Effect Of Human Non-small Cell Lung Cancer Cells On The Function Of Mouse Blood-brain Barrier Model In Vitro And The Expression Of Epsin1?ZO-1 In Mouse Brain Microvascular Endothelial Cells

PART1Effect of human non-small cell lung cancer cells on the function of mouse blood-brain barrier model in vitroobjective: establish blood-brain barrier model (BBBM) in vitro, and investigate the effect of non-small cell lung cancer(A549) cells on the function of BBBM. Method:In the morning we seed the pericytes on the lower surface of Transwell membrane,and the astrocytes were seeded in 12-well plate. 8 hours latter put the transwells into the wells,and the brain microvascular endothelia cells were planted on the upper surface of the transwell membrane, so that the blood-brain barrier model were established. Then we perform leakage test and transendothelial electrical resistance(TEER) test to identify if the models are established successfully . The successful models were assigned to control group and experimental group, and the A549 cells were planted into the luminal chamber of the experimental group BBBMs. The coculture systems were incubated in the incubator, at 24h, 48h, 72h later, we performed the TEER test and horseradish peroxidase permeability test, to evaluate the function of BBBM. The samples of permeability test were collected when the test progress to 2 hours and 6 hours. According to the concentration of horseradish peroxidase in the samples, the apparent permeability coefficient (Papp) was calculated.Result: 1. When the constructions were performed, some of them formed BBBM successfully in 3days, all models are formed in 4 days. 2. After the a549 cells were planted into the luminal chamber of the BBBM, (1) Compare to the control group:one day later, TEER of the experimental group have no significant changes (p>0.05);two days later, TEER of the experimental group reduced with statistic difference(p<0.05); three days later,TEER of the experimental group reduced with significant statistic difference (p<0.01). (2) compare to the control group: one day later, Papp of the experimental group tend to reduce both of 2 hours and 6 hours test, but there is no statistic difference(p>0.05); two days later, Papp of the experimental group increased ,2 hours test with statistic difference (p<0.05), but 6 hours test have no statistic difference (p>0.05). 3 days later, Papp of the experimental group increased, both of 2 hours and 6 hours test have significant statistic difference. Conclusion: The function of mouse blood-brain barrier model in vitro could be affected by human non-small cell lung cancer cells. Coculture system in vitro could be used to research the mechanism of the interactive between tumor cells and BMEC in the process of brain metastasis.PART 2Effect of human non-small cell lung cancer cells on the expression of epsinl and ZO-1 in mouse brain microvascular endothelial cells objective: Investigate the effect of non-small cell lung cancer(A549) cells on the expression of epsinl and ZO-1 in mouse brain microvascular endothelial cells(bEnd.3 ) . Method: The mouse brain microvascular endothelial cells were inoculated into the 75cm2 culture flasks,4 days latter, when the cells fuse into a monolayer and the tight junction formed, we signed the flasks into bEnd.3 alone group, bEnd.3+HBEC group, bEnd.3+A549 group random. The human bronchial epithelial cells were seeded into the bEnd.3+HBEC group, and non-small cell lung cancer cells were seeded into the bEnd.3+A549 group. After co-cultured with 48h,bEnd.3 were selected by flow cytometry, and the PE anti-mouse endothelial cell selective adhesion molecule was used as sorting antibody. The selected bEnd.3 were inoculated into the 12 hole plate. The number of cells per unit area(×400 visual field )was counted by DAPI staining at 12h, 36h and 4D, to evaluate the cell proliferation rate. At 12h ,we performed epsinl immmunofluorescence staining , to observe the expression of epsinl , the morphology and size of bEnd.3 were measured too .At 4 days when the bEnd.3 again fused into monolayer, we performed immunofluorescence staining of epsinl and ZO- 1, to observe the expression of ZO-1 and epsinl .Result: 1. Among the three kinds of cells(mouse brain microvascular endothelial cells, human bronchial epithelial cells and human non-small cell lung cancer cells), PE anti-mouse ESAM binding with mouse brain microvascular endothelial cells has an absolute specificity. The purity of sorted cells is 96.86% in bEnd.3+HBEC group , and 96.36% in bEnd.3+A549 group. 2. After co-culture and flow cytometry sorting, the cell viability of bEnd.3 was not significantly affected,there was no significant difference in cell proliferation rate between bEnd.3 alone group and bEnd.3+HBEC group, compared with the other two groups, the cell proliferation rate of bEnd.3+A549 group increased significantly after co-culture and sorting.3. The expression of epsinl in bEnd.3 alone group and bEnd.3+HBEC group had no significant difference (P>0.05 ) , when compared with these two groups, the expression of epsinl in BEnd.3+A549 group increased with statistically significant(P<0.01). The differences between the two time points within group are statistically significant , in all the three groups. Indicate that the expression of epsinlin bEnd.3 was enhanced when the cells were proliferating. 4. immunofluorescence staining of ZO-1 in bEnd.3 alone group and bEnd.3+HBEC group, fluorescence showed dense banded located between adjacent cells, connected to each other to form a grid,in BEnd.3+A549 group the fluorescent bands were loose and broadened, and the fluorescence was seen in the cytoplasm.Conclusion: Co-culture of human non-small cell lung cancer cells and mouse BMECs can change the morphology and molecular expression of BMECs, the cells became round and smaller, the expression of epsinl increased, ZO-1 ectopic expression to intracellular. May be, these alterations are related to the invasion through BBB of NSCLC cells and the angiogenesis of metastatic tumor.