CAJ | 학술논문

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4 h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；U 251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hBM EC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 hBM EC＋ PC＋ U251非接触共培养模型的形态结构及屏障功能均比单纯hBM EC和hBM EC＋ 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。
목적：이용인뇌미혈관내피세포（hBMEC）、뇌주세포（PC）화U251효질류세포（이하간칭U251）비접촉공배양체외구건은정적삼련혈류（뇌）병장（BTB/BBB）모형。방법3충세포전대배양체일정수목，응용 Transwell삽조체외공배양구건BTB/BBB모형，경4 h시루시험、도치현미경등관찰형태학개변、면역형광기술관찰Claudin-5、Occludin표체정황급모형대랄근과양화물매（HRP）통투성시험평개모형병장공능。결과 hBMEC정현출전형적아란석양외관，비접촉공배양후정단층사형생장，출현기특유적“선와”상，형태정장사형；PC외형불규칙，구유중첩생장특성；U 251정전형적종류세포생장특성。3충불동방법구건적모형액면시루실험균출현일정액면차；면역형광현시hBM EC세포간형성련속이치밀적련접，단강약불등。3충모형대HRP통투성현시：단순 hBMEC 위43．490％±3．572％、hBMEC ＋ U251위36．540％±1．475％、hBMEC ＋ PC ＋ U251위26．460％±2．372％，차이유통계학의의（t＝19．330，P＜0．05）。결론 hBM EC＋ PC＋ U251비접촉공배양모형적형태결구급병장공능균비단순hBM EC화hBM EC＋ U251경우월，구비경완선적BTB/BBB기본특정화공능，위금후체외연구BTB/BBB적공능화조공궤제급약물사선제공일충신형체외모형공구。
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"swirl pool"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 .