| Background:Perfluorooctanoic acid (PFOA) is the representative compound of the Perfluorinated compounds.Because of its stable chemical property, good hydrophobic ity and oleophobicity,it has been used widely since1950s in many civil and industrial production areas,such as firefoam,textiles and industrial cleaning agents. PFOA has fluorocarbon covalent bond structure, this structural properties make it very difficult to degrade, so can exist persistently in environmental media and enriched by the organism. The environment survey results show that PFOA contamination existed in various types of water bodies worldwidely and all the wildlife blood. Population survey data show that higher levels of PFOA contamination are detected in human blood worldwidely.Meanwhile in recent years, non-stick pan and sports brand clothing coating are lighted containing PFOA,so that biological toxicity of PFOA receive increasing attention. Studies have shown that PFOA can cause liver cancer, liver oxidative stress, can affect the fatty acid binding and promote liver cell apoptosis, it can lead to myocardial injury by changing rat myocardial cell membrane action potential and calcium channel to intracellular calcium overload. And PFOA can also cause male testicular cancer, female breast and ovarian lesions. It can lower serum IgG and IgM levels, reduce immune function of T cells and B cells. The animal studies find that perfluoroocantane sulfoate(PFOS), which has similar physical and chemical nature with PFOA, can cause convulsions and irritability of rats. It suggested that PFOA may also cause neurotoxicity, but relevant research data is relatively few. Due to the sensitivity and non-renewable of the central nervous system, the neurotoxicity study of PFOA is particularly important.Objective:To explore the effect mechanism of PFOA on central nervous system from the start of effect of PFOA on BBB permeability and intracellular free calcium concentration in astrocyte. And to reveale the adapter mode of as on neurotoxic chemicals. The results of this study can be used in a variety of cells in the nervous system on [Ca2+]i measuring, and provide technical modules for constructing3-staged models in the topic of vitro screening model of the nerve poison.Methods:(1) Separate brain micro vascular endothelial cells(BCECs) and astrocytes from Wistar rats which born within24h, culture them in vitro, and identificate the purity of BCECs and astrocytes with immunochemical detection of VIII factor antibody and GFAP antibody.(2)Inoculate BCECs and astrocytes into cell flapper and12-well culture plates and co-culture to build BBB toxicity screening model, carry out leak test when BCECs merge, then measure the transendothelial electrical resistance (TEER) of cell flapper which leak test is positive, select cell flappers with stable TEER values flapper to implement the transmembrane experiment. (3) Measure the IC50of PFOA to BCECs and astrocytes with resazurin,then determine PFOA exposure dose.(4) Measure BBB permeability coefficient (Pe) of PFOA and control group with luciffer yellow transmembrane experimental to reckon the effect ofPFOA on BBB permeability.(5) Divided into high, medium and low dose group and the control group depending on PFOA exposure doses, measure the [Ca2+]i in astrocyte after exposed to PFOA by Fura-2/AM fluorescence probe. Speculate the impact on [Ca2+]i in astrocyte of PFOA by comparing the difference between each group.Results:(l)The purity of BCECs and astrocyte reach95%by immunocytochemistry test that meet the experimental requirements.(2) BCECs reach confluent state after5-6days, the leakage test is positive, TEER value is stable, so this BBB model can be used in permeability toxicity test.(3) The IC50of PFOA on BCECs and astrocytes are1.0mmol/L and0.83mmol/L.(4) The Pe value of PFOA exposure group is (1.37±0.16) x10-3cm/min, the Pe value of control group is (1.04±0.09)×10-3cm/min, the Pe value of PFOA exposure group is greater than the control group,and the difference is statistically significant.(5) The [Ca2+]i in astrocytes tend to increase with the higher exposure doses and the longer exposure time of PFOA, the difference with the control group is statistically significant.Conclusion:PFOA can increase BBB permeability, there is the potential neurotoxic effects.PFOA can also elevate the [Ca2+]i in astrocytes of rat,it is possible to bring out neurotoxicity by impacting calcium homeostasis in astrocytes. PFOA has potential neurotoxicity by screening with2-staged model. |
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