神经源性丝氨酸蛋白酶抑制剂在大鼠小胶质细胞缺氧/复氧模型上的作用

 目的 探讨神经源性丝氨酸蛋白酶抑制剂(neuroserpin,NSP)是否可以改变组织型纤溶酶原激活物(tissue plasminogen activator,tPA)对于小胶质细胞(microglia,MG)活化的影响。 方法 体外原代培养的大鼠皮层MG并鉴定。建立缺氧 复氧模型并鉴定。首先,使用ELISA试剂盒测定MG在缺氧/复氧(hypoxia/reoxygenation,H/R)状态下自身释放tPA的含量,细胞被分为正常组、PBS对照组和OGD3h复氧不同时间组(1、3、5、7、24 h)。其次,研究NSP在MG缺氧/复氧时的作用,本部分实验分3组,分别为缺氧/复氧干预组、tPA干预组及tPA+NSP干预组。以上各组均设3个复孔,实验重复3次。采用倒置相差显微镜直接观察盖玻片上细胞形态学变化,CCK 8法检测各组间细胞增殖率,ELISA试剂盒测定IL 1β和NO含量。结果 MG在H/R时合成tPA,OGD3h复氧3 h时释放量最多。H/R时MG激活,炎性因子IL 1β和NO的释放增多。tPA干预MG后,细胞活化并增殖、IL 1β和NO的释放增多。各NSP预处理组细胞活化、增殖和炎性因子的释放增多均没有tPA或H/R干预组明显。结论 MG在H/R时合成tPA。H/R早期即诱发了MG的活化。tPA可以进一步诱发MG的增殖和活化。H/R和tPA的干预均引起IL 1β和NO的释放。NSP可减轻H/R和tPA干预引起的MG活化和增殖,减少MG中IL 1β和NO的释放。

Effects of neuroserpin on the microglias undergoing hypoxia/reoxygenation in rats

Objective To study the effects of tissue plasminogen activator (tPA) on microglias in different cultured conditions, and then to investigate whether neuroserpin (NSP) could decrease these effects.Methods We used primary cultured cortical microglias (MG) of rats and built the oxygen deprivation reoxygenation model. Firstly, we tested the tPA protein levels released by MGs undergoing hypoxia/reoxygenation (H/R) by the way of using ELISA kit. The cells were randomly devided into normal group, PBS group and OGD3h rexogenation group. Secondly, we studied the effects of NSP on MGs undergoing H/R. The cells were randomly devided into OGD3h reoxgenation group, tPA intervention goup and tPA plus NSP intervention group. Each group had 3 readings once, and the experiment was performed 3 times. Then we observed the changes of morphology by inverted microscopy, the proliferation rate by CCK 8 and the inflammatory cytokines IL 1β and NO in culture supernatants by ELISA kit.Results MGs could release tPA undergoing hypoxia/reoxygenation. TPA protein expression reached peak at the time point of 3 hours after reoxygenation. MGs could be activated. IL 1β and NO could be released undergoing the intervention of tPA or hypoxia/reoxygenation. NO but not IL 1β had a time dependent increase after oxygen deprivation in cultured MGs. The intervention of NSP could impact these effects caused by tPA or hypoxia/reoxygenation.Conclusions MGs could release tPA undergoing hypoxia/reoxygenation. TPA could induce the proliferation and activation of the MGs. H/R and tPA could cause MGs release IL 1β and NO. NSP could reduce the effects of tPA or H/R on the microglias. It could decrease the liberation of IL 1β and NO released by MGs.