胶质瘤血瘤屏障体外模型的形态学观察

目的 探讨胶质瘤血瘤屏障体外模型的形态学特征。方法 利用电镜技术观察血管内皮细胞Ecv304和C6胶质瘤细胞共培养(混合共培养、Transwell共培养、Transwell膜两面共培养)后的内皮细胞的孔窗、内皮细胞之间的连接及内皮细胞与肿瘤细胞的相互关系、瘤细胞的“血管周足”等的形态学特征；并与4例人脑胶质瘤组织标本的血瘤屏障进行比较。结果 电镜下发现ECV304细胞经与C6细胞按3种不同方式共培养汇合后均为无孔窗型内皮细胞，细胞间出现紧密连接；Transwell共培养的膜上C6细胞未见伸出伪足突向膜的微孔；经膜两面共培养的瘤细胞则可通过Transwell的微孔突向内皮细胞侧，但瘤细胞未伸出伪足包绕内皮细胞或突入内皮细胞间隙，瘤细胞的“血管周足”不完整，后两者与脑胶质瘤组织标本的血瘤屏障的形态特征相类似。结论 在体外经Transwell膜两面共培养的ECV304和C6胶质瘤细胞的系统可在一定程度模拟体内的血瘤屏障的形态学特征。     

人脑胶质瘤中LN、FN及p53基因蛋白的免疫组织化学与其侵袭微生态系统的相关性探讨

目的探讨人脑胶质瘤中层粘连蛋白(LN)、纤维连接蛋白(FN)及突变型p53基因蛋白的免疫组织化学与肿瘤侵袭微生态系统(tumorous invasion microecosystem,TIMES)的相关性. 方法利用透射电镜观察TIMES中微血管特征和免疫组织化学SP法评价LN、FN、p53的表达情况,并作比较分析. 结果 1.脑胶质瘤微血管基底膜(base membrane,BM)连续,多数呈局限性或广泛性增厚,并与LN、FN染色结果相一致,也与p53染色与否有关,p53阳性染色者BM增厚较p53阴性者明显.2.LN、FN在所有胶质瘤微血管BM及内皮细胞上呈阳性染色,恶性程度越高,BM上LN、FN染色阳性越强,血管管壁越厚(P<0.01和P<0.05),而瘤细胞未见染色.LN在脑内转移瘤BM和内皮细胞上却未见染色,但可见散在瘤细胞浆膜染色;FN在脑内转移瘤上的染色则与脑胶质瘤相类似.3. 45例胶质瘤中p53阳性染色21例,其p53阳性染色与否也与BM上LN、FN染色结果存在密切正相关(P<0.01).p53阳性染色率在脑内转移瘤和恶性胶质瘤中无统计学差别(P>0.05). 结论脑微血管内皮细胞上LN、FN的过分表达可能是脑胶质瘤TIMES中BM形态学增厚的原因之一,p53对TIMES的影响也与微血管的内皮细胞功能状态有关.血管内皮细胞可能是TIMES的调控者之一.     

阪崎杆菌侵袭人脑微血管内皮细胞对细胞骨架的影响

目的观察阪崎杆菌侵袭人脑血管内皮细胞后,细胞骨架中微丝和微管的变化。方法用荧光染色技术观察阪崎杆菌侵袭人脑微血管内皮细胞后,细胞骨架中的微丝和微管的变化,观察微丝和微管的抑制剂对阪崎杆菌侵袭人脑微血管内皮细胞的影响。结果阪崎杆菌侵袭人脑微血管内皮细胞后,细胞骨架结构发生明显的改变,微丝和微管部分断裂,模糊,散在分布,微丝抑制剂(细胞松弛素D)和微管抑制剂(秋水仙素)可以抑制阪崎杆菌侵袭人脑微血管内皮细胞。结论阪崎杆菌侵袭人脑血管内皮细胞破坏细胞骨架结构,微丝和微管抑制剂抑制阪崎杆菌侵袭。     

VEGF高表达的胶质瘤细胞对体外共培养微血管内皮细胞的作用

目的以VEGF阴性表达的正常鼠肝细胞系BRL3A为对照 ,研究VEGF高表达的恶性胶质瘤细胞系C6对体外共培养的肺微血管内皮细胞的作用。方法建立体外C6 ,BRL3A与微血管内皮细胞共培养方法 ,观察不同培养体系中内皮细胞活体形态的改变、毛细血管管腔样结构形成的数量、用生长曲线和流式细胞仪观察内皮细胞的增殖状况 ;利用免疫细胞化学的方法检测共培养后的内皮细胞上和血管新生有关的生长因子和受体的表达变化。结果发现与胶质瘤细胞C6共培养时 ,可引起内皮细胞的增殖速度加快 ,细胞周期中S期和G2-M期百分率与对照组相比明显增加(P<0.05),内皮细胞呈现大量的毛细血管管腔样结构 ;而与BRL3A共培养内皮细胞呈现相对静止的状态(P<0.01) ,未观察到形态学的明显变化。免疫细胞化学结果显示与C6共培养的内皮细胞Flk_1、Flt_1蛋白表达增加(P<0.05) ,而与BRL3A共培养的内皮细胞Flk_1、Flt_1蛋白表达下降(P<0.01)。结论胶质瘤细胞C6可使共培养的肺微血管内皮细胞转化为血管新生活跃的状态 ,其可能原因为C6合成分泌的VEGF上调Flk_1、Flt_1的表达。     

中枢神经系统中具有脊索样特征肿瘤的临床病理分析

目的探讨中枢神经系统中具有脊索样特征肿瘤的临床病理特征、诊断及鉴别诊断。方法回顾性分析81例脊索瘤、9例脊索样脑膜瘤、4例脊索样胶质瘤及2例上皮样血管内皮瘤的临床资料、组织学特征,并复习相关文献。结果脊索样特征肿瘤镜下瘤细胞呈条索状或巢状分布于黏液样间质中。脊索瘤好发于人体中轴,多见于颅底及骶尾部,镜下呈分叶状结构,可见胞质含空泡的液滴细胞及星芒状细胞。免疫表型:CK、EMA、vimentin均阳性,绝大多数S-100阳性,GFAP均阴性,PAS染色阳性。脊索样脑膜瘤好发于幕上,与脑膜组织关系密切,镜下可见脊索样成分与典型脑膜瘤结构混杂,间质炎性细胞浸润,可见淋巴滤泡。免疫表型:vimentin、EMA、PR均阳性。脊索样胶质瘤好发于第三脑室,女性多见,镜下可见部分瘤细胞胶质细胞分化,胶质原纤维突起,核分裂象罕见,间质大量淋巴细胞、浆细胞浸润但不形成淋巴滤泡,可见Russell小体,瘤周组织星形细胞反应性增生。免疫表型:GFAP、vimentin均阳性,CK阴性。上皮样血管内皮瘤具有上皮样细胞和血管内皮细胞的组织学特征,镜下见卵圆形或多角形瘤细胞形成原始血管腔,内含红细胞,肿瘤细胞轻度异型性,电镜下可见W-P小体。免疫表型:CD31、CD34、vimentin均阳性。结论脊索瘤、脊索样脑膜瘤、脊索样胶质瘤及上皮样血管内皮瘤具有共同的脊索样特征,发生于中枢神经系统时,其鉴别诊断主要依据临床、形态学特征及免疫表型综合分析。     

RNA干扰沉默神经轴突导向因子受体4基因增加血肿瘤屏障通透性

目的研究神经轴突导向因子受体(Robo4)对血肿瘤屏障(BTB)通透性的影响。方法建立了体外BTB模型,应用Real-time PCR和Western blot检测Robo4在正常人脑微血管内皮细胞和胶质瘤微血管内皮细胞中的表达变化。设计合成针对Robo4基因的小干扰RNA,转染至人脑微血管内皮h CMEC/D3细胞,下调体外血肿瘤屏障模型内皮细胞中Robo4的表达,跨内皮电阻测量系统和辣根过氧化物酶渗透试验分析血肿瘤屏障通透性变化;Western blot和免疫荧光法检测h CMEC/D3细胞中紧密连接相关蛋白occludin和ZO-1的表达和分布变化。结果和正常人脑微血管内皮细胞相比,Robo4在胶质瘤微血管内皮细胞中的表达显著上调。下调体外血肿瘤屏障模型内皮细胞Robo4的表达后,TEER值显著降低,辣根过氧化物酶透过率显著增高;同时胶质瘤微血管内皮细胞中紧密连接相关蛋白occludin和ZO-1的表达显著降低,在细胞膜上呈不连续分布。结论 RNA干扰沉默Robo4表达能够显著降低紧密连接相关蛋白occludin和ZO-1的表达,增加BTB通透性。     

大鼠脑微血管内皮细胞的原代培养及缓激肽的作用靶细胞探讨

目的建立稳定的原代大鼠脑微血管内皮细胞培养方法,并在体外探讨不同剂量缓激肽的作用靶细胞,进一步阐释缓激肽开放血脑和血肿瘤屏障的机理。方法运用免疫荧光测定原代培养的脑血管内皮细胞、星形胶质细胞及C6胶质瘤细胞在不同剂量缓激肽作用前后的细胞内钙离子变化,根据给药前后的荧光改变来确定不同剂量缓激肽的作用靶点细胞。结果小剂量缓激肽(终浓度:1μmol/L)可以引起C6胶质瘤细胞内的钙离子水平升高,而只有大剂量(终浓度:10μmol/L~1mmol/L)缓激肽才能触发星形胶质细胞内的钙离子水平升高,脑微血管内皮细胞对大、小剂量缓激肽均无任何反应。结论缓激肽的直接作用靶点是胶质细胞及C6胶质瘤细胞,缓激肽调节脑血管内皮细胞通透性的作用可能需要某些细胞间信使的参与。     

胶质瘤细胞对血脑屏障水通道表达的影响

目的:探索胶质瘤细胞对血脑屏障水通道表达的影响及其病理生理意义。方法:通过内皮细胞系与胶质细胞体外共培养的方法建立体外血脑屏障模型。利用重水及高效液相分析的方法观察胶质瘤细胞作用对血脑屏障水转运的影响。运用半定量RT-PCR的方法分析胶质瘤细胞作用后血脑屏障内皮细胞及胶质细胞水通道1和水通道4的表达变化。结果:胶质瘤细胞可以诱导血脑屏障内皮细胞水通道1的异常表达并降低胶质细胞水通道4的表达水平。同时,胶质瘤细胞明显增强了体外血脑屏障模型对水由内皮细胞腔面至基底面的扩散。结论:胶质瘤性脑水肿不一定是血浆等大分子物质通透性增加的结果。血脑屏障中水通道的表达变化是胶质瘤性脑水肿发生的重要分子机制。     

ClC-3在人胶质瘤中的表达及分布

目的探讨ClC-3在人脑胶质瘤中的表达及其生物学意义。方法应用免疫组织化学染色技术检测24例人胶质瘤以及4例脑转移癌手术标本中ClC-3蛋白的表达;RT-PCR检测ClC-3蛋白表达阳性的手术标本中其mRNA表达。结果4例正常脑组织ClC-3蛋白的表达为阴性;而蛋白表达阳性的19例胶质瘤及4例脑转移癌中,ClC-3主要位于瘤细胞和微血管内皮细胞的胞浆或胞膜上。蛋白表达阳性的手术标本中16例胶质瘤和4例脑转移癌检测到ClC-3mRNA的表达。少突胶质细胞瘤Ⅲ级中ClC-3的mRNA和蛋白表达明显高于其Ⅱ级。结论ClC-3在人胶质瘤及脑转移癌组织中普遍表达,并且可能与少突胶质细胞瘤病理分级相关。     

人白内障球结膜微血管的电镜观察

目的 :观察人球结膜微血管的超微结构 ,认识白内障时球结膜微血管的电镜改变 ,为微循环和眼科研究工作提供参考。方法 :观察对象为 5例老年性白内障、1例糖尿病性白内障 ,术前一般状态良好。在白内障手术时取球结膜标本。电镜常规固定、包埋、定位、超薄切片、用电镜观察。结果 :球结膜毛细血管多由 2～ 3个内皮细胞围绕而成。核多为长椭圆形 ,核周缘有轻度凸凹。近腔侧胞浆细胞器较多 ,微丝和微管十分发达。基底膜、周细胞与一般毛细血管相似。 6例白内障球结膜毛细血管的内皮细胞、基底膜、周细胞、管腔都有不同程度的变化。其电镜改变可以分为三类 :( 1)代偿反应性增强改变 :微丝、微管十分发达、核周深凹、胞体突入管腔内、内皮细胞突起穿越基底膜、白细胞穿壁 ;( 2 )可恢复性退变 :线粒体退变、核质周集、胞浆出现空泡、基底膜分层、弥散、小量出血 ;( 3)严重不可恢复性改变 :核质固缩、腔内红细胞融合、血红蛋白溢出弥散、管壁断裂、腔内充满退变、裂解结构 ( 2例 ) ,并通过断裂处出至基底膜外。结论 :白内障病人球结膜微血管的超微结构有程度不等的改变     

血管内皮细胞在肿瘤侵袭微生态系统中作用的研究进展

目的介绍血管内皮细胞在肿瘤侵袭微生态系统中的作用及研究进展。方法系统复习肿瘤侵袭微生态系统研究的背景、组成要素、与肿瘤微环境的区别及血管内皮细胞对其影响的相关文献,并予以归纳总结。结果肿瘤侵袭微生态系统是指肿瘤侵袭过程的细胞群落与其栖息宿主环境所构成的一个自然的整体功能系统,属于细胞和分子水平的生态系统,它包括肿瘤细胞本身在内,具有生态系统的规律;而肿瘤的微环境则与肿瘤侵袭微生态系统不同,它是肿瘤细胞赖以生存的所有外界条件的总称,是一种结构体系,不包括肿瘤细胞本身,不具有生态系统的规律。肿瘤侵袭微生态系统组成要素包括肿瘤细胞群落和宿主细胞群落以及它们的栖息环境(基质成分),而血管内皮细胞群落是最主要的宿主细胞群落,其对肿瘤侵袭微生态系统的作用主要表现在肿瘤细胞群落和内皮细胞群落之间的营养联系上,这种营养联系形成了肿瘤微生态系统的物质循环,包括营养循环和细胞基质循环。肿瘤侵袭转移是肿瘤细胞群落和血管内皮细胞群落不断重建营养联系的结果。结论血管内皮细胞通过和肿瘤细胞之间的营养联系对肿瘤侵袭微生态系统进行调控,切断肿瘤细胞群落和血管内皮细胞群落之间的营养联系是肿瘤治疗的关键。     

小鼠肾小体发育中的细胞形态学变化

目的：探讨小鼠肾小体发生和发育过程中各种细胞的形态变化。方法：应用组织培养、后肾移植和光、电镜技术对发育不同阶段的肾小体进行观察。结果：胚龄13d后肾尚未出现肾小体时,经培养5d,或移植到受鼠5d后出现了成熟的肾小体的结构。电镜下观察表明：上皮基膜先于内皮基膜出现,随后两层膜融合。早期内皮细胞无核部分是连续的,无小孔结构。在发育过程中,小孔结构增多。早期足细胞有两种类型,明型和暗型,细胞基部伸出小突起并穿过基膜与内皮细胞紧贴。结论：血管球来源于内源性后肾细胞。发育过程中足细胞与内皮有结构上的接触,上皮基膜先于内皮基膜出现,足细胞有两型。     

TNF-α在热疗降低胶质瘤侵袭性过程中的作用

 目的 探讨肿瘤坏死因子-α(TNF-α)在热疗抑制肿瘤侵袭性过程中的作用。方法 热处理大鼠恶性胶质瘤细胞(C6细胞)和胶质瘤大鼠后,放射免疫法监测培养液和脑胶质瘤组织内TNF-α的浓度;免疫组化法检测经热疗/ TNF-α/生理盐水处理过的胶质瘤组织内增殖细胞核抗原(PCNA)蛋白的表达。利用Transwell构建肿瘤侵袭模型,通过结晶紫染色法检测肿瘤侵袭性。电镜观察C6恶性胶质瘤大鼠肿瘤血管内皮细胞的凋亡。结果 热疗可增加C6细胞培养液和胶质瘤大鼠肿瘤组织内的TNF-α含量及降低胶质瘤侵袭性,均于热疗后120min时达高峰(P＜0.01)。热疗与TNF-α单独作用于胶质瘤大鼠后,均可引起胶质瘤大鼠肿瘤血管内皮细胞的凋亡。且TNF-α引起内皮细胞的凋亡水平与热处理后C6细胞培养液中TNF-α含量一致。结论 热疗可能是通过增加TNF-α引起肿瘤血管内皮细胞凋亡而抑制了肿瘤侵袭性。     

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.     

Blood group isoantigens in human benign and malignant vascular tumors

Summary Paraffin material of 31 benign and malignant vascular tumors was investigated with respect to their blood group isoantigen (BG) content by the mixed cell agglutination reaction (MCAR). In capillary hemangioma, BG was found in endothelial cells as well as in solid buds. Benign hemangioendothelioma found in children differed from that found in adults in that in juvenile cases only endothelial cells expressed BG whereas in adult cases BG isoantigenity was present in endothelial cells as well as in intercapillary cellular elements. In pericytomas only endothelial cells were BG positive, whereas the tumor cells lacked BG. Similar results were obtained with glomus tumors. All but one hemangiosarcoma were BG negative. In one case, however, which probably resembled a true malignant hemangioendothelioma (Stout and Lattes, 1967) the tumor cells contained BG in conspicuous amounts.     

Ultrastructural observations on the capillaries of human thyroid tumours.

Ultrastructure of the capillaries of malignant and benign thyroid tumours has been examined. The material consisted of biopsies from six cases of thyroid papillary carcinoma, one case of follicular (foetal type) adenoma and six cases of nodular adenomatous goitre. In the group of nodular adenomatous goitre and in the follicular adenoma, the capillary wall was made up of fenestrated endothelium similar to that of capillaries of normal human thyroid. The fenestrae occupied a large area of the endothelial wall. Micro- and macropinocytotic vesicles were frequent in the endothelial cytoplasm. In the thyroid carcinomas the papillary structures always contained numerous capillaries with fenestrated endothelium. The microfollicular area and the solid tumoral areas of the papillary carcinoma showed occasional capillaries with fenestrated endothelium, but many capillaries were lined with continuous endothelium. The capillaries in all the specimens were surrounded externally by a continuous basement membrane which was frequently bilaminate or multilaminate. This study indicates that capillaries with fenestrated endothelium are characteristic of thyroid tumours which arise from follicular cells.     

体外肿瘤细胞对血管内皮细胞影响的观察

目的 观察肿瘤细胞与血管内皮细胞的关系及其在体外直接接触培养时内皮细胞的形态变化。方法 免疫磁珠阳性选择法分离培养大鼠肺血管内皮细胞,生长至汇合状态时加入不同的肿瘤细胞(小鼠树突状细胞肉瘤细胞DCS、人胃癌细胞MGC-803、小鼠肺腺癌细胞LA795)共培养,光镜、扫描电镜、免疫组织化学、transwell法及荧光分子传递等方法观察内皮细胞的形态、功能的变化。结果培养的内皮细胞部分呈卵石样,部分呈长梭形。长至汇合状态的内皮细胞再加入肿瘤细胞后,内皮细胞形态发生明显变化,汇合细胞中间出现许多圆形管腔样结构;肿瘤细胞往往分布在新形成的腔隙内;肿瘤细胞条件培养基可支持内皮细胞的生长,促进内皮细胞的运动;luciffer yellow分子可直接从肿瘤细胞传递到内皮细胞。结论 肿瘤细胞与内皮细胞可进行通讯,肿瘤细胞可直接引起内皮细胞结构和功能表型的改变。     

Fetal and neonatal rat intestinal capillaries: A TEM study of changes in the mural structure

The development of intestinal mucosal capillaries was studied in Wistar rats from the fourteenth day of gestation through the second postnatal day with the transmission electron microscope. The microvessels develop first as continuous capillaries without a basal lamina and become fenestrated during later stages of gestation. In the thickened areas of the cytoplasm of endothelial cells at early stages, there is a paucity of pinocytotic vesicles; but some vesicles are observed which are larger than those in the adult. Later in gestation these larger vesicles can still be seen, but the frequency of their occurrence decreases with the maturation of the vessels. The intercellular junctions vary in length and shape, and the space between the outer leaflets of the apposed cell membranes is usually 20 nm. Surrounding the abluminal surface of the endothelium, the incipient basal lamina appears attenuated and incomplete until at least the second day of neonatal life.     

Precise glioma targeting of and penetration by aptamer and peptide dual-functioned nanoparticles

The treatment of a brain glioma is still one of the most difficult challenges in oncology. To effectively treat brain glioma and reduce the side effects, drugs must be transported across the blood brain barrier (BBB) and then targeted to the brain cancer cells because most anti-tumor drugs are highly toxic to the normal brain tissue. A cascade delivery strategy was developed to perform these two aims and to achieve enhanced and precisely targeted delivery. Herein, we utilize a phage-displayed TGN peptide and an AS1411 aptamer, which are specific targeting ligands of the BBB and cancer cells, respectively and we conjugate them with nanoparticles to establish the brain glioma cascade delivery system (AsTNP). In vitro cell uptake and three-dimensional tumor spheroid penetration studies demonstrated that the system could not only target endothelial and tumor cells but also penetrate the endothelial monolayers and tumor cells to reach the core of the tumor spheroids, which was extremely important but mostly ignored in glioma therapy. In vivo imaging further demonstrated that the AsTNP provided the highest tumor distribution and tumor/normal brain ratio. The distribution was also reconfirmed by fluorescent images of the brain slides. As a result, the docetaxel-loaded AsTNP presents the best anti-glioma effect with improved glioma bearing survival. In conclusion, the AsTNP could precisely target to the brain glioma, which was a valuable target for glioma imaging and therapy.