發(fā)布時(shí)間：2018-08-22 19:02

【摘要】：目的旨在檢測(cè)選擇性線粒體分裂抑制劑-1 (Mdivi-1)對(duì)大鼠急性脊髓損傷(acute spinal cord injury, ASCI)后線粒體損傷和細(xì)胞凋亡的影響,為ASCl的繼發(fā)性損傷機(jī)制以及臨床靶點(diǎn)藥物治療的研究提供重要線索。方法SD大鼠隨機(jī)分為四組：假手術(shù)組(Sham組),Sham+Mdivi-1預(yù)處理組(1.20mg/kg, Sham+ Mdivi-1組),單純脊髓損傷組(SCI組)和SCI+Mdivi-1預(yù)處理組(1.20 mg/kg, SCI+Mdivi-1組)。SCI組和SCl+Mdivi-1組的大鼠采用Allen's方法制備ASCl模型,Sham+Mdivi-1組和SCI+Mdivi-1組大鼠在脊髓打擊之前15 min經(jīng)尾靜脈給予Mdivi-1。 SCI組大鼠分別于術(shù)后2、4,8,16和24小時(shí)(h)五個(gè)時(shí)間點(diǎn)處死。Sham組,Sham+Mdivi-1組和SCl+Mdivi-1組的大鼠于脊髓暴露或者損傷16h后處死。首先,采用western blotting檢測(cè)脊髓損傷后大鼠各時(shí)間點(diǎn)線粒體內(nèi)細(xì)胞色素C (Cytochrome C, Cyt C)的釋放情況,然后選擇ASCl或者暴露后16 h作為檢測(cè)Mdivi-1作用效果的時(shí)間點(diǎn),采用透射電鏡技術(shù)檢測(cè)各組大鼠線粒體形態(tài)變化,采用JC-I標(biāo)記法檢測(cè)各組大鼠脊髓組織中線粒體膜電位(mitochondrial membrane potentials, MMP)變化,采用可見(jiàn)分光光度計(jì)檢測(cè)各組大鼠脊髓組織中線粒體內(nèi)三磷酸腺苷(Adenosine triphosphate, ATP),丙二醛(Malondialdehyde, MDA)和還原型谷胱甘肽(Reduced Glutathione, GSH)的變化,采用Western Blot檢測(cè)各組大鼠脊髓組織線粒體上動(dòng)力蛋白1(Dynamin-related protein 1, Drp1), Cyt C, Bcl-2相關(guān)X蛋白(Bcl-2-associatedX protein, Bax)和B細(xì)胞淋巴瘤基因-2(B-cell lymphoma 2, Bcl-2)以及細(xì)胞內(nèi)Caspase-3表達(dá)的影響,采用免疫熒光標(biāo)記技術(shù)檢測(cè)大鼠脊髓組織內(nèi)caspase-3的表達(dá)水平,采用熒光TUNEL法檢測(cè)各組大鼠脊髓組織中細(xì)胞凋亡情況。結(jié)果1、Western Blot檢測(cè)結(jié)果顯示,與Sham組比較,SCI組中線粒體內(nèi)Cyt C從4 h開(kāi)始明顯降低,16 h達(dá)到最低值,然后逐漸上升至24 h(P0.01)；而細(xì)胞漿中的Cyt C從4h開(kāi)始升高,16 h達(dá)高峰,然后逐漸下降(P0.01)。ASCI后16 h線粒體外膜上Drp1, Bax和Bcl-2較Sham組明顯升高(P0.01),細(xì)胞內(nèi)Caspase-3表達(dá)較Sham組明顯增多(P0.01)。與SCI組16 h相比,SCI+Mdivi-1組線粒體內(nèi)Cyt C表達(dá)增高(P0.01),而細(xì)胞質(zhì)內(nèi)Cyt C表達(dá)降低(P0.01)；線粒體外膜上Drp1和Bax表達(dá)明顯減少(P0.01),細(xì)胞內(nèi)Caspase-3表達(dá)明顯減少(P0.01),而線粒體外膜上Bcl-2無(wú)明顯變化；Sham組和Sham+Mdivi-1組之間以上指標(biāo)的表達(dá)無(wú)明顯變化。2、透射電鏡檢測(cè)結(jié)果顯示,與Sham組比較,ASCI后16 h線粒體數(shù)目明顯增多,而截面積明顯減小(P0.01)；與SCI組16 h比較,SCI+Mdivi-1組線粒體數(shù)目明顯減少,而截面積明顯增大(P0.01)；但是,Sham組和Sham+Mdivi-1組之間以上指標(biāo)的表達(dá)無(wú)明顯變化。3、JC-1標(biāo)記法檢測(cè)結(jié)果顯示,ASCl后16 h MMP較Sham組明顯降低(P0.01)；與SCI組16 h相比,SCl+Mdivi-1組MMP明顯增高(P0.01)；但是,Sham組和Sham+Mdivi-1組之間MMP無(wú)明顯變化。4、可見(jiàn)分光光度計(jì)檢測(cè)結(jié)果顯示,ASCl后16 h線粒體內(nèi)ATP和GSH水平較Sham組明顯降低(P＜0.01), MAD水平明顯升高(P0.01)；與SCI組16 h相比,SCl+Mdivi-1組線粒體內(nèi)ATP和GSH水平明顯升高(P＜0.01), MAD水平明顯降低(P0.01)；但是,Sham組和Sham+Mdivi-1組之間線粒體內(nèi)ATP, GSH和MAD的水平無(wú)明顯變化。5、免疫熒光技術(shù)結(jié)果顯示：ASCl后16 h Caspase-3陽(yáng)性細(xì)胞百分?jǐn)?shù)明顯增多(P0.01)；與SCI組16 h相比,SCl+Mdivi-1組Caspase-3陽(yáng)性細(xì)胞百分?jǐn)?shù)明顯減少(P0.01)。但是,Sham組和Sham+Mdivi-1組之間細(xì)胞內(nèi)Caspase-3陽(yáng)性細(xì)胞百分?jǐn)?shù)無(wú)明顯變化。6、熒光TUNEL法檢測(cè)結(jié)果顯示,ASCI后16 h細(xì)胞凋亡百分?jǐn)?shù)較Sham組明顯增加(P0.01)；與SCI組16 h相比,SCl+Mdivi-1組細(xì)胞凋亡百分?jǐn)?shù)明顯降低(P0.01)。但是,Sham組和Sham+Mdivi-1組之間細(xì)胞凋亡百分比無(wú)明顯變化。結(jié) 論Mdivi-1明顯保護(hù)了ASCI后MMP和ATP水平,抑制了線粒體氧化損傷和細(xì)胞凋亡線粒體途徑的激活。
[Abstract]:Objective To investigate the effects of selective mitochondrial splitting inhibitor-1 (Mdivi-1) on mitochondrial damage and apoptosis in rats with acute spinal cord injury (ASCI), and to provide important clues for the study of secondary injury mechanism and clinical target drug therapy of ASCl. Methods SD rats were randomly divided into four groups: sham operation. Group A (Sham group), Sham+Mdivi-1 preconditioning group (1.20 mg/kg, Sham+Mdivi-1 group), SCI+Mdivi-1 preconditioning group (SCI group) and SCI+Mdivi-1 preconditioning group (1.20 mg/kg, SCI+Mdivi-1 group). Rats in SCI and SCl+Mdivi-1 groups were given ASCl model by Allen's method, and rats in Sham+Mdivi-1 and SCI+Mdivi-1 groups were given via caudal vein 15 minutes before spinal cord injury. Rats in the Mdivi-1 SCI group were sacrificed at five time points, 2,4,8,16 and 24 hours (h), respectively. Rats in the Sham group, Sham+Mdivi-1 group and SCl+Mdivi-1 group were sacrificed 16 hours after spinal cord exposure or injury. First, the release of cytochrome C (Cytochrome C, Cyt C) in mitochondria was detected by Western blotting at each time point after spinal cord injury. Then, ASCl or 16 hours after exposure were selected as the time point to detect the effect of Mdivi-1. The morphological changes of mitochondria were detected by transmission electron microscopy (TEM), the changes of mitochondrial membrane potentials (MMP) in spinal cord tissue were detected by JC-I labeling method, and the changes were detected by visible spectrophotometer. Mitochondrial adenosine triphosphate (ATP), malondialdehyde (MDA) and reduced glutathione (GSH) levels in the spinal cord of rats in each group were measured by Western Blot. Dynamin-related protein 1 (Drp1), Cyt C and Bcl-2 phases in the spinal cord of rats in each group were detected by Western Blot. To investigate the effects of Bcl-2-associated X protein (Bax) and B-cell lymphoma gene-2 (Bcl-2) and intracellular Caspase-3 expression, immunofluorescence labeling technique was used to detect the expression of Caspase-3 in rat spinal cord tissues. Fluorescence TUNEL method was used to detect the apoptosis of spinal cord tissues. The results of stern Blot assay showed that compared with Sham group, Cyt C in mitochondria of SCI group decreased significantly from 4 h to 16 h, reached the lowest value, then gradually increased to 24 h (P 0.01); Cyt C in cytoplasm increased from 4 h to 16 h, reached the peak, and then decreased gradually (P 0.01). Drp 1, Bax and Bcl-2 in mitochondria extracellular membrane increased significantly 16 h after ASCI compared with Sham group. Compared with the SCI group, the expression of Cyt C in mitochondria of SCI+Mdivi-1 group was higher (P 0.01), but the expression of Cyt C in cytoplasm was lower (P 0.01), the expression of Drp 1 and Bax in mitochondria outer membrane was significantly lower (P 0.01), and the expression of Caspase-3 in cells was significantly lower (P 0.01). The results of transmission electron microscopy showed that the number of mitochondria and the cross-sectional area of mitochondria increased significantly at 16 h after ASCI compared with Sham group (P 0.01); the number of mitochondria decreased significantly and the cross-sectional area increased significantly at 16 h after ASCI compared with SCI group (P 0.01). However, there was no significant change in the expression of the above indexes between Sham group and Sham+Mdivi-1 group. The results showed that the levels of ATP and GSH in mitochondria were significantly lower at 16 h after ASCl than those in Sham group (P There was no significant change in the levels of GSH and MAD. Immunofluorescence assay showed that the percentage of Caspase-3 positive cells increased significantly at 16 h after ASCl (P 0.01); the percentage of Caspase-3 positive cells in SCl+Mdivi-1 group was significantly lower than that in SCI group at 16 h (P 0.01). The percentage of apoptosis in SCl+Mdivi-1 group was significantly lower than that in SCI group (P 0.01). However, there was no significant change in the percentage of apoptosis between Sham group and Sham+Mdivi-1 group. The level of MP and ATP inhibited mitochondrial oxidative damage and activation of mitochondrial pathway.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R651.2

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