体外模拟SAH状况下IL-6对大鼠蛛网膜细胞极化状态的影响

Effects of IL-6 on polarization of rat arachnoid cells in mimic subarachnoid hemorrhagic condition in vitro

目的初步研究蛛网膜下腔出血(SAH)状况下IL-6对大鼠蛛网膜细胞极化状态的影响。方法细胞免疫化学法(ICC)鉴定培养的大鼠蛛网膜细胞对CK8+18、Vimentin和CD68的表达;采用体外培养的大鼠蛛网膜细胞,经极化诱导剂粒细胞巨噬细胞集落刺激因子(GM-CSF)和巨噬细胞集落刺激因子(M-CSF)分别刺激后,采用ELISA法检测极化相关因子IL-12、IL-6和IL-10的变化,观察其是否发生极化;然后用凝血酶作为激活剂活化蛛网膜细胞在体外模拟SAH状况,再采用IL-6刺激活化的蛛网膜细胞和正常(非活化)蛛网膜细胞,实时荧光定量PCR(qRT-PCR)法观察CD68、CK8+18、IL-12和IL-23、精氨酸酶-I(Arg-I)的RNA(相对)表达水平。结果 (1)培养细胞的蛛网膜细胞标志CK18+18和Vimentin、巨噬细胞标志CD68表达阳性。(2)GM-CSF可诱导蛛网膜细胞IL-6和IL-12p40表达增高,而M-CSF未能诱导IL-10表达变化。(3)凝血酶可诱导蛛网膜细胞发生高Arg-I的M2极化。(4)IL-6刺激剂量大则CD68表达早持续时间短的时间模式。(5)IL-6刺激剂量与蛛网膜细胞极化的时间模式明显不同,实验组1、3、7、14d时IL-23表达均增高,对照组IL-23、IL-12p40和Arg-I在刺激14d才增高。结论培养的蛛网膜细胞具有单核巨噬细胞的特性;蛛网膜细胞仅能被极化诱导剂诱导M1极化,未发生M2极化,与外周明显不同;在模拟SAH状况下蛛网膜细胞呈持续的M1型极化,低IL-10的M2型极化状态,显示其极化能力不良,这将不利于蛛网膜下腔中溶血产物的清除和相关SAH后并发症的发生、发展;SAH状况下蛛网膜细胞经IL-6刺激后发生M1/M2极化,其时间和表现模式多样,为IL-6作为"预测SAH预后的标志因子"提供了新证据。

Objective To investigate the effect of interleukin-6（IL-6）on the polarization state of rat arachnoid（Ar）cells in mimic subarachnoid hemorrhage（SAH）condition in vitro.MethodsThe mimic condition in vitro was induced by activation of thrombin on cultured rat arachnoid cells.Expression of CK8＋18,Vimentin and CD68 in rat arachnoid cells was detected by immunocytochemistry（ICC）.The changes of IL-12,IL-6 and IL-10 expression were detected by ELISA method after Ar cells were stimulated with GM-CSF and M-CSF.The mR NA expression of CD68,CK8＋18,IL-12,IL-23 and Arg-I in stimulated Ar cells and non-activated Ar cells was detected by real-time fluorescence quantitative RT-PCR（qRT-PCR）.Results The expressions of Ck8＋18,Vimentin and CD68 in Ar cells and CD68 expression in macrophages were positive.The expression of IL-6 and IL-12p40 increased after GM-CSF stimulation,while the expression of IL-10 was not changed after M-CSF stimulation in Ar cells.Thrombin induced high Arg-I M2 polarization in Ar cells.When stimulated by the large dose IL-6,the CD68 was expressed early and shortly.The expression of IL-23 was increased in the SAH group at the 1d,3d,7d and 14d,while the expression of IL-23,IL-12p40 and Arg-I in the control group only increased 14d after stimulation.Conclusion The cultured Ar cells have the characteristics of Macrophages.Ar cells only can be induced to M1 polarization,not to M2 polarization under mimic SAH condition,the poor polarizability may be unfavorable for the elimination of hemolytic products in subarachnoid space resulting in SAH related complications.After IL-6 stimulation Ar cells show M1/M2 polarization,which provides new evidence for IL-6 as a prognostic factor of SAH.