| Background:In the past ten years,the incidence and mortality of malignant tumors in China have been increasing year by year,and the burden of cancer has become increasingly serious,which has become one of the most important public health problems that seriously endanger the health of our people.According to the latest national cancer statistics released by the National Cancer Center in January 2019,the incidence and mortality of brain tumors in China ranks 9th and 8th in the incidence and mortality of malignant tumors nationwide.Central Nervous System?CNS?cancer treatment is mainly based on surgery,radiotherapy and chemotherapy.Because of its unique Blood-Brain Barrier?BBB?structure,compared with other types of tumors,most drugs are difficult to act on the lesion through BBB,resulting in poor treatment of CNS tumors.With the continuous development of experimental techniques in recent years,the research of in vitro BBB models has been deepened.More and more scholars have applied the in vitro BBB model to study the effects of drugs on BBB,permeability,and transport mechanisms,making it a new primary screening means for drug of CNS.Glioma is the most important malignant tumor in adult CNS tumors.It is a typical vascular-dependent malignant tumor.Vascular endothelial growth factor?VEGF?and its receptors are highly expressed and their expression levels are high.It is closely related to the degree of malignancy and grade of glioma.VEGF and its receptors mainly regulate endothelial cell differentiation and angiogenesis,affecting the role of other pro-angiogenic factors,and play an important role in the invasion and dissemination of glioma.An anti-angiogenic drug can specifically bind to VEGF,and by blocking the binding of VEGF to the corresponding receptor on vascular endothelial cells,thereby inhibiting the activation of its downstream signaling pathway,resulting in VEGF failing to function normally and inhibiting tumor blood vessels.Growing work.It can induce tumor vascular normalization,improve vascular permeability,and improve the therapeutic effect of radiotherapy and chemotherapy.The anti-angiogenic therapy of recombinant human endostatin?rh-ES?is characterized by a broad target,which not only acts on the VEGF/VEGFR2 signaling pathway,but also includes HIF-1?and MMPs,bFGF,integrin,multi-step multi-steps affecting angiogenesis.It is suggested that rh-ES may become a new treatment for high-grade glioma treatment.In a number of studies,rh-ES has been shown to reduce glioma-induced brain edema while improving vascular permeability.Compared with radiotherapy alone,combined rh-ES treatment can alleviate brain edema caused by brain metastasis of non-small cell lung cancer,suggesting that rh-ES may act on tumor cells through BBB.Whether the drug can pass the BBB or not,the role of the intracranial is the key to the problem.Objective:The in vitro BBB model was constructed by cell culture technique;the in vitro BBB model was used to evaluate the in vitro BBB permeability of the angiogenic drug rh-ES.Methods:A non-contact co-culture group of human umbilical vein endothelial cells Huvecs and rat glioma cell C6 was established through a Transwell chamber with a microporous PET membrane,and a single-cell Huvecs group and a blank control group were established,and the cells were observed growth state by an electron microscope.The permeability of the in vitro BBB model was evaluated by 4h leakage test,transendothelial cell electrical impedance?TEER?test,and fluorescein sodium permeability test.Using the in vitro BBB model to evaluate the permeability of fluorescence-labeled rh-ES.Results:1.Microscopic observation showed that the endothelial cells in the upper chamber of transwell were"stone paving"both in the co-culture group and the single cell group,and the adjacent endothelial cells were basically fused,without gaps,and the formed single-layer barrier structure was dense.2.The results of the 4h leakage test showed that the co-culture group and the single-cell group could maintain a clear liquid surface difference of more than 0.5cm inside and outside the chamber,which proved that the in vitro BBB model had a certain blocking effect on the liquid.3.The TEER test results showed that the TEER value of the co-culture group was significantly higher than that of the single-cell group.The two groups reached the highest value on the 7th day.The TEER value of the co-culture group was?161.187±1.071??·cm2 on the 7th day,single cell group.The TEER value was?151.103±0.910??·cm 2 on the7th day,and the difference was statistically significant?P<0.001?.The co-culture group has a higher TEER value than the single-cell group and has a higher barrier effect on small ions.The co-culture group is superior to the single-cell culture group.4.The results of sodium fluorescein permeability test showed that the permeability coefficient Pe of fluorescein sodium was?0.39±0.07?×10-3 cm·min-1 in the co-culture group.The co-culture group had low permeability to sodium fluorescein and the model was successfully established.5.Fluorescently labeled rh-ES was applied to the co-culture model at 15 min,30 min,and 60 min.Pe was divided into?2.2±0.9?×10-5cm·min-1,?1.8±0.9?×10-5 cm·min-1,?2.3±0.2?×10-5cm·min-1.Conclusion:1.The non-contact co-culture group formed by Huvecs and C6 has higher trans-endothelial electrical impedance resistance than the Huvecs group.The co-culture group is superior to the single-cell culture group.2.Huvecs and C6 form a non-contact co-culture group.The endothelial cells grow densely.The 4h leakage test can maintain a clear liquid level difference of>0.5cm,with high TEER value,low permeability of fluorescein sodium,and a certain barrier.Function to meet the requirements of subsequent experiments.3.Using FITC fluorescent labeling rh-ES,combined with fluorescence detection technology to detect the permeability of the drug to the co-culture model,rh-ES has a certain permeability to the in vitro BBB,but its The permeability is low and further in vivo experimental support is still needed. |
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