Establish The Vitro Model Of Blood Brain Barrier By Co-culture Human Brain Micro Vascular Endothelial Cells With Astrocytes

The blood brain barrier(BBB)is a barrier structure between the central nervous system and blood circulatory system which has substances restrictive and regulation ability to maintain the microenvironmental stability of the central nervous system.The BBB system is mainly composed of brain microvascular endothelial cells and type I astrocytes footplate and vascular basement membrane.The complete BBB has special enzyme system,low level of endocytosis,and selective transport function.Streptococcus suis is a group of gram-positive bacteria which diameter between 1?2 ?m in single or oval shape,resonating with short chain shapes in liquid culture.According to the difference of capsule antigen,Streptococcus suis are divided into several serotypes and a considerable number of undefined strains.Streptococcus suis can cause meningitis,however,present studies cannot reveal how it penetrates the blood-brain barrier and cause meningitis,so a vitro model at both molecular and cellular level to show how Streptococcus suis penetrates the BBB is urgently in need.With the development of cell culture and material technology in recent years,it has become possible to study transmembrane transport,ionic signal pathways and protein expression mechanism in endothelial cells by simulating vivo environment.By using such cell culture technique,we can conditionally rebuild the BBB structure in vitro and observe the changes of interactions under the help of immunofluorescence and electron microscope between bacteria and cells,and which is helpful for the further study of brain blood barrier.In this study,we used Transwell cell culture chamber as a carrier to rebuild the BBB model in vitro.We designed three types of BBB model using human brain microvascular endothelial cells and human normal astrocytes.We evaluate the effect of the BBB model by measuring TEER(Trans-endothelial electrical resistance)value and the permeability of FLU(sodium Fluorescein).The TEER results showed that in the fifth day after seeding cells,the co-culture barrier model can reach 143?cm2 while single hBMEC barrier model is 137 ?cm2 and single HA-1800 barrier model is 124?cm2.FLU permeability results showed that the hBMEC barrier model is 67.8%while the co-culture barrier is 42.6%on average.Subsequently,we use Streptococcus suis type 2 strain ZY05719 compared to Escherichia coli strain DH5a,Streptococcus suis type CHz strain CZ130302 compared to its attenuated strain A50K to penetrate the BBB model.Statistical results showed that ZY05719 had a stronger penetration ability than DH5a which is significantly different.Furthermore,DH5a could not even penetrate the co-culture barrier model within 4 hours.Meanwhile,the penetration ability of CZ130302 is stronger than 450K.The above results has proved that our BBB cell models can form similar structures and the ability to restrict substances in vitro,and also have an obvious resistance to Streptococcus suis,in addition,reflect the differences on bacterial penetration ability to the vitro BBB model.Our study used Streptococcus suis to penetrate the vitro BBB model to simulate meningitis on the basis of cell co-culture which has few reports in China.This cell model not only build a vitro visualization platform for studying interactions between Streptococcus suis and BBB cells,but also provide theoretical support.