CAJ | 학술논문

目的应用原代培养的大鼠脑微血管内皮细胞(brain microvascular endothelial cell,BMVEC)与脑周细胞共培养建立可模拟在体状态的稳定体外血脑屏障(blood-brain barrier,BBB)模型.方法原代分离、纯化培养大鼠BMVEC和周细胞,通过免疫细胞化学染色方法鉴定分离的细胞,应用Transwell插槽(孔径0.4μm)共培养构建体外BBB模型,经4h渗漏试验、紧密连接蛋白鉴定、跨内皮电阻检测以及通透性试验评价其屏障功能,比较共培养模型与单纯BMVEC模型膜两侧电阻值差异以及对小分子荧光素钠(sodium fluorescein,Na-F)通透性的差异.结果融合的BMVEC单层呈现典型的鹅卵石样外观,脑周细胞呈典型的不规则外形并具有重叠生长等特性.免疫双标法鉴定显示,脑周细胞阳性表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和神经元-胶质抗原2(neuron-glial antigen2,NG2)；共培养模型内皮细胞融合后,液面渗漏试验呈阳性；免疫细胞化学染色显示,内皮细胞间形成连续而致密的紧密连接；与BMVEC模型相比,共培养模型跨内皮细胞电阻[( 190.762±10.326)Ω/cm2对(96.503±8.012)Ω/cm2；t=- 24.489,P＜0.01]显著增高,通透性显著降低(为单内皮模型的56.149％ ±3.572％；t=19.330,P＜0.01).结论原代分离大鼠BMVEC 和周细胞共培养体外模型的形态、结构及屏障功能具备BBB的基本特征,为研究BBB提供了一种有用工具.
목적 응용원대배양적대서뇌미혈관내피세포(brain microvascular endothelial cell,BMVEC)여뇌주세포공배양건립가모의재체상태적은정체외혈뇌병장(blood-brain barrier,BBB)모형.방법 원대분리、순화배양대서BMVEC화주세포,통과면역세포화학염색방법감정분리적세포,응용Transwell삽조(공경0.4μm)공배양구건체외BBB모형,경4h삼루시험、긴밀련접단백감정、과내피전조검측이급통투성시험평개기병장공능,비교공배양모형여단순BMVEC모형막량측전조치차이이급대소분자형광소납(sodium fluorescein,Na-F)통투성적차이.결과 융합적BMVEC단층정현전형적아란석양외관,뇌주세포정전형적불규칙외형병구유중첩생장등특성.면역쌍표법감정현시,뇌주세포양성표체α-평활기기동단백(α-smooth muscle actin,α-SMA)화신경원-효질항원2(neuron-glial antigen2,NG2)；공배양모형내피세포융합후,액면삼루시험정양성；면역세포화학염색현시,내피세포간형성련속이치밀적긴밀련접；여BMVEC모형상비,공배양모형과내피세포전조[( 190.762±10.326)Ω/cm2대(96.503±8.012)Ω/cm2；t=- 24.489,P＜0.01]현저증고,통투성현저강저(위단내피모형적56.149％ ±3.572％；t=19.330,P＜0.01).결론 원대분리대서BMVEC 화주세포공배양체외모형적형태、결구급병장공능구비BBB적기본특정,위연구BBB제공료일충유용공구.
Objective To establish a stable in vitro model of blood-brain barrier (BBB) simulating in vivo state using the primary-cultured rat brain microvascular endothelial cells (BMVECs) and pericytes.Methods The primary rat BMVECs and pericytes were isolated,purified and cultured.The isolated cells were identified by immunocytochemical staining method.An in vitro model of BBB was constructed using Transwell inserts (pore size 0.4 μm) coculture.Its barrier function was evaluated by the 4-hour leakage test,tight junction protein identification,transendothelial resistance detection,and permeability test.The difference between the cocultured model and simple BMVEC model across the membrane resistance values,and the permeability difference of the small molecule sodium fluorescein (Na-F) were compared.Results Confluent BMVEC monolayers demonstrated a typical cobblestone appearance and the pericytes displayed irregular shape and overlapping growth.Immunodouble labeling technique identification showed that the pericytes positively expressed α-srmooth muscle actin (α-SMA) and neuron-glial antigen 2 (NG2); after the fusion of cocultured model endothelial cells,the surface leakage test became positive; immnocytochemical staining shows that a continuous and dense tight junction formed between the endothelial cells; compared to the BMVEC model,the transendothelial electrical resistance of the cocultured model increased significantly (190.762 ± 10.326 Ω/cm2 vs.96.503 ± 8.012 Ω/cm2; t=- 24.489,P ＜0.01),and the permeability decreased significantly (56.149％ ± 3.572％ of the single endothelial model; t =19.330,P ＜ 0.01 ).Conclusions The primary isolated rat BMVECs and pericytes cocultured the morphology,structure and barrier function of in vitro model have the basic characteristics of BBB,and they have provided a useful tool for the research of BBB.