本文關(guān)鍵詞：NF-κB信號通路在小鼠腦缺血再灌注損傷細(xì)胞凋亡中的作用及機(jī)制　出處：《貴州醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腦缺血再灌注 細(xì)胞凋亡 NF-κB信號通路 Bcl-2 C-myc

【摘要】：目的:初探核因子-κB(nuclear factor-kappa B,NF-κB)信號傳導(dǎo)通路對小鼠腦缺血再灌注損傷(cerebral ischemia reperfusion injury,CIRI)后發(fā)生細(xì)胞凋亡的作用及機(jī)制。方法:通過無創(chuàng)微動(dòng)脈夾夾閉小鼠雙側(cè)頸總動(dòng)脈建立小鼠腦缺血再灌注損傷動(dòng)物模型。根據(jù)缺血后再灌注時(shí)間的不同,將各模型組分為3h、6h、12h、1d、3d、7d、14d、21d,共8組。TTC染色確定腦缺血損傷區(qū)域;HE染色觀察細(xì)胞形態(tài)變化;Nissl染色觀察神經(jīng)元功能改變;免疫組織化學(xué)染色SV法檢測NF-κB p65/RelA表達(dá)變化;TUNEL法檢測腦缺血再灌注過程中不同時(shí)間節(jié)點(diǎn)缺血區(qū)細(xì)胞凋亡數(shù)量;通過原位雜交法和western blotting檢測各時(shí)間節(jié)點(diǎn)損傷區(qū)域NF-κB信號通路的靶基因/蛋白Bcl-2和C-myc mRNA及蛋白表達(dá)的變化情況;運(yùn)用NF-κB抑制劑吡咯烷二硫代氨基甲酸鹽(pyrrolidine dithiocarbamate,PDTC)特異性抑制NF-κB信號通路后,采用TUNEL檢測法和原位雜交檢測建模后1d、7d、14d各組與相對應(yīng)時(shí)間點(diǎn)模型組比較,神經(jīng)細(xì)胞凋亡數(shù)和Bcl-2 mRNA、C-myc mRNA變化情況。結(jié)果:TTC染色:各模型組在海馬所在腦冠狀層面出現(xiàn)不同程度蒼白缺血灶,且以缺血再灌注后7d最為明顯。HE染色:假手術(shù)組海馬CA3區(qū)神經(jīng)細(xì)胞形態(tài)及排列情況與正常組相比無明顯差異;CIRI后各模型組在該區(qū)域可出現(xiàn)神經(jīng)細(xì)胞腫脹(3h)、膠質(zhì)細(xì)胞增生和炎性細(xì)胞增多(6h)、胞內(nèi)空泡(12h)、組織水腫(1d出現(xiàn),7d明顯)、細(xì)胞排列疏松紊亂(14d)、胞核變形、核固縮(21d)等病理形態(tài)改變。Nissl染色:隨病程的進(jìn)展,出現(xiàn)有細(xì)胞腫脹(3h)、尼氏小體數(shù)量減少或消失(12h出現(xiàn)尼氏小體減少,隨后逐漸加重,21d部分神經(jīng)元尼氏小體消失)、組織水腫(1d出現(xiàn),7d嚴(yán)重)、神經(jīng)元變性壞死成空泡狀(21d)等病理變化。免疫組化:正常組及假手術(shù)組低表達(dá)NF-κB p65/RelA;3h出現(xiàn)NF-κB p65/RelA表達(dá)增高,并在隨后的7d內(nèi)均逐漸增高至峰值,且第3d起可觀察到NF-κB p65/RelA移位入核增加;14d及21d時(shí),胞質(zhì)及胞核內(nèi)NF-κB p65/RelA表達(dá)量逐漸下降,但仍高于正常組及假手術(shù)組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。應(yīng)用PDTC后,不同時(shí)間點(diǎn)海馬CA3區(qū)內(nèi)神經(jīng)細(xì)胞胞核內(nèi)的陽性表達(dá)明顯減少,且平均光密度值與同時(shí)間點(diǎn)的模型組相比均明顯下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。TUNEL檢測法、原位雜交及western blotting結(jié)果顯示:各模型組海馬CA3區(qū)凋亡細(xì)胞數(shù)量、NF-κB p65/RelA蛋白表達(dá)量、Bcl-2 mRNA和C-myc mRNA陽性細(xì)胞數(shù)量及二者蛋白表達(dá)量較正常組、假手術(shù)組增加,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。PDTC抑制NF-κB信號通路后,各抑制組與對應(yīng)時(shí)間點(diǎn)模型組相比,Bcl-2 mRNA陽性細(xì)胞數(shù)減少[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陽性細(xì)胞數(shù)[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陽性細(xì)胞數(shù)[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,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:通過夾閉雙側(cè)頸總動(dòng)脈能夠成功復(fù)制小鼠腦缺血再灌注損傷動(dòng)物模型;NF-κB細(xì)胞信號通路參與了小鼠腦缺血再灌注損傷的病理過程;CIRI發(fā)生后的早期(3h)即出現(xiàn)有凋亡細(xì)胞數(shù)的增加,并在其后較長一段時(shí)間(21d)內(nèi)細(xì)胞凋亡過程仍存在著持續(xù)性的進(jìn)展;NF-κB信號通路在CIRI病程進(jìn)展中對神經(jīng)細(xì)胞凋亡起抑制作用,其機(jī)制可能通過Bcl-2誘導(dǎo)和C-myc抑制共同發(fā)揮調(diào)節(jié)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 鎺掑垪鐤忔澗绱婁貢(14d),鑳?yōu)鏍稿彉迮?鏍稿浐緙，

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