应用U251胶质瘤细胞、脑内皮细胞和周细胞体外构建血瘤/脑屏障模型的研究  

Establishment of an blood-brain/tumor barrier modal in vitro by coculturing brain endothelial cells,pericytes and U251

作　　者：杨海洋[1] 蔡理[2] 龙江[1] 何吉洪 

机构地区：[1]昆明医科大学第一附属医院神经外科,昆明650032 [2]南华大学附属第一医院神经外科,湖南衡阳421001

出　　处：《重庆医学》2014年第20期2613-2616,2619,共5页Chongqing medicine

基　　金：2011年云南省应用基础研究面上项目(2011FZ116)

摘　　要：目的利用人脑微血管内皮细胞(hBMEC)、脑周细胞(PC)和U251胶质瘤细胞(以下简称U251)非接触共培养体外构建稳定的三联血瘤(脑)屏障(BTB/BBB)模型。方法 3种细胞传代培养达一定数目,应用Transwell插槽体外共培养构建BTB/BBB模型,经4h试漏试验、倒置显微镜等观察形态学改变、免疫荧光技术观察Claudin-5、Occludin表达情况及模型对辣根过氧化物酶(HRP)通透性试验评价模型屏障功能。结果 hBMEC呈现出典型的鹅卵石样外观,非接触共培养后呈单层梭形生长,出现其特有的"漩涡"状,形态呈长梭形;PC外形不规则,具有重叠生长特性;U251呈典型的肿瘤细胞生长特性。3种不同方法构建的模型液面试漏实验均出现一定液面差;免疫荧光显示hBMEC细胞间形成连续而致密的连接,但强弱不等。3种模型对HRP通透性显示:单纯hBMEC为43.490%±3.572%、hBMEC+U251为36.540%±1.475%、hBMEC+PC+U251为26.460%±2.372%,差异有统计学意义(t=19.330,P<0.05)。结论 hBMEC+PC+U251非接触共培养模型的形态结构及屏障功能均比单纯hBMEC和hBMEC+U251更优越,具备更完善的BTB/BBB基本特征和功能,为今后体外研究BTB/BBB的功能和调控机制及药物筛选提供一种新型体外模型工具。Objective To establish a stable in vitro model of blood-brain/tumor barrier （BBB/BTB） simulating in vivo state u-sing the human brain endothelial cells（hBMEC） ,brain pericytes （PC） and U251 glioma cells（U251） .Methods An in vitro model of BBB/BTB was constructed using Transwell inserts（pore size 1 .0 μm）coculture while three cell subculture reaches a certain num-ber .Its barrier function was evaluated by the 4-hour leakage test ,inverted microscope to observe morphological changes ,horseradish peroxidase permeability test and tight junction protein Claudin-5 and Occludin identification expression observed by immunofluores-cence technology .Results hBM EC monolayers demonstrated a typical cobblestone-like appearance ,Non-contact spindle cocultured monolayer growth appeared its unique＆quot;swirl pool＆quot;shape ,fusiform morphology ;the pericytes displayed irregular shape and overlap-ping grow ;U251 showed typical tumor cell growth .Fluid leakage test were interviewing a certain level difference in three different ways to build models ;immnocytochemical staining showed a continuous and dense tight junction formed between the endothelial cells but intensity ranging ;horseradish peroxidase permeability of three different models showed that pure hBMEC （43 .490 ± 3 .572）% ,hBMEC＋U251（36 .540 ± 1 .475）% ,hBMEC＋ PC＋U251（26 .460 ± 2 .372）% was significantly lower ,the difference was statistically significant（t=19 .330 ,P〈0 .01） .Conclusion hBMEC＋ PC＋ U251 cocultured the morphology ,tructure and barrier function have more advantages than pure hBMEC and hBMEC＋U251 ,have the basic characteristics and function of BTB/BBB ,and they have provided a new tool reseach function ,regulation mechanisms and drug screening the BTB/BBB in future .

关 键 词：血脑屏障 内皮细胞 周细胞 U251胶质瘤细胞 

分 类 号：R739.4[医药卫生—肿瘤]