外增殖快速,具有多向分化潜能,是目前组织工程种子细胞的研究热点。近年来越来越多的研究表明脂肪干细胞在一定条件下可被诱导分化为内皮细胞,促进血管生成。 目的：观察兔脂肪干细胞体外分离培养及诱导分化为血管内皮细胞的生物学特性。 方法：取兔附睾处脂肪,采用胶原酶消化分离获得脂肪干细胞,体外培养至第3代后加入血管内皮细胞生长因子与碱性成纤维细胞生长因子联合诱导分化,对诱导前后细胞进行形态学观察、生长曲线测定、免疫组织化学染色及流式细胞仪表型检测。 结果与结论：兔脂肪干细胞生长旺盛,第3代兔脂肪干细胞呈成纤维细胞样,生长曲线呈“S”型,15代以内细胞形态未见明显变化。免疫荧光法检测Vimentin阳性,流式细胞仪检测CD44表达阳性,CD31表达阴性;诱导后细胞CD31表达阳性,CD44表达阴性。第3代兔脂肪干细胞向血管内皮细胞诱导分化21 d显微镜下呈铺路石样形态,血管内皮细胞第Ⅷ因子相关抗原染色细胞阳性,透射电镜下可见W-P小体。提示脂肪干细胞在体外可诱导分化为血管内皮细胞,可为组织工程血管提供理想的种子细胞。     

切应力作用下与血管平滑肌细胞联合培养的内皮细胞的形态学改变

内皮细胞与平滑肌细胞是血管壁的主要组成细胞。血管平滑肌细胞与流体切应力是影响血管内皮细胞形态和功能的重要因素。形态学作为内皮细胞研究中最直观的指标,一直受到重视。单独培养的内皮细胞丧失了在体条件下的长梭形、沿流体方向平行排列的特征,呈现“铺路石”样外观。与平滑肌细胞联合培养的内皮细胞呈长梭形,与在体条件下的内皮细胞形态较为接近;但排列无极性,这可能是因为内皮细胞缺少了与其腔面直接作用的流体应力的缘故。为了了解切应力对联合培养的内皮细胞的影响,本文建立了用于内皮细胞和平滑肌细胞联合培养的应力测试系…     

三维培养内皮细胞形成血管样结构的实验研究

目的 :观察三维培养内皮细胞在生长因子血小板源性生长因子 (plateletderivedgrowthfactor,PDGF)作用下血管样结构的形成。方法 :制备三维培养胶原基质和细胞培养。结果 :对照组内皮细胞在胶的表面呈单层生长 ,未见其向深层生长及管腔结构形成。PDGF组内皮细胞在胶内呈多层浸润生长 ,伸展、变形呈扁平状 ;内皮细胞与胶原纤维之间以半桥粒连接 ,内皮细胞之间以桥粒连接 ,形成毛在管样管腔结构 ,管腔样结构大小不一 ,其间有多个散在的内皮细胞 ;部分内皮细胞内可见空泡样变 ,边界由胞质与胞膜组成 ,细胞核变形 ,凹陷 ,可见切迹 ,或呈薄片状 ,空泡样变及细胞核形态的变化在形成管状结构的内皮细胞中尤为明显 ;内皮细胞管腔面及游离面内皮细胞均有微绒毛突起。结论 :三维培养技术简单易行 ,是揭示在体血管形成的最简单模型 ;PDGF有利于三维培养内皮细胞在有展性的胶原基质面形成血管样结构。     

小鼠脑微血管内皮细胞株bEnd.3的细胞特征及基因表达谱分析

目的:对一种小鼠脑组织来源的血管内皮细胞株bEnd.3的主要细胞特征进行研究并做血管内皮细胞生物学功能基因芯片分析。方法:利用倒置显微镜、扫描和透射电子显微镜观察细胞形态特征;免疫细胞化学法显示血管内皮细胞特异性表面标志;ELISA定量检测PGE₂;流式细胞术、MTT法研究增殖和凋亡、血管内皮细胞基因芯片研究正常培养bEnd.3基因表达谱。结果:bEnd.3具备多种典型的微血管内皮细胞(MVEC)特征:细胞呈多边形或梭型、呈铺路石样生长;存在管腔样结构(TLS)和毛细血管样网络,细胞表面有丰富的微绒毛;vW因子、CD34阳性;CD31、CD36、CD105等也有不同程度的表达;细胞高水平分泌前列腺素E₂(PGE₂);多种与正常血管功能密切相关的基因也有不同程度的表达。结论:bEnd.3保留了MVEC的基本特性,可用于心脑血管疾病、肿瘤等重大疾病的发病机制的基础与临床研究。     

体外三维血管生成模型的建立及肿瘤细胞分泌的MCP-1/VEGF对该模型的影响

目的:建立体外三维血管生成模型,观察肿瘤细胞分泌的单核细胞趋化蛋白1(MCP-1)/血管内皮生长因子(VEGF)对该模型血管生成的影响。方法:分别将人脐静脉内皮细胞(HUVECs)及小鼠内皮细胞SVEC4-10EE2包被于磁珠表面,与纤维蛋白原混合后接种于含有凝血酶的细胞培养板中进行培养,包被在磁珠上的内皮细胞在含有纤维蛋白凝聚物的三维空间中生长成脉管样结构,以建立体外血管生成的三维模型。在模型中分别加入肿瘤细胞(人乳腺癌细胞MDA-MB-231和小鼠髓样乳腺癌细胞E0771)共培养,观察肿瘤细胞在体外对血管生成的影响。用酶联免疫吸附法(ELISA)分别检测肿瘤细胞培养上清液中影响血管生成的MCP-1和VEGF的浓度,并在内皮细胞-肿瘤细胞共培养体系中加入MCP-1受体C-C趋化因子受体2(CCR2)的拮抗剂以及VEGF受体的抑制剂司马沙尼(SU5416),观察其对血管生成的影响。收集肿瘤细胞刺激后的肿瘤细胞培养上清液作为条件培养液,加入到内皮细胞HUVECs或SVEC4-10EE2单独培养的血管生成系统中,在加入或不加入CCR2的拮抗剂以及SU5416的情况下分别观察其对血管生成的影响。结果:在不加入任何影响血管生成的物质时,内皮细胞HUVECs和SVEC4-10EE2均能在体外培养基中形成多细胞组成的脉管样结构,即建立起体外三维血管生成模型;加入肿瘤细胞(MDA-MB-231和E0771)共培养,可显著促进脉管样结构形成(P0.05)。ELISA检测结果显示,肿瘤细胞上清液中MCP-1和VEGF水平显著增高(P0.05),MCP-1具有体外促进三维培养体系中脉管生成作用,而在加入MCP-1受体CCR2的拮抗剂以及VEGF受体的抑制剂SU5416之后,可显著抑制血管形成(P0.05)。条件培养液可促进内皮细胞体外脉管生成,而这一作用可以被CCR2的拮抗剂以及SU5416阻断(P0.05)。结论:成功建立了体外三维血管生成模型,肿瘤细胞共培养可以显著促进内皮细胞脉管形成,机制可能与其分泌的MCP-1和VEGF有关。     

原发性心脏血管肉瘤临床病理及遗传学特征

目的探讨原发性心脏血管肉瘤(primary cardiac angiosarcoma, PCAS)临床病理及遗传学特点。方法收集河南省人民医院确诊的PCAS共9例, 采用免疫组织化学和二代测序技术检测蛋白和基因突变情况。结果本组患者男性7例, 女性2例;年龄18～53岁;6例位于右心房, 2例位于心包, 1例位于右房室沟。9例伴心包积液, 3例伴胸腔积液, 4例伴肺多发转移。细胞学见肿瘤细胞呈腺样、乳头状排列, 上皮样形态, 酷似腺癌细胞;组织学见肿瘤组织呈高-中等分化, 见不规则血管腔样结构, 瘤细胞鞋钉样或乳头状, 部分区域呈片状、束状排列, 细胞胖梭形, 核深染不规则, 异型性明显, 病理性核分裂象易见;免疫标记显示肿瘤细胞强表达CD34、CD31、ERG。伴多发肺转移结节和浆膜腔积液病例存在TP53错义突变(p.R273C), 且肿瘤细胞p53蛋白强阳性。结论 PCAS早期即存在浆膜腔积液和肺多发转移, 组织形态呈高-中等分化, 细胞学极易误诊为腺癌, 免疫组织化学有助于鉴别诊断;且TP53突变可能与肿瘤的高侵袭性生物学行为相关。     

Differential effects of interleukin-1 and formylmethionylleucylphenylalanine on chemotaxis and human endothelium adhesivity for A549 tumor cells

We investigated the effects of formylmethionylleucylphenylalanine (FMLP), interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) on tumor cell chemotaxis and tumor cell/endothelial cell adhesion. Chemotaxis of A549 human lung carcinoma cells was measured as the number of tumor cells which migrated across a nitrocellulose filter in a Boyden chamber. Tumor cell/endothelial cell adhesion was measured as the number of 125IUdR tumor cells adherent to monolayers of endothelial cells. Confluent monolayers of human umbilical endothelial cells were incubated from 10 to 240 minutes with FMLP, monocyte-derived interleukin-1, or recombinant IL1 alpha or IL1 beta. The endothelial cells were washed and then incubated with 125IUdR-tumor cells. Thirty minutes later the number of adherent tumor cells was assessed isotopically. Our results demonstrate that (a) interleukin-1 but not FMLP, has chemotactic activity for tumor cells, and (b) both FMLP and interleukin-1 enhance tumor cell adhesion to the endothelium independent of any chemotactic activity. Furthermore, we demonstrate that IL1 alpha and IL1 beta have different effects on tumor cell/endothelial cell adhesion, and raise the possibility that IL1 alpha but not IL1 beta is continuously synthesized and stored within the endothelium. We postulate that IL1 alpha and IL1 beta influence tumor cell/endothelial cell adhesion independent of chemotaxis through the expression of adhesive receptors on the endothelial cell surface.     

Tumor cell-platelet interactions in vitro and their relationship to in vivo arrest of hematogenously circulating tumor cells

Aggregation of rat platelets was induced in vitro by homologous rat Walker 256 carcinosarcoma cells and the extent of tumor cell-platelet interactions examined ultrastructurally. By 30s there was surface contact between unstimulated platelets and tumor cell microvilli. By midphase aggregation (2–3 min) tumor cells became enmeshed within expanding platelet aggregates. Tumor cell microvilli and platelet pseudopodia interdigitated as aggregation progressed. During the later stages of aggregation (6–10 min) tumor cells formed large processes which penetrated deep into the aggregate. Platelet activation (i.e. degranulation) occurred in gradient fashion and was concentrated near tumor cell membrane sites involved in process formation. At these later stages tumor cells near the aggregate periphery were found to have engulfed platelets or platelet fragments.Tumor cell-platelet interactions in the pulmonary microvasculature were also studied in vivo following injection of murine Lewis lung carcinoma, 16C mammary adenocarcinoma, and B16 amelanotic melanoma tumor cells into the tail vein. Platelets demonstrated a biphasic association with arrested tumor cells with peak interactions occurring at 10–30 min and 4–24h. Ultrastructurally, tumor cells exhibited newly formed processes which interdigitated with the platelet aggregate. Such processes formed only in areas of contact with platelets and not in areas of contact with endothelial cells or other blood elements (i.e. erythrocytes, polymorphonuclear leukocytes). Numerous tumor cell mitochondria were concentrated in the areas of greatest platelet-tumor cell process activity. At early time intervals (2–10 min), intravascular platelet degranulation was observed primarily in platelets associated with tumor cell processes. Tumor cells also were found to have engulfed platelet fragments in vivo.     

Transmission and scanning-electronmicroscopy of experimental liver metastases derived from intrasplenically growing primary tumor

Liver metastasis formation from intrasplenically growing Lewis lung tumor was studied with transmission and scanning electron microscopy. Tumor cells arrested in the sinusoids formed desmosome-like junctions with endothelial cells and crossed the endothelial lining at intercellular gaps and by transcellular diapedesis. The metastatic foci had no newly formed vessels, and the stroma was provided by non-parenchymatous liver cells. Morphology suggested different outcome for tumor cell-host cell interactions at different stages of tumor growth. The host cell activity to destroy tumor cells was present only at the early stage but disappeared later, when the tumor cells were ready to phagocyte normal cells. The reason for this could be the immunosuppressive effect of the primary tumor. Results emphasize the importance to study metastasis formation in primary tumor-bearing hosts.     

Altered angiogenesis in the tumor microenvironment

Tumor blood vessels play an important role in tumor progression and metastasis. Thus, targeting the tumor blood vessels is an important strategy in cancer therapy. Tumor blood vessels generally arise from pre-existing vessels and have been thought to be genetically normal. However, they have been shown to differ from their normal counterparts, e.g. with regard to the morphological changes. We isolated tumor endothelial cells (TEC) from mouse tumor xenografts and showed that they were abnormal. TEC up-regulate many genes, proliferate more rapidly and migrate more than normal endothelial cells (NEC). Furthermore, the TEC in our study were cytogenetically abnormal. We concluded that TEC can acquire cytogenetic abnormalities while in the tumor microenvironment. In order to develop ideal antiangiogenic therapies, understanding the crosstalk between blood vessels and the tumor microenvironment is important. This review considers the current studies on TEC abnormalities and discusses the possible mechanism by which the tumor microenvironment produces abnormal TEC.     

Effects of lipopolysaccharide and Mannheimia haemolytica leukotoxin on bovine lung microvascular endothelial cells and alveolar epithelial cells

Bovine respiratory disease resulting from infection with Mannheimia haemolytica commonly results in extensive vascular leakage into the alveoli. M. haemolytica produces two substances, lipopolysaccharide (LPS) and leukotoxin (LKT), that are known to be important in inducing some of the pathological changes. In the present study, we examined bovine pulmonary epithelial (BPE) cell and bovine lung microvascular endothelial cell monolayer permeability, as measured by trans-well endothelial and epithelial cell electrical resistance (TEER), after incubation with LPS, LKT, or LPS-activated neutrophils. Endothelial cell monolayers exposed to LPS exhibited significant decreases in TEER that corresponded with increased levels of proinflammatory cytokines, apoptosis, and morphological changes. In contrast, BPE cells exposed to LPS increased the levels of production of inflammatory cytokines but displayed no changes in TEER, apoptosis, or visible morphological changes. Both cell types appeared to express relatively equal levels of the LPS ligand Toll-like receptor 4. However, TEER in BPE cell monolayers was decreased when the cells were incubated with LPS-activated neutrophils. Although the incubation of BPE cells with LKT decreased TEER, this was not reduced by the incubation of LKT with a neutralizing antibody and was reversed when LKT was preincubated with the LPS-neutralizing compound polymyxin B. Because BPE cells did not express the LKT receptor CD11a/CD18, we infer that contaminating LPS was responsible for the decreased TEER. In conclusion, LPS triggered changes in endothelial cells that would be consistent with vascular leakage, but neither LPS nor LKT caused similar changes in epithelial cells, unless neutrophils were also present.     

人脑胶质瘤侵袭微生态系统的形态学观察

目的　探讨人脑胶质瘤侵袭微生态系统的形态学特征。　方法　利用光镜及透射电镜观察 12例手术切除的脑胶质瘤标本中微血管内皮细胞和基底膜及其与肿瘤细胞的相互关系。　结果　光镜下可见侵袭性生长的瘤细胞可在血管周形成巢状结构。电镜下不同胶质细胞起源的胶质瘤中的微血管内皮细胞基本上为无孔型 ,偶见窗孔形成。内皮细胞下基质完整 ,基底膜呈局限性或广泛性增厚 ,有的伴有多层基板样结构形成。基底膜靠近瘤细胞侧常出现大小不等、数量不一的虫蚀样空洞 ,含有较大空洞的基底膜常突向瘤细胞侧。有的血管外腔扩张 ,腔内可见瘤细胞以单个形式侵袭 ,少见瘤细胞伸出伪足突入基底膜及穿透内皮细胞间隙现象 ,瘤细胞与基底膜相互接触处呈光滑平整的界面。　结论　无孔型内皮细胞、肿瘤微血管基底膜结构的局限性或广泛性增厚和虫蚀样空洞的改变 ,及少见瘤细胞伸出伪足突入其间 ,可能是不同胶质细胞起源的人脑胶质瘤侵袭微生态系统形态学上共有的特征     

基底硬度对肝细胞与肝癌细胞迁移行为的影响

目的通过比较不同硬度基底对肝细胞和肝癌细胞迁移特征的影响,研究肿瘤细胞迁移行为变化的成因。方法运用免疫荧光染色、形态学分析法和Transwell侵袭实验,观察不同硬度基底上HCCLM3和L02细胞的形态特征及对细胞运动能力进行检测和定量分析。结果 (1)相对于极软(0.5 kPa)和极硬(玻璃)基底,HCCLM3和L02细胞在较软基底(4 kPa)上具有较高的迁移速率和迁移净距离,且L02细胞表现出高的迁移效率。(2)HC-CLM3和L02细胞在不同硬度基底上均方位移趋势一致,较软基底上L02细胞具有较高的方向持续能力。(3)0.5和1 mg/mL三维胶原基质中,HCCLM3细胞穿透基底膜的个数分别显著多于L02细胞穿透基底膜的个数;加入40μg/mL水解酶抑制剂GM6001后,HCCLM3细胞穿透基底膜的个数显著增加,而L02细胞穿透基底膜的个数显著减少。结论 (1)二维较软基底上,L02细胞因有较高的方向持续能力而表现出高的迁移效率。(2)三维胶原基质中,HCCLM3细胞以不同迁移模式适应周围环境,从而表现出更大的侵袭能力。     

New invasion assay using endothelial cells grown on native human basement membrane

A new invasion assay is introduced using endothelial cells grown on native human basement membrane (BM). The source of the BM was human amnion. The amnion is a uniform tissue composed of an epithelial layer resting on a continuous basement membrane overlying an avascular collagenous stroma. The epithelium was removed exposing the basement membrane (BM) surface. Human umbilical cord endothelium or bovine capillary endothelium were cultivated on the BM surface. Human squamous carcinoma cells were inoculated onto the BM surface in the presence or absence of the endothelial monolayer. Tumor cells attached readily to both the endothelial monolayer or the BM surface alone. Tumor cells which invaded the basement membrane and underlying collagenous connective tissue were collected on a Millipore filter applied to the opposite side of the amnion. Tumor cells invaded the devitalized amnion connective tissue in the absence of endothelium. The presence of either bovine or human endothelium significantly reduced the rate of tumor cell invasion. This system should be useful for further quantitative studies of the interaction between endothelium and tumor cells with regard to the mechanism of invasion.     

YIGSR,a synthetic laminin peptide,inhibits the enhancement by cyclophosphamide of experimental lung metastasis of human fibrosarcoma cells

Tumor cells must attach themselves to basement membranes, through which they degrade and migrate, in order to spread to distant sites. If vascular endothelial cells are damaged by pretreatment with anticancer drugs and the subendothelial basement membranes are exposed, the attachment of malignant cells to basement membranes and subsequent metastasis formation in some tissues may be enhanced. In this study, the pretreatment of endothelial cell monolayers and mice with cyclophosphamide (CPA) was respectively shown to promote the adhesion of HT1080 human fibrosarcoma cells to endothelial cell monolayersin vitro and lung colonizationin vivo. YIGSR, a synthetic laminin pentapeptide, inhibited the enhancement of lung colonization by CPA when it was co-injected intravenously with tumor cells. This inhibitory effect of YIGSR may be due to a reduction in the adhesion of HT1080 cells to injured blood vessel walls since YIGSR inhibited both the adhesion of HT 1080 cells to CPA-treated endothelial cell monolayers and the invasion through basement membranesin vitro.     

Human Retinoblastoma: A Morphological Study of Apoptotic, Leukocytic, and Vascular Elements

Retinoblastoma (Rb), derived from retinal neuroepithelial progenitor cells, is the most common intraocular malignancy of childhood. This study examined 10 human Rb biopsy specimens with light and electron microscopy for histopathological features not previously described in detail, including cell death, leukocytic infiltration, and the tumor vasculature. Rb is a solid well-vascularized tumor with regions of viable tumor cells surrounding vessels, interspersed with zones of necrosis; apoptotic cells were seen in all specimens. Mononuclear phagocyte series (MPS) cells and lymphocytes often colocal-ized, adjacent to tumor vessels, and MPS cells frequently invested the perivascular space. Lymphocytes were rarely seen within areas of viable tumor. Tumor vessels at early stages of formation resembled normal developing retinal vessels. While junctions were often seen between endothelial cells, disruption of these junctions and endothelial fenestrae was sometimes evident. Muller cells and astrocytes extended processes around tumor cells and blood vessels, and contributed to the formation of the vascular glia limitans, which in some mature vessels was disrupted and discontinuous. Overall, this study provides further morphological details of cell death within Rb, particularly apoptotic involution, and describes the presence of a vascular-associated leukocytic infiltration in Rb. Evidence of compromise of the normal blood-retinal barrier (BRB) within the Rb tumor vessels is presented.