内源性调节性T细胞在SAH后脑血管痉挛及脑损伤中的作用

1、 内源性调节性t细胞在sah后脑血管痉挛及脑损伤中的作用汇报人：张金卉汇报人：张金卉 导师：孙保亮导师：孙保亮研究的目的及意义研究的目的及意义sah后脑血管痉挛、炎症反应及后脑血管痉挛、炎症反应及神经功能预后的改善是一个非常复杂的病理 、生理、临床过程。虽然进行了大量研究，为止其 发生机制仍未完全明了。研究小鼠体内内源性调节性小鼠体内内源性调节性t细细对对sah后脑血管痉挛、后脑血管痉挛、炎症反应及症反应及神经功能预后的改善仍将是今后一个时期的热点。随着研究的逐渐深入，将会对内源性调节性t细胞移植sah的临床治疗及神经功能预后带来深远的影响。国内外相关文献国内外相关文献 the kinetic

2、s of lymphocyte subsets andmacrophages in subarachnoid space after subarachnoid hemorrhage in rats it has been suggested that humoral immunity plays a role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage, there has been no quantitative assay for cellular immunity. we studied

3、the kinetics of immune cells in the subarachnoid space after subarachnoid hemorrhage in the rat. a serial response of immunoreactive cells, which resembles that of the chronic allergic reaction observed in autoimmune diseases or delayed-type hypersensitivity, exists in the subarachnoid space after s

4、ubarachnoid hemorrhage. the present results suggest that the initial response in cellular immunity, which is followed by humoral immunity and eicosanoid reactions, plays a role in eliciting the development of cerebral vasospasm.蛛网膜下腔出血后脑血管痉挛蛛网膜下腔出血后脑血管痉挛分子机制的研究进展分子机制的研究进展与no代谢有关的cvsk 通道活性的改变文氧合血红蛋白的

5、作用本磷酸二酯酶一v的作用本no和et一1的平衡失调与与no代谢无关的代谢无关的cvsras蛋白的作用前列环素与血栓烷a2失衡自由基与脂质过氧化物增多血管加压素的作用ca 代谢紊乱statin-induced t-lymphocyte modulation and neuroprotection following experimental subarachnoid hemorrhage statins in fl uence immune system activities through echanisms independent of their lipidlowering proper

6、ties. t cells can be subdivided based on cytokine secretion patterns into two subsets: t-helper cells type 1 (th1) and type 2 (th2). independent laboratory studies have shown statins to be potent inducers of a th2 switch in immune cell response and be neuroprotective in severalmodels of central nerv

7、ous system (cns) disease. this study was the fi rst to evaluate the immune modulating effects of statins in subarachnoid hemorrhage (sah). the present study elucidated the potential role of a th2 immune switch in statin provided neuroprotection following sah.matrix metalloproteinase-9 concentration

8、in the cerebral extracellular fluid of patients during the acute phase of aneurysmal subarachnoid hemorrhage elevated cerebral interstitial pro-mmp-9 relates toearly brain injury in asah patients. a larger prospectivestudy should be performed to confirm whether patients with prolonged elevation or a

9、 second peak of cerebral pro-mmp-9 would be more likely to develop dci and to confirm whether mmp-9, a mediator of neurovascular injury, would be worth to consider as a predictor of vasospasm.trehalose treatment suppresses inflammation,oxidative stress, and vasospasm induced by experimental subarach

10、noid hemorrhage subarachnoid hemorrhage (sah) frequently results in several complications, including cerebral vasospasm, associated with high mortality. although cerebral vasospasm is a major cause of brain damages after sah, other factors such as inflammatory responses and oxidative stress also con

11、tribute to high mortality after sah.trehalose is a non-reducing disaccharide in which two glucose units are linked by ,-1,1-glycosidic bond, and has been shown to induce tolerance to a variety of stressors in numerous organisms. in the present study, we investigated the effect of trehalose on cerebr

12、al vasospasm, inflammatory responses, and oxidative stress induced by blood in vitro and in vivo. trehalose has suppressive effects on several pathological vents after sah,including vasospasm, inflammatory responses, and lipid peroxidation. trehalose may be a new therapeutic approach for treatment o

13、f complications after sah.properties within the cerebrospinal fluid after subarachnoid hemorrhage in vivo and in vitro to functionally characterize pro-inflammatory and vasoconstrictive properties of cerebrospinal fluid after aneurysmal subarachnoid hemorrhage (sah) in vivo and in vitro. we function

14、ally characterized inflammatory and vasoactive properties of patients csf after sah in vivo and in vitro. this pro-inflammatory milieu in the subarachnoid space might play a pivotal role in the pathophysiology of early and delayed brain injury as well as vasospasm development following sah.研究内容研究内容小

15、鼠体内内源性调节性t细胞对蛛网膜下腔出血（sah）后脑血管痉挛的影响小鼠体内内源性调节性t细胞移植sah后神经功能预后的改善作用小鼠体内内源性调节性小鼠体内内源性调节性t细胞移植对细胞移植对sah后炎症反应后炎症反应的影响的影响 每项研究内容均分4组1、tregs删除组2、sah模型组3、假手术组4、pbs腹腔注射组体重、窝别、喂养方法相同的小鼠随机分组每组1015只小鼠；每只小鼠编号后按计算机随机表分4组，双盲（研究者、负责资料收集和分析的人员也不了解分组情况）较好地避免了偏倚。 研究方法研究方法实验实验动物处理动物处理1、tregs删除删除腹腔注射0.25mg cd25特异性抗体小鼠加入肝

16、素抗凝hanks 液洗涤、els裂解注射后第2天取外周血加入标记的抗细胞表面特异性抗原的抗体pbs 洗两遍4 下避光反应20-30分钟，洗涤2 次加入标记的抗细胞表面特异性抗原的抗体facs calibur 检测cd4+cd25+t细胞300l染色缓冲液重悬细胞cd4+cd25+t细胞几乎减少至0%为调节性t细胞删除成功cell quest 收集细胞并分析流式结果2、sah模型制作模型制作感觉有阻力时再继续前进3mm, 总进入长度约为10mm(已刺破动脉壁）然后迅速将线栓拔出，动脉血灌入，造成蛛网膜下腔出血将制备的线栓通过切口经颈动脉插入颈内动脉，一直送至大脑前动脉与大脑中动脉的分叉处（类似大

17、脑中动脉阻断的方法）首先制作线栓（5-0单丝尼龙线，头端磨成子弹头状，总长度15mm，直径0.2mm）3、假手术：假手术：遇到遇到 阻力即停止继续插入，并迅速退出线栓阻力即停止继续插入，并迅速退出线栓4、pbs腹腔注射腹腔注射：经腹腔注射与：经腹腔注射与0.25mg cd25特异性抗体等体积的特异性抗体等体积的pbs。 一、一、内源性调节性内源性调节性t细胞对蛛网膜下腔出血细胞对蛛网膜下腔出血（sah）后脑血管痉挛的）后脑血管痉挛的检测内容检测内容text基底动脉形态学检测脑动脉匀浆液中一氧化氮合酶mrna和蛋白含量；no含量；cgmp含量局部脑血流量局部脑血流量循环内皮细胞检测循环内皮细胞检

18、测基底动脉形态学检测脑动脉匀浆液中一氧化氮合酶mrna和蛋白含量；no含量；cgmp含量局部脑血流量局部脑血流量循环内皮细胞检测循环内皮细胞检测检测内容局部脑血流量（局部脑血流量（regional cerebral blood flow, rcbf）检测：）检测：颅骨开窗，在立体定向仪控制下，在颅骨开窗，在立体定向仪控制下，在sah前及前及sah后后12小时内（或小时内（或6小时）用激光多普勒血流计动态测量动物顶叶皮层小时）用激光多普勒血流计动态测量动物顶叶皮层rcbf。基底动脉形态学检测：基底动脉形态学检测：1、立体显微镜下观察基立体显微镜下观察基底动脉（底动脉（ba）、大脑中）、大脑中动脉

19、（动脉（mca）和大脑前）和大脑前动脉（动脉（aca）并记录它）并记录它们的直径们的直径2、冰冻切片上显微镜下冰冻切片上显微镜下观察基底动脉形态学变化，观察基底动脉形态学变化，用图像分析仪测定基底动用图像分析仪测定基底动脉内径、外径与管腔横截脉内径、外径与管腔横截面积面积改用改用10明胶和墨水混明胶和墨水混合液灌注合液灌注13.5 mlmin)15s于于sah后后2天、天、2周，经周，经心脏灌注，心脏灌注，10福尔马福尔马林灌注（林灌注（5.5 mlmin）min 后后循环内皮细胞检测：循环内皮细胞检测：于于sahsah后后3天、天、2周周取静脉血标本，用取静脉血标本，用因子相关抗原免疫荧光染

20、色因子相关抗原免疫荧光染色检测循环内皮细胞（检测循环内皮细胞（circulating endothelial cell, cec），以反映），以反映血管内皮细胞的损伤。血管内皮细胞的损伤。于于sahsah后后3天、天、2周，周，取动物脑底取动物脑底willis动脉环及其相连的动脉制备匀动脉环及其相连的动脉制备匀浆液浆液 pcr、western blot等等检测脑检测脑动脉匀浆液中一氧化氮合酶动脉匀浆液中一氧化氮合酶（nitric oxide synthase, nos, nos）mrna和蛋白含量和蛋白含量 硝酸酶还原法测定硝酸酶还原法测定nono含量含量 放射免疫分析法测定放射免疫分析法测定

21、cgmp含量含量二、二、内源性调节性内源性调节性t细胞移植细胞移植sah后神经功能后神经功能预后的改善作用预后的改善作用运动评分共运动评分共12分分感觉评分（感觉评分（515分）分）认知功能障碍评估认知功能障碍评估振动觉（振动觉（13分）分）触觉（触觉（13分）分）视觉（视觉（13分）分） 对刺激的触激反应（对刺激的触激反应（13分）分）嗅觉（嗅觉（13分）分）感觉评分感觉评分运动评分运动评分 平衡和平衡和协调协调能力（能力（3 3分）分） 随意活 动（3分） 运动评分攀爬能力（攀爬能力（3分）分） 肢体活动对称性（3分） 认知功能障碍评估认知功能障碍评估在2次测试中的后一次测试中，将平台移去

22、，让动物游泳30秒，计算动物到达目标象限（原来放置平台的位置）所花费的时间和游泳速度。在术后3天和2周，对大鼠每天测试4次术后第2天在无平台情况下将大鼠放于morris水迷宫进行驯化二次数据表示为动物找到沉水的平台所需要的时间（潜伏期）如树突状细胞、小胶质细胞及嗜中性粒细胞（gr1抗体染中性粒细胞），染中心粒细胞时用gfap（胶质细胞）、cd31（血管内皮细胞）mmp-9+血管长度（mmp-9与cd31双标记）检测toll样受体tlr2和tlr4在基因和蛋白水平的表达检测其下游信号分子如trak1、traf6、nf-b、tnf-、il-1、il-6、mcp-1等在蛋白水平的表达中性粒细胞计数、淋巴细胞等计数血液标本检测有关免疫和炎