本文關(guān)鍵詞：褪黑素對(duì)大鼠蛛網(wǎng)膜下腔出血后繼發(fā)性腦損傷及認(rèn)知功能影響機(jī)制的實(shí)驗(yàn)研究　出處：《蘇州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蛛網(wǎng)膜下腔出血 Morris水迷宮 褪黑素 認(rèn)知功能 TUNEL 蛛網(wǎng)膜下腔出血 褪黑素 Western blot 免疫組化 EMSA ELISA

【摘要】：第一部分褪黑素對(duì)大鼠蛛網(wǎng)膜下腔出血后繼發(fā)性腦損傷及認(rèn)知功能的影響 目的：研究褪黑素（Melatonin,MT）對(duì)大鼠蛛網(wǎng)膜下腔出血（subarachnoidhemorrhage,SAH）后繼發(fā)性腦損傷及認(rèn)知功能障礙的影響。 方法：任選80只成年雄性SD大鼠，隨機(jī)分為以下四個(gè)實(shí)驗(yàn)組：正常組、SAH組、安慰劑治療組和褪黑素治療組，將SD大鼠股動(dòng)脈非肝素化動(dòng)脈血通過1.0ml注射器注入大鼠視交叉前池，從而建立實(shí)驗(yàn)性蛛網(wǎng)膜下腔出血?jiǎng)游锬Ｐ�。在褪黑素治療組，模型建立后的第2小時(shí)、24小時(shí)及36小時(shí)，腹腔注射褪黑素溶液（150mg/kg）。在安慰劑治療組，模型建立后的第2小時(shí)、24小時(shí)及36小時(shí)，腹腔注射生理鹽水。觀察SD大鼠SAH后神經(jīng)行為功能的變化，同時(shí)進(jìn)行神經(jīng)行為功能評(píng)分。SAH動(dòng)物模型建立完畢第48小時(shí)處死部分大鼠，取出大鼠額顳底皮層腦組織做標(biāo)本，采用TUNEL熒光染色與FLUORO-JADE B熒光染色檢測(cè)大鼠額顳底皮層腦組織神經(jīng)元細(xì)胞凋亡及壞死比率，剩余未處死SD大鼠在SAH動(dòng)物模型建立完畢第48小時(shí)開始進(jìn)行Morris水迷宮試驗(yàn)，檢測(cè)SD大鼠的認(rèn)知功能障礙。 結(jié)果：1.與正常組相比，SAH組SD大鼠的神經(jīng)認(rèn)知功能水平降低，活動(dòng)能力及飲食能力降低（P0.05）；褪黑素組SD大鼠與安慰劑組SD大鼠相比，神經(jīng)認(rèn)知功能明顯改善，食欲及活動(dòng)能力明顯好轉(zhuǎn)（P0.01）；SAH組SD大鼠和安慰劑組SD大鼠之間無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。2. TUNEL染色結(jié)果顯示：與正常正常組比較，SAH組SD大鼠神經(jīng)元細(xì)胞的凋亡百分顯著增高（P0.01），褪黑素治療組SD大鼠神經(jīng)元細(xì)胞凋亡百分比明顯低于安慰劑治療組SD大鼠（P0.01），而安慰劑治療組與SAH組SD大鼠神經(jīng)元細(xì)胞的凋亡百分比比較無(wú)統(tǒng)計(jì)學(xué)差異（P0.05）。3. Fluoro-Jade B熒光染色檢測(cè)各組大鼠神經(jīng)元細(xì)胞的壞死情況，結(jié)果顯示：與正常組相比，SAH組 SD大鼠神經(jīng)元細(xì)胞壞死的百分比顯著增高（P0.01），褪黑素治療組SD大鼠神經(jīng)元 細(xì)胞壞死的百分比顯著低于安慰劑治療組SD大鼠（P0.05），而安慰劑治療組SD大鼠與SAH組SD大鼠神經(jīng)元細(xì)胞壞死的百分比無(wú)統(tǒng)計(jì)學(xué)差異（P0.05）。5.在Morris水迷宮試驗(yàn)測(cè)試中，與正常組相比，SAH組SD大鼠及安慰劑組SD大鼠的空間學(xué)習(xí)記憶功能明顯下降（P0.01），且SAH組SD大鼠及安慰劑組SD大鼠之間無(wú)統(tǒng)計(jì)學(xué)差異（P0.05）。褪黑素組SD大鼠與安慰劑組SD大鼠相比，SD大鼠的空間學(xué)習(xí)記 憶能力有顯著提高（P0.01）。 結(jié)論：褪黑素（Melatonin,MT）能夠降低SD大鼠蛛網(wǎng)膜下腔出血后繼發(fā)性神經(jīng)元細(xì)胞凋亡及壞死水平，同時(shí)明顯改善SD大鼠SAH后的空間學(xué)習(xí)等認(rèn)知功能障礙。 第二部分褪黑素對(duì)大鼠蛛網(wǎng)膜下腔出血后TLR4介導(dǎo)的炎性信號(hào)通路相關(guān)蛋白表達(dá)影響的研究 目的：建立大鼠蛛網(wǎng)膜下腔出血（SAH，subarachnoid hemorrhage）模型，觀察褪黑素治療后，TLR4介導(dǎo)的炎性信號(hào)通路相關(guān)蛋白表達(dá)的變化，如高遷移率族蛋白B1（HMGB1），Toll樣受體4（TLR4），核轉(zhuǎn)錄因子（NF-κB），髓樣分化因子88（MyD88），白介素-1β（IL-1β），腫瘤壞死因子α（TNF-α），白介素-6（IL-6），和誘導(dǎo)型一氧化氮合酶等。 方法：任選80只成年雄性SD大鼠，隨機(jī)分為以下四個(gè)實(shí)驗(yàn)組：正常組、SAH組、安慰劑治療組和褪黑素治療組，將SD大鼠股動(dòng)脈非肝素化動(dòng)脈血通過1.0ml注射器注入大鼠視交叉前池，從而建立實(shí)驗(yàn)性蛛網(wǎng)膜下腔出血?jiǎng)游锬Ｐ�。在褪黑素治療組，模型建立后的第2小時(shí)、24小時(shí)及36小時(shí)，腹腔注射褪黑素溶液（150mg/kg）。在安慰劑治療組，模型建立后的第2小時(shí)、24小時(shí)及36小時(shí)，腹腔注射生理鹽水。觀察SD大鼠SAH后神經(jīng)行為功能的變化，同時(shí)進(jìn)行神經(jīng)行為功能評(píng)分。SAH動(dòng)物模型建立完畢第48小時(shí)處死各組大鼠，取出大鼠額顳底皮層腦組織做標(biāo)本，用Western blot、EMSA、PCR、ELISA及免疫組化等方法，分析測(cè)定TLR4介導(dǎo)的炎性信號(hào)通路相關(guān)蛋白表達(dá)的變化。 結(jié)果：Western blot結(jié)果顯示：高遷移率族蛋白B1（HMGB1），Toll樣受體4蛋白（TLR4），核轉(zhuǎn)錄因子（NF-κB P65，NF-κB P50），髓樣分化因子88（MyD88），，誘導(dǎo)型一氧化氮合酶（iNOS）在正常組樣本中呈低水平表達(dá)。與正常組相比，SAH組樣本上述蛋白的表達(dá)水平明顯增加（P0.01），但是在單純SAH組的表達(dá)水平與安慰劑治療組并無(wú)明顯差別（P0.05）。在褪黑素治療組，上述蛋白的表達(dá)水平明顯低于安慰劑治療組（P0.05）。EMSA結(jié)果顯示：通過凝膠射線顯跡顯示，正常組NF-κB結(jié)合活性水平較低，而SAH組及安慰劑治療組NF-κB結(jié)合活性水平較正常組明顯上升（P0.01）。同時(shí)在褪黑素治療組，NF-κB結(jié)合活性水平較安慰劑治療組明顯下調(diào)（P0.05）。免疫組化結(jié)果：正常組中表達(dá)TLR4、NF-κB和iNOS的陽(yáng)性細(xì)胞比率很低。SAH組及安慰劑治療組中表達(dá)TLR4、NF-κB和iNOS的陽(yáng)性細(xì)胞比率明顯上升（P0.01）。褪黑素治療組TLR4、NF-κB和iNOS的陽(yáng)性細(xì)胞比率較安慰劑治療組明顯下降（P0.05）。實(shí)時(shí)定量PCR結(jié)果：正常組TNF-α，IL-1β，IL-6的mRMA表達(dá)水平較低，而SAH組及安慰劑治療組上述蛋白mRNA表達(dá)水平明顯增高（P0.01），且SAH組與安慰劑治療組上述蛋白mRNA表達(dá)水平無(wú)明顯差異（P0.05）。同時(shí)褪黑素治療組上述蛋白mRNA表達(dá)水平較安慰劑治療組有明顯下降（P0.01）。ELISA結(jié)果顯示：正常組IL-1β，TNF-α和IL-6濃度較低，SAH組及安慰劑治療組IL-1β，TNF-α和IL-6因子濃度大幅上升（P0.01），而褪黑素治療組IL-1β，TNF-α和IL-6因子濃度有相當(dāng)程度的降低（P0.05）。 結(jié)論：褪黑素（Melatonin,MT）在大鼠蛛網(wǎng)膜下腔出血模型中顯著降低TLR4介導(dǎo)的炎性反應(yīng)信號(hào)通路相關(guān)蛋白如HMGB1、TLR4、NF-κB、MyD88、IL-1β、TNF-α、IL-6和iNOS的表達(dá)及轉(zhuǎn)錄水平。
[Abstract]:The effect of melatonin on secondary brain injury and cognitive function after subarachnoid hemorrhage in rats
Objective: To study the effects of Melatonin (MT) on secondary brain injury and cognitive impairment after subarachnoid hemorrhage (subarachnoidhemorrhage, SAH) in rats.
Methods: select 80 adult male SD rats were randomly divided into four experimental groups: normal group, SAH group, placebo group and melatonin treatment group, SD rats femoral artery non heparinized arterial blood pool before crossing rat optic by 1.0ml injection, so as to establish the experimental animal model of subarachnoid space subarachnoid hemorrhage in the treatment group. Melatonin, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of melatonin solution (150mg/kg). In the placebo group, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of saline. Changes of neurobehavioral function in SD rats after SAH were observed. At the same time the neurobehavioral function evaluation.SAH animal model has been established. Some rats were sacrificed forty-eighth hours, remove the rat frontotemporal bottom cerebral cortex specimens, using TUNEL staining and FLUORO-JADE B staining were used to detect the rat frontal and temporal cortex at the end The rate of apoptosis and necrosis of neurons in brain tissue was not detected in SD rats. After forty-eighth hours of establishment of SAH animal model, Morris water maze test was carried out to detect cognitive dysfunction in SD rats.
Results: 1. compared with the normal group, SAH group, SD rats the level of cognitive function decreased, decreased activity and eating ability (P0.05); melatonin group SD rats compared with the placebo group SD rats, neurocognitive function improved appetite and activity improved significantly (P0.01); there was no statistically significant between group SAH and SD the placebo group rats and SD rats (P0.05) and.2. TUNEL staining showed that: compared with normal group, SAH group of neurons of SD rats significantly increased the apoptosis percentage (P0.01), melatonin treatment group SD rat neuronal apoptosis percentage was significantly lower than the placebo group SD rats (P0.01). But no significant differences between the placebo group and the percentage of apoptotic neurons in SAH SD rats (P0.05) necrosis neurons of rats were detected.3. Fluoro-Jade B fluorescent staining, the results showed: compared with normal group, S AH group
The percentage of necrosis of neuron cells in SD rats increased significantly (P0.01), and the neurons of SD rats in the melatonin treatment group
The percentage of cell necrosis was significantly lower than that of the placebo group SD rats (P0.05), but no significant difference between the placebo treatment group the percentage of SD rats and SAH rats group SD neuron necrosis (P0.05).5. in the Morris water maze test, compared with the normal group, SAH group, SD rats and placebo group SD in the spatial learning and memory function decreased significantly (P0.01), and between SAH group and placebo group SD rats SD rats showed no significant difference (P0.05). Melatonin group SD rats compared with the placebo group SD rats, SD rats spatial learning notes
The memory ability was improved significantly (P0.01).
Conclusion: Melatonin (MT) can reduce the secondary neuronal apoptosis and necrosis level after subarachnoid hemorrhage in SD rats, and significantly improve spatial learning and cognitive dysfunction in SD rats after SAH.
The effect of the second part of melatonin on the expression of TLR4 mediated inflammatory signaling pathway related protein after subarachnoid hemorrhage in rats
Objective: to establish a rat model of subarachnoid hemorrhage (SAH subarachnoid hemorrhage) model, to observe the effect of melatonin treatment, changes in the expression of inflammatory signal pathway mediated by TLR4, such as high mobility group protein B1 (HMGB1), Toll like receptor 4 (TLR4), a nuclear transcription factor (NF- K B), myeloid differentiation factor 88 (MyD88), interleukin -1 beta (IL-1 beta), tumor necrosis factor alpha (TNF- alpha), interleukin -6 (IL-6), and inducible nitric oxide synthase.
Methods: select 80 adult male SD rats were randomly divided into four experimental groups: normal group, SAH group, placebo group and melatonin treatment group, SD rats femoral artery non heparinized arterial blood pool before crossing rat optic by 1.0ml injection, so as to establish the experimental animal model of subarachnoid space subarachnoid hemorrhage in the treatment group. Melatonin, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of melatonin solution (150mg/kg). In the placebo group, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of saline. Changes of neurobehavioral function in SD rats after SAH were observed. At the same time the neurobehavioral function evaluation.SAH animal model has been established. Rats were killed forty-eighth hour, remove the rat frontotemporal bottom cerebral cortex specimens with Western, blot, EMSA, PCR, ELISA and immunohistochemistry method, determination of TLR4 mediated Changes in the expression of inflammatory signaling pathway related proteins.
Results: Western blot showed that high mobility group protein B1 (HMGB1), Toll like receptor 4 protein (TLR4), a nuclear transcription factor (NF- - B P65, NF- P50 K B), myeloid differentiation factor 88 (MyD88), inducible nitric oxide synthase (iNOS) in normal group was the low expression level. Compared with the normal group, the expression level of SAH protein significantly increased the sample group (P0.01), but the expression level in SAH group and placebo treatment groups had no significant difference (P0.05). The treatment group in the expression level of melatonin, the protein was significantly lower than that in the placebo group (P0.05).EMSA results by ray tracing gel showed normal NF- kappa B binding activity of low level, while the SAH group and the placebo group NF- kappa B binding activity level increased significantly compared with the normal group (P0.01). At the same time in the treatment group, melatonin, NF- kappa B binding activity was significantly lower than that of placebo group (P0.05 ). The results of immunohistochemistry: the expression of TLR4 in the normal group, the expression of TLR4 positive cell ratio of NF- kappa B and iNOS low.SAH group and placebo group, positive cell ratio of NF- kappa B and iNOS was significantly increased (P0.01). Melatonin treatment group TLR4, positive cell ratio of NF- kappa B and iNOS were the placebo treatment group decreased significantly (P0.05). Real time quantitative PCR results: in normal group, TNF- alpha, IL-1 beta, IL-6 mRMA expression level was low, while the SAH group and the placebo group the protein expression level of mRNA was significantly higher (P0.01), and SAH group and the placebo group showed no significant difference between the mRNA protein expression level (P0.05). At the same time melatonin treatment group the protein expression level of mRNA compared with the placebo treatment group decreased significantly (P0.01).ELISA results showed: normal group IL-1 beta, TNF- alpha and low concentrations of IL-6, SAH group and placebo group IL-1 beta, TNF- alpha and IL-6 factor concentration increased significantly (P0. 01), while the concentration of IL-1 beta, TNF- alpha and IL-6 in the melatonin treatment group decreased to a considerable degree (P0.05).
Conclusion: Melatonin (MT) significantly reduces the expression and transcription level of TLR4 mediated inflammatory response related proteins such as HMGB1, TLR4, NF- kappa B, MyD88, IL-1, beta, TNF-, IL-6 and TNF- in rat subarachnoid hemorrhage models.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R651.15

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 侯金才;劉建勛;張鵬;韓笑;李丹;王秀輝;;黃芩苷對(duì)缺氧/復(fù)氧小膠質(zhì)細(xì)胞TLR4通路的影響[J];中華中醫(yī)藥雜志;2012年03期

2 王超;路華;楊智勇;;TLR4信號(hào)通路在腦卒中后腦損傷中的研究進(jìn)展[J];國(guó)際神經(jīng)病學(xué)神經(jīng)外科學(xué)雜志;2010年06期

3 王超;楊智勇;路華;王東;張勝平;;Toll樣受體4信號(hào)通路在蛛網(wǎng)膜下腔出血后早期腦損傷中的機(jī)制研究[J];國(guó)際神經(jīng)病學(xué)神經(jīng)外科學(xué)雜志;2012年01期

4 李瑩;張興毅;;TLR4與腦血管神經(jīng)疾病的研究進(jìn)展(綜述)[J];安徽衛(wèi)生職業(yè)技術(shù)學(xué)院學(xué)報(bào);2013年06期

5 吳卓超;宋英;宋必衛(wèi);;NF-κB介導(dǎo)線粒體依賴的神經(jīng)細(xì)胞凋亡途徑[J];癌變.畸變.突變;2014年01期

6 ;Translational medicine in China Ⅰ:Perspectives from Chinese physicians and scientists[J];Science China(Life Sciences);2011年12期

7 馮蕊;李樹清;;缺血后適應(yīng)調(diào)控樹

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