缺血后处理对脑缺血再灌注大鼠TLR4-TRIF信号转导途径的影响

发布时间：2018-05-15 09:11

本文选题：脑缺血 + 脑卒中　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:缺血后处理(ischemic post-conditioning,IPO)能够激发机体内源性保护作用,减轻缺血再灌注(ischemia reperfusion,I/R)后的炎症反应,但具体作用机制目前尚不明确。本课题旨在探讨IPO对局灶性脑缺血再灌注大鼠Toll样受体4(Toll-like receptor 4,TLR4)-Toll/IL-1受体结构域接头分子(Toll-interleukin 1 receptor domain-containing adapter-inducing interferon-b,TRIF)信号转导途径的影响,进一步阐述IPO的神经保护机制,为临床应用IPO治疗缺血性卒中提供理论依据。方法:研究选用130只成年健康雄性Sprague-Dawley大鼠并随机分为假手术组(sham组)30只,模型组和干预组各50只,sham组根据手术后,模型组和干预组根据再灌注后时间点随机分为6h、12h、24h、48h和72h 5个亚组,sham组每个亚组各6只,模型组和干预组每个亚组各10只。后两组采用Zea-longa线栓法建立大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型。sham组仅手术暴露颈总动脉及分叉处,不阻断大脑中动脉。在再灌注开始时即模型建立后2h对干预组大鼠进行IPO处理(即术后2 h将栓线拔出至头部位于颈总动脉分叉处,10s后再将栓线置入初始位置10s,如此重复6次)。对模型组大鼠仅拔出栓线,不给予IPO处理。于再灌注后6h、12h、24h、48h、72h等时间点,采用Menzies法进行神经功能缺损评分;2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色观察脑梗死体积;原位末端标记法(terminal-deoxynucleoitidyl transferase mediated nick end labeling,TUNEL)法检测缺血侧脑组织细胞凋亡;实时定量荧光PCR(Real-time quantitative PCR,qPCR)检测大鼠缺血侧颞、顶叶皮质TLT4-TRIF信号转导途径的特异性结构分子及关键细胞因子TLR4、TRIF、TRIF相关的接头分子(TRIF-related adaptor molecule,TRAM)、干扰素调节因子3(interferon regulatory factor 3,IRF-3)及β干扰素(interferon-β,INF-β)等的m RNA表达;免疫组织化学(immunohistochemistry)检测大鼠缺血侧脑组织中TLR4、TRIF、TRAM、IRF-3及IFN-β等结构分子及关键细胞因子蛋白阳性细胞的分布与表达;免疫印迹法(western blotting)定量检测大鼠缺血侧颞、顶叶皮质中TLR4、TRIF、TRAM、IRF-3及IFN-β等结构分子及关键细胞因子的蛋白表达。结果:模型组、干预组大鼠都出现不同程度的神经功能缺损及缺血侧大脑半球梗死。相比模型组,干预组大鼠神经功能缺损评分得到显著改善(t=2.963~5.262,P0.05),脑梗死体积明显减少(t=3.341~3.875,P0.05),凋亡细胞计数明显减少(t=2.332~3.643,P0.05)。模型组和干预组TLR4、TRIF、TRAM、IRF-3及IFN-βm RNA和蛋白表达于再灌注6h时已显著升高。qPCR结果显示干预组TLR4、TRIF、TRAM、IRF-3及IFN-βm RNA表达较模型组各对应时间点显著降低(t=2.240~6.587,P0.05),免疫组织化学检测结果显示TLR4、TRIF、TRAM、IRF-3及IFN-β蛋白阳性细胞主要位于缺血侧额、颞叶皮质,干预组阳性细胞计数较模型组各对应时间点显著降低(t=2.256~8.180,P0.05)。Western blotting检测结果显示干预组TLR4、TRIF、TRAM、IRF-3及IFN-β蛋白表达较模型组各对应时间点显著降低(t=2.943~8.227,P0.05)。结论:本研究证实IPO能够通过抑制细胞凋亡、减少脑梗死体积发挥神经保护作用,改善MCAO大鼠的神经功能。IPO还可能通过抑制TLR4-TRIF信号转导途径减少通路下游IRF-3、IFN-β等炎症相关因子的表达,减轻I/R后的局灶性炎症反应,进而激发内源性神经保护作用。
[Abstract]:Objective: ischemic post-conditioning (IPO) can stimulate the endogenous protective effect of the body and reduce the inflammatory response after ischemia reperfusion (ischemia reperfusion, I/R), but the specific mechanism is not yet clear. The aim of this study is to explore Toll like receptor 4 (Toll-like receptor 4, TLR) in focal cerebral ischemia-reperfusion rats (Toll-like receptor 4, TLR). 4) the effect of -Toll/IL-1 receptor domain junction molecule (Toll-interleukin 1 receptor domain-containing adapter-inducing interferon-B, TRIF) signal transduction pathway, further expounds the neuroprotective mechanism of IPO, and provides a theoretical basis for the clinical application of IPO to treat ischemic stroke. Methods: 130 adult healthy male Sprague-D were selected. AWLEY rats were randomly divided into 30 rats in sham operation group (Group sham), model group and intervention group, and group sham were randomly divided into 5 subgroups of 6h, 12h, 24h, 48h and 72h, 6 in each group of sham group, 10 in each group of the model group and in each subgroup of intervention group after the operation. The latter two groups were treated with Zea-longa thread. The rat model of middle cerebral artery occlusion (MCAO) model was established in group.Sham only to expose the common carotid artery and the bifurcation, and did not block the middle cerebral artery. At the beginning of the reperfusion, the model was established by 2H to the rats in the intervention group (that is, 2 h after the operation was pulled out to the head of the common carotid artery, and the 10s after the operation. " Then the thrombus line was placed in the initial position 10s, so repeated 6 times). The rats in the model group were only pulled out of the thrombus line and did not give IPO treatment. After the reperfusion, 6h, 12h, 24h, 48h, 72h and other time points were measured by Menzies method, and the volume of cerebral infarction was observed by 2,3,5- chlorination of three phenyl tetrazolium (2,3,5-triphenyltetrazolium chloride, TTC). In situ terminal labeling (terminal-deoxynucleoitidyl transferase mediated nick end labeling, TUNEL) detection of apoptosis in ischemic brain tissue; real-time quantitative fluorescent PCR (Real-time quantitative PCR, qPCR) detection of rat ischemic side temporomandibular, parietal cortex, specific structural molecules and key cytokines R4, TRIF, TRIF related joint molecules (TRIF-related adaptor molecule, TRAM), interferon regulatory factor 3 (interferon regulatory factor 3, IRF-3) and interferon beta (interferon- beta, beta), etc. The distribution and expression of the positive cell and key cell factor protein positive cells; Western blotting were used to detect the protein expression of TLR4, TRIF, TRAM, IRF-3 and IFN- beta in the temporal and temporal cortex of the rat, and the protein expression of the key cytokines in the parietal cortex. Results: the model group, the rats in the intervention group had different degrees of nerve function defect and the rats in the intervention group. Compared with the model group, the neurological deficit score of the rats in the intervention group was significantly improved (t=2.963~5.262, P0.05), the volume of cerebral infarction decreased significantly (t=3.341~3.875, P0.05), the number of apoptotic cells decreased significantly (t=2.332~3.643, P0.05). The model group and the intervention group were TLR4, TRIF, TRAM, IRF-3 and IFN- beta m and protein expression and protein expression. The TLR4, TRIF, TRAM, IRF-3 and IFN- beta m RNA expression in the intervention group was significantly lower than that of the model group (t=2.240~6.587, P0.05) in the intervention group. The results of immunohistochemical detection showed that TLR4, TLR4, and beta protein positive cells were mainly located in the ischemic side, temporal lobe cortex, and the intervention group. The cell count decreased significantly at the corresponding time points of the model group (t=2.256~8.180, P0.05).Western blotting detection results showed that the expression of TLR4, TRIF, TRAM, IRF-3 and IFN- beta protein in the intervention group was significantly lower than the corresponding time points in the model group (t=2.943~8.227, P0.05). Conclusion: this study confirmed that IPO can reduce the volume of cerebral infarction by inhibiting apoptosis and reducing the volume of cerebral infarction. To improve the neuroprotective effect and improve the neural function of MCAO rats,.IPO may also reduce the expression of inflammatory related factors such as IRF-3, IFN- beta, and so on by inhibiting the TLR4-TRIF signal transduction pathway, and reduce the focal inflammatory response after I/R, and then stimulate endogenous neuroprotective effect.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3

【参考文献】