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目的:建立体外血脑屏障模型(blood-brain barrier model,BBBM),观察非小细胞肺癌(non-small cell lung cancer,NSCLC)A549细胞对BBBM功能的影响。方法:早上将周细胞(pericytes,PC)接种到Transwell膜的外侧面,星形胶质细胞(astrocytes,AS)接种到12孔板内,8 h后将Transwell置入12孔板内,再将小鼠脑微血管内皮细胞(brain microvascular endothelial cells,BMEC)接种到transwell膜的内侧面,建立血脑屏障模型。模型建立后每天进行试漏试验及跨内皮电阻(transendothelial electrical resistance,TEER)测定,鉴定是否成模。成模后分为对照组与实验组,将A549细胞接种到实验组BBBM内室,共培养体系置于培养箱,于24 h、48 h、72 h后进行电阻测定及辣根过氧化物酶渗透试验,以评估BBBM的功能。分别于渗透试验进行至2 h、6 h时取样,根据样本中辣根过氧化物酶浓度计算表观渗透系数(apparent permeability coefcient,Papp)。结果:(1)部分血脑屏障模型在建立后3 d成模,4 d可全部成模。(2)A549细胞加入BBBM内室共培养。(a)实验组TEER值与对照组比较:1 d后无显著变化(P>0.05);2 d后降低出现统计学差异(P<0.05);3 d后降低具有统计学差异(P<0.01);(b)渗透试验与对照组比较:1 d后2 h及6 h渗透试验实验组Papp均降低,但无统计学差异(P>0.05);2 d后实验组Papp升高,2 h渗透试验差异具有统计学意义(P<0.05),6 h渗透试验差异无统计学意义(P>0.05);3 d后实验组Papp升高,2 h及6 h渗透试验均具有显著统计学差异(P<0.01)。结论:小鼠体外血脑屏障模型的功能可被人类非小细胞肺癌细胞影响。体外共培养体系可用于研究肺癌脑转移过程中肿瘤细胞与脑微血管内皮细胞间相互作用的分子机制。
OBJECTIVE: To establish an in vitro blood-brain barrier model (BBBM) to observe the effect of non-small cell lung cancer (NSCLC) A549 cells on BBBM function. Methods: Pericytes (PC) were inoculated on the lateral surface of Transwell membrane in the morning and astrocytes (AS) were inoculated into 12-well plates. Transwell was placed in 12-well plates after 8 h, Mouse brain microvascular endothelial cells (BMECs) were inoculated into the inner surface of the transwell membrane to establish a blood-brain barrier model. After the model was established, the daily test was conducted and the transendothelial electrical resistance (TEER) was measured to determine whether the model was established. Divided into the control group and the experimental group after modeling, A549 cells were inoculated into the experimental group BBBM inner chamber, co-culture system placed in the incubator at 24 h, 48 h, 72 h after resistance determination and horseradish peroxidase Penetration test to assess BBBM function. Samples were taken at 2 h and 6 h after the infiltration test. The apparent permeability coefficient (Papp) was calculated according to the concentration of horseradish peroxidase in the sample. Results: (1) Some blood-brain barrier models were established 3 days after establishment and all became model 4 days later. (2) A549 cells were added to BBBM internal chamber for co-culture. (a) There was no significant difference in the TEER value between the experimental group and the control group after 1 day (P> 0.05); after 2 days, there was a significant difference (P <0.05); after 3 days, there was a significant difference ); (b) Compared with the control group, the infiltration test showed that the Papp of experimental group was lower than that of the control group at 1 and 2 h, but Papp was no significant difference (P> 0.05) (P <0.05). There was no significant difference in 6 h osmotic test (P> 0.05). After 3 days, the Papp increased, and the osmotic pressure at 2 h and 6 h were significantly different (P <0.01). Conclusion: The function of mouse model of BBB can be affected by human non-small cell lung cancer cells. In vitro co-culture system can be used to study the molecular mechanism of the interaction between tumor cells and brain microvascular endothelial cells in the process of brain metastasis of lung cancer.