發(fā)布時間：2018-03-01 08:12

  本文關(guān)鍵詞： 中低溫低流量 動物模型 腦缺血/再灌注損傷 缺血預適應 腦保護 線粒體凋亡信號通路　出處：《南京醫(yī)科大學》2014年博士論文　論文類型：學位論文

【摘要】：[目的] 制作成年雄性SD大鼠中低溫低流量(MHLF)的腦損傷模型,進行表型分析,觀察成年雄性SD大鼠模型中使用缺血預適應的策略進行腦保護的結(jié)果,并進一步研究其通過線粒體相關(guān)凋亡通路及其上游的信號通路發(fā)揮腦保護作用的機制。 [方法] 180只健康的成年雄性SD大鼠(8-10周齡,體重76-85g)隨機的分為三組：假手術(shù)組、手術(shù)組和缺血預適應組,每組60只。手術(shù)組在SD大鼠的肛溫降至(25±0.5)℃時阻斷兩側(cè)頸總動脈120min后再重新開放,模擬臨床上腦部中低溫低流量的狀態(tài)。假手術(shù)組除不阻斷兩側(cè)頸總動脈外,其余的操作均相同。缺血預適應組在120分鐘雙側(cè)頸動脈阻斷前行缺血預適應,即阻斷雙側(cè)勁總動脈2分鐘后再灌注5分鐘,反復循環(huán)4次,之后與手術(shù)組一樣,阻斷兩側(cè)頸動脈120分鐘再重新開放。另取成年雄性健康SD大鼠(8-10周齡,體重76-85g)30只,隨機的分為三組：假手術(shù)組、手術(shù)組和缺血預適應組,每組10只。利用激光多普勒血流儀監(jiān)測假手術(shù)組、手術(shù)組和缺血預適應組(n=10)大鼠的局部腦血流量(rCBF),并記錄在降溫前、降溫至(25.0±0.5)℃MHLF0～5min, MHLF10～15min、MHLF30～35min、MHLF55～60min、MHLF65～70min、 MHLF75～80min、MHLF85～90min、MHLF105～110min、MHLF115～120min和再灌注后0-5min,復溫等十二個時間段的局部腦部血流量。對于前述180只雄性SD大鼠,我們根據(jù)再灌注后的時間隨機的把每組60只雄性SD大鼠再分成5個亞組,每組12只：分別為再灌注后1h,6h,24h,72h和7d。通過TUNEL法檢測三組SD大鼠腦組織的細胞凋亡水平,利用免疫組織化學與RT-PCR法檢測各亞組對應的時間點中細胞色素C和caspase-3等線粒體凋亡通路相關(guān)蛋白的表達水平以及mRNA等表達變化情況,利用western blot檢測細胞色素C、caspase-3與caspase-9以及其上游BCL-2與BAX以及AKT等蛋白的變化。 [結(jié)果] 成年雄性SD大鼠的局部腦血流量(rCBF)在手術(shù)組和缺血預適組的MHLF期間均下降了約84%左右。其手術(shù)組的rCBF、內(nèi)環(huán)境等指標的變化趨勢與臨床MHLF中腦血流灌注水平,內(nèi)環(huán)境變化趨勢等基本一致。缺血預適應組的SD大鼠在再灌注6h,24h,72h和7d等各時間點死亡率均低于手術(shù)組,其腦組織病理改變較輕,腦組織損傷顯著低于手術(shù)組,同時,TUNEL染色顯示凋亡細胞數(shù)量在再灌注后6h,24h,72h和7d等各時間點較手術(shù)組均顯著下降(P0.05or P0.01),并且免疫組化提示與手術(shù)組相比較,缺血預適應明顯抑制了上述四個對應時間點的線粒體凋亡信號通路相關(guān)蛋白,細胞色素C與caspase-3的釋放與活化等(P0.05or P0.01)。RT-PCR顯示線粒體凋亡通路中的細胞色素C與caspase-3的mRNA表達水平在再灌注24h與72h表達水平較手術(shù)組明顯減少(P0.05or P0.01)。 Western blot的結(jié)果顯示在再灌注24h后細胞色素C與caspase-3的蛋白表達與上述結(jié)果一致,caspase-9在缺血預適應組的表達在再灌注24h后較手術(shù)組也顯著減少(P0.05or P0.01)。在調(diào)控的上游基因中,缺血預適應組的BCL-2與AKT等蛋白的表達較之于手術(shù)組增強,而BAX的表達較手術(shù)組減弱(P0.05or P0.01)。 [結(jié)論] 本研究中制作的中低溫低流量SD大鼠的腦損傷模型,其病理生理變化近似于臨床上主動脈夾層手術(shù)中MHLF的腦部缺血再灌注的過程,模型制作成功。缺血預適應通過減少細胞DNA的斷裂、阻止線粒體凋亡通路中細胞色素C的釋放以及抑制了caspase-3、caspase-9等的活性從而發(fā)揮了腦保護的作用；同時,本實驗研究發(fā)現(xiàn)其上游BCL-2、BAX以及AKT等基因與蛋白的相關(guān)變化,顯示缺血預適應通過PI3K/Akt信號通路的這些節(jié)點抑制了線粒體凋亡通路的活性從而產(chǎn)生了腦保護作用的機制。缺血預適應可能成為臨床上防治中低溫低流量術(shù)后神經(jīng)系統(tǒng)損傷新的治療方法,而AKT蛋白可能成為新的分子治療靶點。
[Abstract]:[Objective]
Production of adult male SD rats in low temperature and low flow rate (MHLF) of the brain injury model, phenotype analysis, observation of adult male SD rats were used in the model of ischemic preconditioning strategy of brain protection results, and further study the neuroprotective signaling through mitochondria related apoptosis pathway and its upstream mechanism.
[method]
180 healthy adult male SD rats (8-10 weeks old, weight 76-85g) were randomly divided into three groups: sham operation group, operation group and ischemic preconditioning group, 60 rats in each group. Group fall in rectal temperature of SD rats (25 + 0.5) on both sides of the common carotid artery occlusion after 120min C re open state simulation clinical brain hypothermia with low flow rate. The sham operation group was not blocked on both sides of the common carotid artery, the rest of the operation were the same. The preconditioning group blocked in 120 minutes before the bilateral carotid artery ischemic preconditioning, namely blocking bilateral common carotid arteries for 2 minutes after 5 minutes of reperfusion, repeatedly after 4 cycles, and the operation group, blocked on both sides of the carotid artery for 120 minutes and then re open. Another adult healthy male SD rats (8-10 weeks old, weighing 30 76-85g) were randomly divided into three groups: sham operation group, operation group and ischemic preconditioning group, 10 rats in each group. The use of laser Doppler flowmetry Monitoring of the sham operation group, operation group and ischemic preconditioning group (n=10) and local cerebral blood flow (rCBF), and recorded in the cool, cool to (25 + 0.5) MHLF0 C ~ 5min, MHLF10 ~ 15min, MHLF30 ~ 35min, MHLF55 ~ 60min, MHLF65 ~ 70min, MHLF75 ~ 80min. MHLF85 ~ 90min, MHLF105 ~ 110min, MHLF115 ~ 120min and 0-5min after reperfusion, local blood flow to the brain and rewarming time period twelve. For the 180 male SD rats, according to reperfusion time random each group of 60 male SD rats were divided into 5 subgroups in each group. 12: are 1h after reperfusion, 6h, 24h, 72h and 7d. by the level of apoptosis in brain tissue of rats with TUNEL assay in three SD group, the apoptosis pathway of cytochrome C and caspase-3 mitochondrial protein by immunohistochemistry and RT-PCR method corresponding to each subgroup of time points in the detection of expression level and mRNA Western blot was used to detect the changes in cytochrome C, caspase-3 and caspase-9, and the upstream BCL-2, BAX and AKT.
[results]
Regional cerebral blood flow in adult male SD rats (rCBF) during MHLF surgery group and ischemic preconditioning group were decreased by about 84%. The operation group of rCBF, the change trend of environmental indicators and clinical MHLF cerebral blood flow perfusion level, internal environment change trend of ischemic preconditioning is basically the same. SD rats at reperfusion 6H, 24h, 72h and 7d each time point of death were lower than the surgery group, the pathological changes of the brain is light, brain injury was significantly lower than the surgery group, at the same time, TUNEL staining showed that the number of apoptotic cells after reperfusion, 6h, 24h, 72h and 7d at each time point compared with the operation group were significantly decreased (P0.05or P0.01), and immunohistochemical staining showed that compared with the surgery group, ischemic preconditioning significantly inhibited the mitochondrial apoptosis pathway related proteins of the four at the same time, cytochrome C and caspase-3 release and activation of.R (P0.05or P0.01) T-PCR showed that the expression level of cytochrome C and caspase-3 mitochondrial apoptosis pathway in the expression of mRNA was significantly reduced compared with the operation group at 24h after reperfusion and 72h (P0.05or P0.01) Western blot. The results showed that in the reperfusion of cytochrome C and caspase-3 protein are consistent with the above results the expression of 24h, caspase-9 in ischemic preconditioning in after 24h compared with the operation group also significantly reduced the expression of group (P0.05or P0.01). In the upstream of gene regulation, ischemic preconditioning expression compared with operation group BCL-2 and AKT protein increased, while the expression of BAX decreased compared with the operation group (P0.05or P0.01).
[Conclusion]
Brain injury model in rats with low temperature and low flow SD in this study, the pathophysiological changes similar to clinical MHLF aortic dissection in brain ischemia reperfusion, ischemic preconditioning model was established successfully. The fracture reduction of cell DNA, prevent cytochrome C in the mitochondrial apoptosis pathway and inhibition of release the Caspase-3, caspase-9 activity and thus play the role of cerebral protection; at the same time, this study found that the upstream BCL-2, BAX and AKT related changes in gene and protein, showed that ischemic preconditioning mechanism through PI3K/Akt signal pathway of these nodes inhibits mitochondrial apoptotic pathway activity resulting in cerebral protective effect. Ischemic preconditioning may become clinically in prevention of hypothermia and low flow after injury to the nervous system a new treatment method, while AKT protein may become a new molecular therapeutic target.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R651.15;R-332

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本文編號：1551108