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目的:通过检测小鼠短暂性脑缺血(tMCAO)后不同时间点CD4+CD25+调节性T细胞(Treg)比例及血清TGF-β1浓度变化,探讨其免疫调节功能在缺血性脑卒中炎性损伤中作用。方法:60只昆明小鼠随机分为6组,假手术组(sham组,24 h)10只,缺血再灌注后12 h、24 h、48 h、72 h及5 d五个tMCAO组(n=10/组)。采用腔内线栓法建立小鼠tM-CAO模型;tMCAO组在再灌注后各时间点进行神经功能缺陷评分(NDS),其后处死小鼠行氯化三苯基四氮唑(TTC)染色观察脑梗死容积变化;同期免疫荧光染色观察脾脏中Foxp3表达;提取脾脏单个核细胞,流式细胞术(FCM)测定Treg在单个核细胞中比例;分离血清,ELISA检测血清中TGF-β1浓度。结果:缺血再灌注12 h脑部炎性损伤明显,且逐渐加重,TTC染色示炎症反应中心脑梗死面积同期增大,缺血再灌注48 h达高峰,此后前述表现逐渐减小;随再灌注时间延长,神经功能也逐渐改善。Foxp3在各组小鼠脾脏中均有阳性表达,但存在明显差异;FCM分析发现,缺血再灌注24 h时Treg比例较sham组明显减少(P<0.05),但在72 h时Treg基本恢复正常,5 d时则明显高于sham组(P<0.05)。血清中TGF-β1浓度与Treg有相似表现,再灌注24 h时降低明显,48 h则恢复至sham组水平,而在5 d时高出sham组(P<0.05)。并且Treg与TGF-β1呈正相关。结论:在缺血性脑损伤急性期Treg与TGF-β1明显减少,而在恢复期二者大量增加,且与脑梗死容积变化紧密相关,说明缺血性脑损伤后Treg与TGF-β1功能失衡在急性缺血性脑卒中炎性损伤中发挥着重要作用。
OBJECTIVE: To investigate the changes of CD4 + CD25 + regulatory T cells (Treg) and serum TGF-β1 concentrations at different time points after transient focal cerebral ischemia (tMCAO) in mice and explore the role of immune regulation in ischemic stroke Injury role. Methods: Sixty Kunming mice were randomly divided into six groups: sham operation group (sham group, 24 h), ischemia / reperfusion 12 h, 24 h, 48 h, 72 h and 5 d n = 10 / group). The rat model of tM-CAO was established by intraluminal suture method. The neurological deficit score (NDS) was observed in tMCAO group at each time point after reperfusion, and then the mice were sacrificed and stained with triphenyltetrazolium chloride (TTC) The changes of volume of cerebral infarction were observed. Foxp3 expression in spleen was observed by immunofluorescence staining at the same time. Spleen mononuclear cells were extracted. The proportion of Treg in mononuclear cells was determined by flow cytometry (FCM). Serum levels of TGF-β1 were detected by ELISA. Results: Cerebral inflammatory injury was obvious and gradually increased 12 h after ischemia / reperfusion. TTC staining showed that infarct size in inflammatory reaction center increased at the same period and peaked at 48 h after ischemia-reperfusion, Prolonged infusion time, neurological function also gradually improved. The expression of Foxp3 in the spleens of all the mice was positive, but there was a significant difference. FCM analysis showed that the percentage of Tregs was significantly decreased at 24 h after ischemia / reperfusion (P <0.05), but Treg recovered at 72 h Normal, 5 d was significantly higher than sham group (P <0.05). The concentration of TGF-β1 in serum was similar to that of Treg. The levels of TGF-β1 in serum decreased significantly at 24 h and returned to sham level at 48 h, and sham group at 5 d (P <0.05). And Treg and TGF-β1 was positively correlated. CONCLUSION: Treg and TGF-β1 are significantly decreased during acute phase of ischemic brain injury, while both are significantly increased in recovery phase and closely related to changes in volume of cerebral infarction. This indicates that imbalance of Treg and TGF-β1 after ischemic brain injury Plays an important role in the inflammatory injury of acute ischemic stroke.