【作者】 朱莹；

【导师】 莫新民； 黎杏群；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2007， 博士

【摘要】 研究背景脑出血后留有不同程度的偏瘫、失语等残疾,致使病人生活质量严重下降。神经干细胞(neural stem cells,NSC)的发现给脑血管疾病的治疗带来新的希望,但由于脑出血后内源性神经干细胞发生的数目有限,不足以修复脑出血后神经损伤,因此用外源性神经干细胞移植治疗脑出血有可能取得满意的效果。然而脑出血后血脑屏障破坏,大量血液细胞侵入脑内,其中免疫细胞激活导致细胞因子释放。这种脑内环境的改变既可促进小胶质细胞等细胞表面表达主要组织相容性复合物(MHC)分子,促进抗原递呈及识别,也可诱导神经干细胞MHC分子的表达,脑内原有的“免疫豁免”环境发生了改变,可诱发免疫排斥反应,加重组织损伤。根据免疫反应理论,脑出血血脑屏障损伤情况下,用NSC移植治疗可能发生移植物抗宿主反应(GVHD)。即以TH1为主,TH2、CTL参与的免疫反应。TH1释放许多细胞因子,如IFN-γ、TNF-α等。IFN-γ作用于内皮细胞,选择性诱导黏附分子VCAM-1的表达,白细胞穿越毛细血管壁,游出至脑组织引起炎症反应。血管内皮细胞无论在脑出血的炎症反应或免疫排斥过程中,均起着关键作用。目的本研究旨在通过观察泻火补肾汤对白介素-2(interleukin2,IL-2)和白介素-10(IL-10)在NSC移植后脑出血大鼠脑内及IFN-γ干预大鼠脑微血管内皮细胞(rat cerebral microvascular endothelial cells,RCMECs)中的表达的影响,探讨泻火补肾汤在NSC移植免疫反应中对微血管内皮细胞的保护作用及可能机制。方法1.大鼠随机分为正常组、假手术组、脑出血组、NSC移植组及泻火补肾汤组,Ⅶ型胶原酶注射复制脑出血模型,术后2 d进行NsC移植,第4 d,7 d分两批处死,分别用Western blot及ELISA观察大鼠脑组织及外周血IL-2、IL-10和蛋白表达的变化,用TUNEL观察细胞凋亡情况。2.分离原代鼠脑微血管内皮细胞,随机分为空白组、IFN-γ刺激组、正常血清对照组、泻火补肾汤组,IFN-γ干预4h后分别用正常血清、舍泻火补肾汤血清孵育4h。用RT-PCR及ELISA观察IL-2、IL-10mRNA和蛋白表达水平,用TUNEL观察细胞凋亡情况。结果体内实验1.脑出血进行侧脑室下区神经于细胞移植治疗的最佳治疗时间是出血后第2天。2.在正常鼠脑中,未见TUNEL阳性细胞。脑出血后可见较多TUNEL阳性细胞,主要分布于出血区周围,NSC移植后4天、7天TUNEL阳性细胞表达增加,与脑出血组比较差异显著(P＜0.05);泻火补肾汤干预后阳性细胞较NSC移植组明显减少(P＜0.05),神经干细胞移植后各组7d较4d细胞凋亡减少。3.IL-2的检测:(1)ELISA检测血清中IL-2蛋白表达:4天时,正常组、假手术组血清IL-2有轻度表达,脑出血组、NSC移植组血清IL-2水平较正常组和假手术组明显上升,泻火补肾汤组血清IL-2水平低于脑出血组及NSC移植组:7天时,脑出血组、NSC移植组、泻火补肾汤血清IL-2水平较4d表达减少,NSC移植组IL-2表达水平最高,泻火补肾汤组血清IL-2水平低于脑出血组及NSC移植组。(2)Western bolt检测脑内IL-2蛋白表达:4天时,正常组、假手术组IL-2有轻度表达,脑出血组、NSC移植组、泻火补肾汤组IL-2水平较正常组和假手术组明显上升,NSC移植组表达最强,泻火补肾汤组IL-2表达较NSC移植组减少;7天时各组表达差异比较同4天,各组与4天比较表达减轻,但无统计学差异。4.IL-10的检测:(1)ELISA检测血清中IL-10蛋白表达:4天时,正常组、假手术组血清检测到较高水平的IL-10表达;脑出血组、NSC移植组、泻火补肾汤组大鼠IL-10较正常组、假手术组IL-10表达显著降低,泻火补肾汤组较脑出血组IL-10表达增加;7天时脑出血组、NSC移植组、泻火补肾汤组大鼠IL-10较正常组、假手术组IL-10表达减轻,三组间没有显著性差异。(2)Western bolt检测脑内IL-10蛋白表达情况:4天时,正常组、假手术组IL-10有轻度表达,脑出血组、NSC移植组、泻火补肾汤组IL-10水平较正常组和假手术组明显上升,NSC移植组表达最弱,泻火补肾汤组IL-10表达NSC移植组明显增加;7天时脑出血组与NSC移植组、泻火补肾汤组比较已没有统计学差异。而泻火补肾汤组IL-10表达仍明显强于NSC移植组。体外实验:1.IFN-γ刺激脑微血管内皮细胞模拟免疫损伤模型,确定IFN-γ刺激浓度最佳为40ng/ml,5%泻火补肾汤血清作用4h作为最佳的干预条件。2.IL-2的检测:(1)ELISA检测细胞上清中IL-2蛋白表达:IFN-γ刺激后,IFN-γ组、正常血清组、泻火补肾汤组细胞上清IL-2较空白对照组表达增加,泻火补肾汤干预后与IFN-γ组、正常血清组比较IL-2表达减少。(2)RT-PCR检测结果显示:IL-2:空白组大鼠脑微血管内皮细IL-2mRNA表达;IFN-γ干预后,IL-2mRNA表达显著增加;正常血清组较IFN-γ刺激组显著减少;泻火补肾汤血清孵育后IL-2mRNA表达较正常血清组明显降低。3.IL-10的检测:(1)ELISA检测细胞上清中IL-10蛋白表达:IFN-γ刺激后,IFN-γ刺激组、正常血清组、泻火补肾汤组细胞上清IL-10较空白对照组表达减少,泻火补肾汤干预后能上调IL-10的表达,与IFN-γ刺激组、正常血清组比较IL-2表达增加。(2)RT-PCR空白组大鼠脑微血管内皮细胞有IL-10mRNA表达;IFN-γ干预后,IL-10mRNA表达降低;正常血清组较IFN-γ刺激组表达增加;泻火补肾汤组IL-10mRNA表达较正常血清组明显增加。4.TUNEL检测:NF-γ干预后脑微血管内皮细胞可见大量TUNEL阳性细胞,泻火补肾汤组与正常血清组、IFN-γ干预组比较TUNEL细胞阳性细胞数显著减少。结论1.出血大鼠神经干细胞移植后IL-2表达上调,IL-10表达下调,细胞凋亡增加。泻火补肾汤能下调移植后IL-2的表达,上调IL-10的表达,减少细胞凋亡。2.免疫排斥反应用IFN-γ刺激脑微血管内皮细胞进行模拟Th1免疫损伤模型,IFN-γ干预后脑微血管肉皮细胞的IL-2表达增加、IL-10表达减弱,泻火补肾汤血清能减弱IL-2的表达、增加IL-10的表达,减少脑微血管内皮细胞的凋亡,增加细胞活力。泻火补肾汤对脑出血神经干细胞移植后的免疫排异反应有耐受效果,其机制可能是通过下调IL-2和上调IL-10的表达来实现的。更多还原

【Abstract】 Objective:The study aimed to observe the effects of Xiehuo Bushen decoction on the expression of Interleukin-2,Interleukin-10 in rats brain of neural stern cells(NSCs)-transplanted experimental intracerebral hemorrhage and cultured interferon-gamma(IFN-γ)-induced cerebral microvascular endothe-lial cells(RCMECs),to explore the mechanism of Xiehuo Bushen Decoction(BSXH) in protecting RCMECs in NSCs-transplanted intracerebral hemorrhagic rat brains.Methods:1.Ninety rats were randomly divided into five groups including normal group(n=10),sham-operation group(n=20),model group(n=20),NSC-transplanted group(n=20) and BSXH-treated group(n=20).The intra-cerebral hemorrhage model was induced by injecting 0.4UⅦcollagenase into right globus pallidus(1.4mm posterior, 3.2mm lateral to bregma,and 5.6-mm depth from the cortical surface) with stereotaxic apparatus.BrdU-labled NSCs were transplanted to subventricle zone(SVZ) 2 days later.We observ-ed the expression of IL-2 and IL-10 by enzyme-linked immunosorbent assay(ELISA)、western bolt and reverse transcriptase polymerase chain reaction (RT-PCR),as well as the apoptosis by TdT-mediated dUTP-biotin nick end abeling(TUNEL). 2.The cultured RCMECs were randomly divided into control group, IFN-γgroup,serum control group and XHBS serum group.The expression of IL-2 and IL-10,and apoptosis was assayed by the same technique as those in vivo respectively.Results:1.IL-2,IL-10 mRNA expressed remarkably in cultured RCMECs after induced with IFN-γ(P＜0.01 vs control group).IL-2 expression were decreased and IL-10 expression were increased after treated with BSXH (P＜0.05 vs IFN-γ,group),The cell viability of XHBS serum group were higher than that of IFN-γgroup by MTT(P＜0.05).2.The number of TUNEL positive cells which was very small in control group increased markedly in IFN-γgroup(P＜0.01 vs control group) and decreased in XH BSserum group(P＜0.05 vs serum control group).3.The expression of IL-2 mRNA and protein in RCMECs were increased remarkably after induced by IFN-γ(P＜0.05 vs control group) and decreased in BSXH serum group(P＜0.01 vs serum control group).The expression of IL-10 were gradually up-regulated in IFN-γgroup,serum control group and BSXH serum group,and there is significant difference between IFN-γgroup and BSXH serum group(P＜0.05).4.IL-2 expressed at low level in normal group and sham operation group, it was up-regulated remarkably in model group and NSC transplanted group.The expression of IL-2 was down-regulated significantly in XHBS-treated group(P＜0.05 vs NSCs-transplanted group).IL-10 mRNA also can be seen mildly in normal group and sham operation group (P＜0.05).It was higher in model group than in sham operation group (P＜0.05).The expression was up-regulated markedly in BSXH-treated group(P＜0.05 vs NSC-transplanted group).5.There was no TUNEL positive cells in the rat brains of normal group and some in sham operation group.The number of TUNEL positive cells increased significantly in model group and NSC transplanted group,and there was no difference between the two groups.The number of apoptosis cells decrea-sed markedly in XHBS-treated group(P＜0.05 vs NSC-transplanted group).6.IL-2 expressed at low level in the rat brains of normal group and sham operation group.The expression of IL-2 mRNA and protein in model group and transplanted group increased at 4d(P＜0.01 vs control group and sham operation group).It expressed mainly in neurons and endothelial cells around hematoma.it was down-regulated remarkably in XHBS-treated group(P＜0.05 vs NSC-transplanted group).There was little IL-10 mRNA expressed in normal group and sham operation group.The trend of IL-10 expression incr-eased gradually in model group, transplanted group and XHBS-treated group.The expression of IL-10 from 4d to 7d is ascendant.Conclusions:1.The expression of IL-2 and IL-10 were increased in brains of NSCstransplanted intracerebral hemorrhage rat.Xiehuo Bushen Decoction could depress IFNlammation by up-regulating the expression of IL-10and down-regulating the expression of IL-2.2.IL-10、IL-2 play a important role in brains of NSC-transplanted intrace-rebral hemorrhage rat.Xiehuo Bushen Decoction could inhibit ndothelial apoptosis by depressing the expression of IL-2 and promoting the expression of IL-10.3.Restraining the activation of IL-2 and promoting the expression of IL-10 might be the mechanism of XHBS on lessening IFNlammation to protect RCMECs.更多还原