星形胶质细胞与淋巴细胞在体外血脑屏障中的相互作用

Interactions between astrocytes and lymphocytes under in vitro blood-brain barrier model

目的 本实验旨在探讨多发性硬化（MS）发病中血脑屏障（BBB）中星形胶质细胞（AS）与淋巴细胞（LCs）的相互作用.方法 分离原代鼠脑血管内皮细胞（rBMECs）和SD鼠AS并建立体外BBB模型,对rBMECs与AS共培养和rBMECs常规培养进行跨内皮细胞电阻（TEER）和渗透率的比较.用荧光定量法检测rBMECs单独培养、rBMECs与AS共培养、rBMECs和LCs共培养、rBMECs与AS及LCs三重培养四种模式的神经生长因子（NGF）、基质金属蛋白酶（MMP）-2、MMP-9、白细胞介素（IL）-17,干扰素（IFN）-γ,Foxp3的水平.结果 rBMECs和AS共培养比rBMECs单培养的TEER高,而其LCs渗透率却很低;与rBMECs单培养相比,rBMECs、AS和LCs三重培养可降低NGF、IL-17、IFN-γ的表达（P=0.036）,MMP-2和Foxp3的表达增加（P=0.0216）;与rBMECs和AS共培养相比,rBMECs、AS、LCs三重培养能有效降低NGF、IL-17和IFN-γ的表达（P =0.0451）.与rBMECs和AS的共培养相比,rBMECs、AS、LCs三重培养能增加MMP-2的表达（P =0.021）,而MMP-9在四种模式中表达无差异.结论 在体外BBB中原代鼠脑rBMECs和AS共培养比rBMECs单培养的TEER高,AS与LCs共培养可能增加MMP-2的表达,降低IL-17和IFN-γ的表达.

Objective The study aimed to investigate the impact of interactions between astrocytes and lymphocytes in blood-brain barrier （BBB） in the pathogenesis of multiple sclerosis （MS）.Methods Primary rat brain microvascular endothelial cells （rBMECs） and astrocytes from SD rats were isolated,In vitro BBB model was established.Trans-endothelial electrical resistance （TEER） and permeability were determined.Nerve growth factor （NGF）,matrix metalloproteinase （MMP）-2,MMP-9,interleukin （IL）-17,interferon （IFN）-γ,and forkhead box P3 （Foxp3） were measured by fluorescent real-time quantitative polymerase chain reaction （PCR） in 4 models：rBMECs mono-culture,rBMECs and astrocyte co-Culture,rBMECs and lymphocyte co-Culture,and rBMECs,astrocytes and lymphocyte tri-culture.Results rBMECs and astrocytes co-culture had higher TEER values and lower permeability of lymphocytes than rBMEC mono-culture.Compared with rBMEC mono-culture,rBMEC,astrocytes and lymphocytes tri-cultire had significantly decreased level of NGF,IL-17,IFN-γ（P =0.036）,MMP-2,and Foxp3 （P =0.0216）.In comparison with rBMEC and astrocyte co-culture,the tri-culture had further decreased level of NGF,IL-17,and IFN-γ（P =0.0451）.The expression of MMP-2 was further increased in the tri-culture model compared to that of rBMEC and lymphocyte co-culture（P =0.021）.The expression of MMP-9 was not significantly differentamong the 4 models.Conclusion The study successfully established in vitro BBB model with primary rBMECs and astrocyte co-culture.The research speculated that interaction between astrocytes and lymphocytes might increase the expression of MMP-2 and decrease the expression of IL-17 and IFN-γ.