| Objective:In this study, the primary cells of brain microvascular endothelial cell (BMEC)and astrocyte cell (AC) were co-cultured to bulid in vitro blood brain barrier (BBB)model. The permeability mechanism of Ligustrazine on the BBB model wasinvestigated by measuring the change of transendothelial electrical resistance (TEER),permeability of fluorescein and expression of tight junction proteins Occludin andZO-1.Method:1. The primary AC was isolated using trypsin digest,200mesh sieve filteration ofboth side cerebral cortex of new born SD rat, and the other cell was removed bydifferent removal method. The isolated AC was cultured in DMEM with20%fetalbovine serum which changed every3days. The different states of AC cell growth wereobserved by Inverted microscope. The expression of glial fibrillary acidic protein(GFAP) on AC surface were explored by immunofluorescence staining method, andobserved by fluorescence microscope.2. The microvascular was isolated by twice enzyme digest and gradientcentrifugation of the both side cerebra gray of new born SD rat. Then, the isolatedmicrovascular was plant into1%gelatin pre-coating Primary Sexual cell culture bottle,and cultured in DMEM/F12complete medium added by heparin and endothelial cellgrowth supplement to avoid the effects of other cells. The TEER was measured byMillicell-ERS-2, and the expression of surface Ⅷ factor related antigens wereidentified by immunofluorescence staining method to determine the type of culturedcells.3. The BMEC and AC were double-edged planted on Transwell permeable supportmicroporous membrane. The BBB in vitro model was evaluated by TEER measurement of different times,4h Liquid interview leak test, and analysis of γ-GT and ALP oftypical index of BBB.4. The BBB were divied into contro group and Ligustrazine treatment set as6drugconcentrations (208.69μg/mL、104.35μg/mL、52.17μg/mL、26.09μg/mL、13.04μg/mL、6.52μg/mL). The safe ligustrazine concentrations on BMEC and AC wereidentified by MTT method, and the high, medium and low concentrations weredetermined by aggregated results.5. The TEERs were measured at2h,4h,8h,12h,16h,20h and24h after drugadded. The effect of ligustrazine on permeability of BBB was identified by analysis theTEER differences of TEER at different time and different group TEER at same time.6. The outside and inside culture liquid in Transwell cell was discarded at24hafter ligustrazine added. The PBS with100μg/mL of fluorescein was added in insidecell, and PBS was added in outside cell to maintain the liquid level of both sides. After4h, the liquid of outside cell were collected to detect the absorbance using fluorescencespectrophotometer. The permeance of fluorescein was calculated and compared todetermine the effects of ligustrazine on permeability of BBB.7. The culture liquid of outside Transcell cell was absorbed at24h afterligustrazine added, and the permeance of ligustrazine was measured by HPLC. Thepermeability of different group was compared.8. The total cell protein of control group, high, medium and low concentrationgroups was isolated to measure the expression Occludin、ZO-1using western blotting.The effects of ligustrazine on tight junction proteins of BBB were explored.Result:1. Primary culture of AC: the cell morphology of3d and7d was observed byinverted microscope, and showed long and branching projections, stretching fillingbetween the cell body and its neurite of neighboring nerve cells according with themorphological characteristics of AC. After immunofluorescent staining with GFAP, thecytoplasm and neurite emitted obvious green fluorescence; celluar outline was clear. 2. Primary culture of BMEC: the cell morphology of3d,7d,14d and16d wasobserved by inverted microscope, and showed short spindle, polygonal and typical signsof paving stone. After immunofluorescent staining of Ⅷ factor related antigens, thecell connection was tight; the cytoplasm emitted obvious green fluorescence.3. Identification of the in vitro BBB model: Observing the cell morphology onTranswell by inverted microscope, the BMEC showed dense fusion adhere growth, clearshape, and the bottom AC cell was faintly vislble. The TEER of co-culture group andsingle layer BMEC was increased with time, and co-culture group was more obvious.The TEERs of both group were stable at72h. The4h Liquid interview leak test arepositive. The content of γ-GT and ALP of co-culture cell was significantly higher thansingle layer.4. The screen of safe concentration of ligustrazine:13.04μg/mL～208.69μg/mLof ligustrazine showed activity of AC and BMEC growth. The concentrations of high,medium and low group were208.69μg/mL、52.17μg/mL、13.04μg/mL.5. The permeability mechanism of Ligustrazine on the BBB model：（1）The TEERs of high, medium and low ligustrazine concentration group wereall decreased, especially the high concentration group.（2）The HPLC results showed that the permeability existed in all high, mediumand low concentration groups which the permeances were43.4%、38.5%and36.4%.（3）The fluorescein permeable results showed that the permeances of threetreatment groups were15.7%、12.1%and8%whereas control group was4.9%.（4）The western blotting results of expression of Occludin and ZO-1in eachgroup revealed that two proteins were expressed on in vitro BBB model and thecontents of AC and BMEC, especially the BMEC, and the expression levels of both twoproteins were decreased by ligustrazine added with dose dependent.Conclusion:1. Culture of primary cell and model establishment: The results of cellmorphology and immunofluorescent staining by specific antibody of primary BMEC and AC showed that the primary culture was successful; the TEER and typical enzymemeasurement showed that simulation in vitro BBB model through BMEC and ACco-culture in Transwell was successful.2. Effects of Ligustrazine on the in vitro blood brain barrier model: After addeddifferent dose of ligustrazine, the TEER value of normal BBB was decreased by timeelongation, revealed that ligustrazine can cause opening of tight junction; thepermeability of BBB model was increased by ligustrazine dose added by TEER andtransmittance of fluorescein measurement; the HPLC results showed that ligustrazinecan directly permeate in vitro BBB model, and the permeance were increased by doseincreased.3. Effects of ligustrazine on expression of tight junction Occludin and ZO-1: Thewestern blotting results showed that Occludin and ZO-1were all expressed on AC andBMEC, and the expression level were decreased by ligustrazine added in in vitro BBBmodel. The results suggested that ligustrazine activate the permeability of BBB throughdecreasing the expression of tight junction Occludin and ZO-1. |
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