【作者】 杨锋；

【导师】 冯加纯；

【作者基本信息】 吉林大学 ， 临床医学硕士（神经病学）（专业学位）， 2022， 硕士

【摘要】 目的:1、探究远隔缺血后处理的神经保护作用及对Th1、Th2、Th17、Treg细胞占CD4~+T细胞比例的影响。2、探究远隔缺血后处理对各亚群分化相关转录因子表达的影响。方法:1、根据入组及排除标准,筛选自2021年5月至2021年11月就诊于吉林大学第一医院神经内科,且发病时间距开始post-RIC治疗在3天内的急性前循环脑梗死患者共35例,根据患者意愿分为post-RIC组(14例)及常规治疗组(21例)。同期招募与患者组年龄、性别、高血压、糖尿病病史无差异且无脑梗死及炎症相关疾病的15名志愿者作为非梗死组。常规治疗组给予改善循环、营养神经、抗血小板聚集、稳定斑块及对症治疗。post-RIC组在常规治疗组用药基础上,给予post-RIC治疗,即使用缺血预适应仪对患者双上肢进行加压5分钟/再灌注5分钟,每次进行5个循环,每天治疗两次并连续治疗7天。非梗死组仅针对卒中危险因素进行对症处理。post-RIC组在后处理治疗前及治疗3天、7天后的清晨空腹采取外周静脉血,常规治疗组在相同的时间进行采血,采血时间记为0d、3d、7d,并对患者在治疗前及治疗7天后进行NIHSS评分,非梗死组仅采集一次空腹外周静脉血。记录患者一般临床资料,采用流式细胞分析仪及Flowjo 10软件测定三次采血时Th1、Th2、Th17及Treg细胞占CD4~+T细胞比例,用荧光定量PCR法测定三次采血时外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中各亚群分化相关转录因子mRNA表达水平,包括T-box转录因子(T-box transcription factors,T-bet)、GATA结合蛋白3(GATA binding protein-3,GATA3)、RAR相关孤儿受体γ(RAR-related orphan receptor、RORγt)、叉头样转录因子p3(forhead transcription p3,Foxp3)。2、分析脑梗死患者治疗前Th1、Th2、Th17及Treg细胞占CD4~+T细胞比例及Th1/Th2、Th17/Treg比值与NIHSS评分的相关性,并进一步行简单线性相关分析;比较常规治疗组与非梗死组患者各亚群占CD4~+T细胞比例差异。3、比较常规治疗组与post-RIC组NIHSS评分变化程度(ΔNIHSS评分=治疗前NIHSS评分-治疗后NIHSS评分)差异,以及两组在各次采血时Th1、Th2、Th17、Treg细胞占CD4~+T细胞比例和Th1/Th2、Th17/Treg比值差异。4、比较常规治疗组与post-RIC组各次采血时各亚群分化相关转录因子(T-bet、GATA3、RORγt、Foxp3)mRNA表达水平差异。结果:1、在脑梗死发病3天内,患者外周血Th2细胞占CD4~+T细胞比例与NIHSS评分呈负相关(r=-0.481,P=0.005),Th1/Th2比值与NIHSS评分呈正相关(r=0.389,P=0.028),Th1、Th17、Treg细胞占CD4~+T细胞比例及Th17/Treg比值与NIHSS评分未见明显相关性。2、在三次采血中,常规治疗组患者Th2细胞占CD4~+T细胞比例始终低于非梗死组(P=0.001,P=0.006,P=0.008),Th1/Th2比值始终高于非梗死组(P=0.008,P=0.03,P=0.049);在第一次采血时,Treg细胞占CD4~+T细胞比例低于非梗死组(P=0.022),Th17/Treg比值高于非梗死组(P=0.022);Th1、Th17细胞占CD4~+T细胞比例与非梗死组无明显差异。3、post-RIC组患者ΔNIHSS评分高于常规治疗组(P=0.002)。4、post-RIC组患者外周血Th2细胞占CD4~+T细胞比例比例在3d、7d时均高于常规治疗组(P=0.001,P=0.041),Th1/Th2比值均低于常规治疗组(P=0.05,P=0.006)。5、post-RIC组患者PBMC中GATA3 mRNA表达水平在3d、7d时,显著高于常规治疗组(P=0.032,P=0.035)。结论:1、脑梗死患者发病3天内,外周血Th2细胞占CD4~+T细胞比例与患者病情严重程度呈负相关,Th1/Th2比值与病情严重程度呈正相关。2、脑梗死急性期患者外周炎症水平向促炎方向倾斜,可见Th2、Treg细胞占CD4~+T细胞比例降低,Th1/Th2、Th17/Treg比值升高。3、远隔缺血后处理可能通过提高患者外周血Th2细胞占CD4~+T细胞比例、降低Th1/Th2比值来发挥其神经保护作用。4、远隔缺血后处理可能通过促进GATA3 mRNA表达来提高外周血Th2细胞占CD4~+T细胞比例,降低Th1/Th2比值。更多还原

【Abstract】 Objective: 1、To explore the neuroprotective effect of distant ischemic postconditioning and its effect on the proportion of Th1,Th2,Th17 and Treg cells in CD4~+T cells.2、To investigate the effects of distant ischemic post-treatment on the expression of differentiation-related transcription factors in each subgroup.Methods: 1.According to the inclusion and exclusion criteria,a total of 35 patients with acute anterior circulation cerebral infarction who were admitted to the Department of Neurology,The First Hospital of Jilin University from May 2021 to November 2021 and within 3 days of the onset of post-RIC treatment were selected,and they were divided into the post-RIC group(n = 14)and the conventional treatment group(n = 21)according to their will.During the same period,15 volunteers with no difference in age,gender,hypertension,diabetes history and no cerebral infarction or inflammation-related diseases were recruited as the non-infarction group.The conventional treatment group received improvement of circulation,nutritional nerve,anti-platelet aggregation,stabilization of plaque and symptomatic treatment.In addition to conventional treatment group,patients in the post-RIC group were given post-RIC treatment,i.e.,the upper limbs were pressurized for 5min reperfusion for 5min with ischemia adaptation training instrument,5 cycles per time,twice a day for consecutive 7 days.The non-infarct group only received symptomatic treatment for risk factors.In the post-RIC group,peripheral fasting venous blood was taken in the morning before,3and 7 days after post-treatment,while in the conventional treatment group,blood was collected at the same time,and the blood collection time was recorded as 0d,3d and 7d.NIHSS score was performed on admission and 7 days after treatment.In the noninfarction group,peripheral fasting venous blood was taken only once.The general clinical data of the patients were recorded.Flow cytometry and Flowjo10 software were used to determine the proportion of Th1,Th2,Th17 and Treg subsets in CD4~+T cells during the third blood collection.The mRNA expression levels of transcription factors(T-bet GATA3,RORγ T,Foxp3)related to differentiation in peripheral blood mononuclear cells were determined by real-time quantitative PCR.2.The correlation between the proportion of Th1,Th2,Th17 and Treg cells in CD4~+T cells and NIHSS score in patients with acute cerebral infarction before treatment was analyzed,and simple linear correlation analysis was further performed.The proportion of CD4~+T cells in each subgroup was compared between the conventional treatment group and the non-infarction group.3.The difference in NIHSS score between the conventional treatment group and the post-RIC group was compared,as well as the difference in the proportion of Th1,Th2,Th17 and Treg cells in CD4~+T cells in each blood collection between the two groups.4.The mRNA expression levels of differentiation-related transcription factors(TBET,GATA3,RORγ T,Foxp3)in each subgroup were compared between the conventional treatment group and the post-RIC group.Results: 1、Within 3 days of onset of cerebral infarction,the proportion of Th2 subsets in CD4~+T cells in peripheral blood of patients was negatively correlated with NIHSS score(r =-0.481,P=0.005),the ratio of Th1/Th2 was positively correlated with NIHSS score(r=0.389,P=0.028),and There was no significant correlation between Th1,Th17,Treg or Th17/Treg and NIHSS score.2、In triple blood sampling,the proportion of Th2 subsets in CD4~+T cells in the conventional treatment group was always lower than that in the non-infarction group(P=0.001,P=0.006,P=0.008),and the ratio of Th1/Th2 was always higher than that in the non-infarction group(P=0.008,P=0.03,P=0.049).At the time of the first blood collection,the proportion of Treg subsets in CD4~+T cells in the conventional treatment group was lower than that in the non-infarct group(P=0.022),and the ratio of Th17/Treg was higher than that in the non-infarct group(P=0.022).The proportion of Th1 subsets in CD4~+T cells in the conventional treatment group was not significantly different from that of the non-infarct group.3、The ΔNIHSS score in the post-RIC group was higher than that in the conventional treatment group(P=0.002).4、The proportion of Th2 subsets in CD4~+T cells in peripheral blood of postRIC group was higher than that in the conventional treatment group at 3d and 7d(P=0.001,P=0.041),and the ratio of Th1/Th2 was lower than that in the conventional treatment group(P=0.05,P=0.006).5、GATA3 mRNA expression level in PBMC of patients in the post-RIC group was significantly higher than that in the conventional treatment group at 3d and 7d(P=0.032,P=0.035).Conclusion: 1、The proportion of Th2 subsets in CD4~+T cells in peripheral blood of patients with cerebral infarction within 3 days of onset was negatively correlated with the severity of patients’ disease,while Th1/Th2 ratio was positively correlated with the severity of patients’ disease.2、In acute stage of cerebral infarction,the level of peripheral inflammation tended to be pro-inflammatory,and the proportion of Th2 and Treg subsets in CD4~+T cells decreases,while the ratio of Th1/Th2 and Th17/Treg increases.3 、 The neuroprotective effect of distant ischemic posttreatment may be increased by increasing the proportion of Th2 subsets in CD4~+T cells and decreasing the ratio of Th1/Th2 in peripheral blood of patients.4、Remote ischemic post-treatment may increase the proportion of Th2 subsets in CD4~+T cells and decrease the ratio of Th1/Th2 in peripheral blood by promoting GATA3 mRNA expression.更多还原