NF-κB信号通路在小鼠脑缺血再灌注损伤细胞凋亡中的作用及机制

发布时间：2018-01-12 01:20

  本文关键词：NF-κB信号通路在小鼠脑缺血再灌注损伤细胞凋亡中的作用及机制　出处：《贵州医科大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 脑缺血再灌注 细胞凋亡 NF-κB信号通路 Bcl-2 C-myc

【摘要】：目的:初探核因子-κB(nuclear factor-kappa B,NF-κB)信号传导通路对小鼠脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)后发生细胞凋亡的作用及机制。方法:通过无创微动脉夹夹闭小鼠双侧颈总动脉建立小鼠脑缺血再灌注损伤动物模型。根据缺血后再灌注时间的不同,将各模型组分为3h、6h、12h、1d、3d、7d、14d、21d,共8组。TTC染色确定脑缺血损伤区域;HE染色观察细胞形态变化;Nissl染色观察神经元功能改变;免疫组织化学染色SV法检测NF-κB p65/RelA表达变化;TUNEL法检测脑缺血再灌注过程中不同时间节点缺血区细胞凋亡数量;通过原位杂交法和western blotting检测各时间节点损伤区域NF-κB信号通路的靶基因/蛋白Bcl-2和C-myc mRNA及蛋白表达的变化情况;运用NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate,PDTC)特异性抑制NF-κB信号通路后,采用TUNEL检测法和原位杂交检测建模后1d、7d、14d各组与相对应时间点模型组比较,神经细胞凋亡数和Bcl-2 mRNA、C-myc mRNA变化情况。结果:TTC染色:各模型组在海马所在脑冠状层面出现不同程度苍白缺血灶,且以缺血再灌注后7d最为明显。HE染色:假手术组海马CA3区神经细胞形态及排列情况与正常组相比无明显差异;CIRI后各模型组在该区域可出现神经细胞肿胀(3h)、胶质细胞增生和炎性细胞增多(6h)、胞内空泡(12h)、组织水肿(1d出现,7d明显)、细胞排列疏松紊乱(14d)、胞核变形、核固缩(21d)等病理形态改变。Nissl染色:随病程的进展,出现有细胞肿胀(3h)、尼氏小体数量减少或消失(12h出现尼氏小体减少,随后逐渐加重,21d部分神经元尼氏小体消失)、组织水肿(1d出现,7d严重)、神经元变性坏死成空泡状(21d)等病理变化。免疫组化:正常组及假手术组低表达NF-κB p65/RelA;3h出现NF-κB p65/RelA表达增高,并在随后的7d内均逐渐增高至峰值,且第3d起可观察到NF-κB p65/RelA移位入核增加;14d及21d时,胞质及胞核内NF-κB p65/RelA表达量逐渐下降,但仍高于正常组及假手术组,差异具有统计学意义(P0.05)。应用PDTC后,不同时间点海马CA3区内神经细胞胞核内的阳性表达明显减少,且平均光密度值与同时间点的模型组相比均明显下降,差异具有统计学意义(P0.05)。TUNEL检测法、原位杂交及western blotting结果显示:各模型组海马CA3区凋亡细胞数量、NF-κB p65/RelA蛋白表达量、Bcl-2 mRNA和C-myc mRNA阳性细胞数量及二者蛋白表达量较正常组、假手术组增加,差异具有统计学意义(P0.05)。PDTC抑制NF-κB信号通路后,各抑制组与对应时间点模型组相比,Bcl-2 mRNA阳性细胞数减少[1d:(43.10±3.712)(76.05±2.964);7d:(64.05±4.807)(79.90±3.508);14d:(42.00±3.309)(70.00±4.496)],TUNEL阳性细胞数[1d:(46.20±3.205)(29.90±2.292);7d:(103.95±3.348)(65.50±3.411);14d:(116.10±3.093)(45.55±1.959)]和C-myc mRNA阳性细胞数[1d:(109.00±7.609)(101.40±8.287);7d:(126.45±9.572)(109.25±6.206);14d:(98.00±5.058)(89.20±5.836)]增加,差异具有统计学意义(P0.05)。结论:通过夹闭双侧颈总动脉能够成功复制小鼠脑缺血再灌注损伤动物模型;NF-κB细胞信号通路参与了小鼠脑缺血再灌注损伤的病理过程;CIRI发生后的早期(3h)即出现有凋亡细胞数的增加,并在其后较长一段时间(21d)内细胞凋亡过程仍存在着持续性的进展;NF-κB信号通路在CIRI病程进展中对神经细胞凋亡起抑制作用,其机制可能通过Bcl-2诱导和C-myc抑制共同发挥调节作用。
[Abstract]:Objective: To study on nuclear factor kappa B (nuclear factor-kappa B, NF- K B) signal transduction pathway on cerebral ischemia-reperfusion injury in mice (cerebral ischemia reperfusion injury, CIRI) after effect and mechanism of apoptosis. Methods: by non-invasive micro artery clipping bilateral common carotid artery of mice cerebral ischemia in mice reperfusion injury animal model. According to the reperfusion time, the model group was divided into 3h, 6h, 12h, 1D, 3D, 7d, 14d, 21d, a total of 8 groups were determined by.TTC staining of regional cerebral ischemia injury; morphological changes were observed by HE staining; observe the neuronal function change Nissl staining; immunohistochemistry NF- staining was used to detect p65/RelA expression of kappa B SV method; TUNEL method detect cerebral ischemia and the number of cells at different time nodes in the ischemic area of apoptosis; by in situ hybridization and Western blotting to detect the time node damage area NF- B pathway The change of target gene / protein expression of Bcl-2 and C-myc mRNA and protein; using NF- kappa B inhibitor pyrrolidine dithiocarbamate (pyrrolidine two dithiocarbamate, PDTC) specific inhibition of NF- B signaling pathway was detected by TUNEL method and in situ hybridization detection after 1D, 7d, 14d groups and the corresponding time model group, the number of neuronal apoptosis and Bcl-2 mRNA, C-myc mRNA changes. Results: TTC staining: the model group in hippocampus of brain coronal sections with varying degrees of pale and focal ischemia, 7d after ischemia reperfusion is the most obvious.HE staining: in sham group CA3 area of hippocampus nerve cell morphology and arrangement compared with the normal group had no significant difference; after CIRI of each model group in the region there may be swelling of nerve cells (3H), the proliferation of glial cells and inflammatory cells increased (6h), intracellular vacuoles (12h), tissue edema (1D, 7D), cell 鎺掑垪鐤忔澗绱婁贡(14d),鑳炴牳鍙樺舰,鏍稿浐缂，

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