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目的建立大鼠脑毛细血管内皮细胞(BCECs)和星形胶质细胞共培养模型,评价纳米粒的跨血脑屏障(BBB)转运和对内皮细胞紧密连接的毒性。方法采用复乳/溶媒蒸发法制备载荧光探针6-香豆素的聚乙二醇-聚乳酸纳米粒。首先,分别从新生鼠大脑分离培养BCECs和星形胶质细胞并进行免疫组化鉴定。然后,将BCECs接种于细胞培养池微孔膜的上面,将星形胶质细胞接种于膜下面建立共培养模型。分别测定14C-蔗糖和纳米粒的渗透系数。结果载6-香豆素纳米粒的平均重均粒径为(102.4±6.8)nm,zeta电位为(-16.81±1.05)mV。BCECs的VIII因子表达呈阳性;星形胶质细胞的胶质原纤维酸性蛋白表达呈阳性。模型的跨内皮细胞电阻值为(313±23)Ω.cm2。扫描电镜和透射电镜观察发现,共培养模型中的BCECs形成紧密连接。纳米粒的质量浓度低于200μg.mL-1时,不影响14C-蔗糖渗透系数的改变,表明其不会影响BBB内皮细胞的紧密连接。10μg.mL-1载6-香豆素纳米粒的渗透系数为0.29×10-3cm.m in-1。结论该大鼠BBB模型与体内情况接近,适合用于评价纳米粒的脑内转运和毒性。
OBJECTIVE: To establish a co-culture model of rat brain capillary endothelial cells (BCECs) and astrocytes to evaluate the transmembrane activities of nanoparticles across the blood-brain barrier (BBB) and their toxicity to endothelial cells. Methods The poly (ethylene glycol) -polylactic acid nanoparticles containing fluorescent probe 6-coumarin were prepared by double emulsion / solvent evaporation method. First, BCECs and astrocytes were isolated from neonatal rat brain and identified by immunohistochemistry. Then, BCECs were seeded on the top of a cell culture cell microporous membrane, and ascitic cells were inoculated under the membrane to establish a co-culture model. The permeation coefficients of 14C-sucrose and nanoparticles were determined respectively. Results The average weight-average particle size of 6-coumarin nanoparticles was (102.4 ± 6.8) nm and the zeta potential was (-16.81 ± 1.05) mV. The expression of factor VIII in BCECs was positive; the expression of glial fibrillary acidic protein in astrocytes was positive. The trans-endothelial cell resistance of the model was (313 ± 23) Ω.cm2. Scanning electron microscopy and transmission electron microscopy showed that BCECs in the co-culture model formed tight junctions. The nanoparticle mass concentration below 200 μg · mL-1 did not affect the change of 14C-sucrose permeability, indicating that it did not affect the tight junctions of BBB endothelial cells. The osmotic coefficient of 10μg.mL-1 6-coumarin nanoparticles was 0.29 × 10-3cm.m in-1. Conclusion The rat model of BBB is similar to that in vivo and is suitable for evaluation of intracerebral transport and toxicity of nanoparticles.