【摘要】：目的:初步探討不同時(shí)間窗介入運(yùn)動(dòng)訓(xùn)練對(duì)實(shí)驗(yàn)性腦出血大鼠認(rèn)知功能以及海馬可塑性的影響,為臨床治療提供依據(jù)和指導(dǎo)。方法:1、造模及干預(yù)方法:向大鼠右側(cè)尾狀核內(nèi)注射Ⅰ型膠原酶及肝素鈉建立實(shí)驗(yàn)性腦出血大鼠模型,對(duì)照組大鼠相同部位注入等體積生理鹽水代替膠原酶及肝素鈉,造模后進(jìn)行改良神經(jīng)功能缺損評(píng)分(Modified Neurological Severity Score,m NSS),并于術(shù)后12h進(jìn)行正電子發(fā)射計(jì)算機(jī)斷層顯像/計(jì)算機(jī)體層掃描(Positron Emission Tomography/Computed Tomography PET/CT)檢查,評(píng)分≥8.5分且PET/CT檢查確有血腫存在即為造模成功,并根據(jù)開(kāi)始介入運(yùn)動(dòng)訓(xùn)練的時(shí)間不同將運(yùn)動(dòng)訓(xùn)練組分別分為24小時(shí)(24h)組、3天(3d)組、7天(7d)組,分別于造模成功后24h、3d、7d開(kāi)始運(yùn)動(dòng)訓(xùn)練,訓(xùn)練總時(shí)間為3周,對(duì)照組及模型組不予運(yùn)動(dòng)訓(xùn)練。2、指標(biāo)觀察:(1)訓(xùn)練結(jié)束后分別對(duì)各組大鼠進(jìn)行Y-迷宮試驗(yàn)以評(píng)估大鼠分辨學(xué)習(xí)能力及一次性被動(dòng)回避反應(yīng)試驗(yàn)以評(píng)估記憶保持能力;(2)利用HE染色法觀察腦出血模型大鼠腦出血區(qū)組織病理學(xué)改變;(3)用免疫組織化學(xué)法檢測(cè)腦出血模型大鼠右側(cè)大腦組織海馬CA3區(qū)內(nèi)源性神經(jīng)營(yíng)養(yǎng)因子(Brain derived neurotrophic factor,BDNF)和白細(xì)胞介素-10(Interleukin 10,IL-10)的蛋白表達(dá)量,以平均光敏度值(Optical density,OD值)表示;(4)采用RT-PCR法檢測(cè)腦出血模型大鼠右側(cè)大腦組織海馬CA3區(qū)bax和bcl-2的基因表達(dá),以灰度值表示。3、統(tǒng)計(jì)學(xué)分析:本實(shí)驗(yàn)的數(shù)據(jù)處理使用的是IBM SPSS Statitistics 17.0軟件進(jìn)行分析,實(shí)驗(yàn)數(shù)據(jù)結(jié)果用的均數(shù)±標(biāo)準(zhǔn)差(?x±s)表示,各組大鼠組間檢測(cè)指標(biāo)IL-10、BDNF平均光密度值的比較以及bax m RNA、bcl-2 m RNA、bcl-2 m RNA/bax m RNA灰度比值的比較均采用單因素方差分析(one-way ANOVA)進(jìn)行兩兩比較,以p0.05表示差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:1、學(xué)習(xí)記憶能力檢測(cè):運(yùn)動(dòng)訓(xùn)練組大鼠的分辨學(xué)習(xí)能力評(píng)分均明顯低于模型組,24h組、3d組、7d組達(dá)到學(xué)會(huì)標(biāo)準(zhǔn)所需次數(shù)分別為74.25±10.10,66.25±9.67,77.50±10.88,模型組達(dá)到學(xué)會(huì)標(biāo)準(zhǔn)所需次數(shù)為104.92±12.25(p0.05);運(yùn)動(dòng)訓(xùn)練組中24h組和7d組的分辨學(xué)習(xí)能力評(píng)分無(wú)明顯差異(p0.05),但均顯著高于3d組的分辨學(xué)習(xí)能力評(píng)分(p0.05);運(yùn)動(dòng)訓(xùn)練組大鼠記憶保持能力評(píng)分均明顯優(yōu)于模型組,24h組、3d組、7d組的電擊后步入潛伏期(step-through latency,STL)分別為272.7s,283.1s,242.3s,模型組大鼠的電擊后STL為132.8s(p0.05);運(yùn)動(dòng)訓(xùn)練組中24h組和3d組的記憶保持能力評(píng)分無(wú)明顯差異(p0.05),但均顯著高于7d組的記憶保持能力評(píng)分(p0.05)。2、腦組織形態(tài)觀察:對(duì)照組大鼠腦組織未見(jiàn)右側(cè)尾狀核有血腫形成;造模成功大鼠則可見(jiàn)右側(cè)尾狀核區(qū)有不規(guī)則出血區(qū),呈彌漫性,且術(shù)后1天可見(jiàn)血腫周圍腦組織腫脹明顯,呈淺淡模糊區(qū),而術(shù)后28天可見(jiàn)血腫基本吸收,留下一囊性腔隙,囊腔周圍腦組織腫脹明顯消退,運(yùn)動(dòng)訓(xùn)練組大鼠血腫吸收較模型組稍快。3、HE染色:模型組及運(yùn)動(dòng)訓(xùn)練組較對(duì)照組有典型的病理學(xué)改變。對(duì)照組大鼠右側(cè)尾狀核腦組織結(jié)構(gòu)均勻,染色均一,細(xì)胞輪廓完整清晰,胞漿豐富,胞核居中,未見(jiàn)明顯神經(jīng)膠質(zhì)細(xì)胞增生及新生血管生成。模型組大鼠病灶區(qū)血腫吸收不完全,周圍組織疏松,可見(jiàn)不均勻分布、排列不齊且大小不均勻的空泡,伴有少量神經(jīng)膠質(zhì)細(xì)胞增生及新生血管生成。與模型組比較,24h運(yùn)動(dòng)訓(xùn)練組血腫吸收較完全,周圍組織疏松程度稍改善,仍可見(jiàn)大小不等的空泡,并伴有明顯神經(jīng)膠質(zhì)細(xì)胞增生及新生血管生成。與24h運(yùn)動(dòng)訓(xùn)練組相比,3d組及7d組血腫吸收完全,周圍組織疏松程度明顯改善,空泡明顯減少,新生血管生成及膠質(zhì)細(xì)胞增生程度基本一致。4、BDNF、IL-10蛋白的表達(dá):(1)對(duì)照組大鼠右側(cè)大腦組織海馬CA3區(qū)BDNF蛋白表達(dá)量極少,模型組和各運(yùn)動(dòng)訓(xùn)練組BDNF蛋白表達(dá)量均較對(duì)照組顯著增高(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組BDNF蛋白表達(dá)量均較模型組明顯增高(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組中,3d組和24h組的BDNF蛋白表達(dá)量無(wú)明顯差異(p0.05),但均明顯高于7d組(p0.05);(2)對(duì)照組大鼠右側(cè)大腦組織海馬CA3區(qū)IL-10蛋白表達(dá)量極少,模型組和各運(yùn)動(dòng)訓(xùn)練組IL-10蛋白表達(dá)量均較對(duì)照組顯著增高(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組IL-10蛋白表達(dá)量均較模型組明顯增高(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組中以3d組的IL-10蛋白表達(dá)量最高,而24h組和7d組兩組之間IL-10蛋白表達(dá)量無(wú)明顯差異(p0.05);5、bax m RNA、bcl-2 m RNA的表達(dá):(1)bax m RNA的表達(dá):對(duì)照組大鼠右側(cè)大腦組織海馬CA3區(qū)bax基因表達(dá)量極少,模型組與各運(yùn)動(dòng)訓(xùn)練組bax基因表達(dá)量均較對(duì)照組明顯增加(p0.05);模型組bax基因表達(dá)量較3個(gè)運(yùn)動(dòng)訓(xùn)練組增加更為明顯(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組中,以7d組bax基因表達(dá)量增高最為明顯(p0.05),24h組和3d組的bax基因表達(dá)量無(wú)明顯差異(p0.05)。(2)bcl-2 m RNA的表達(dá):對(duì)照組大鼠右側(cè)大腦組織海馬CA3區(qū)bcl-2基因表達(dá)量較低,模型組與各運(yùn)動(dòng)訓(xùn)練組bcl-2基因表達(dá)量均較對(duì)照組增加(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組bcl-2基因表達(dá)量較模型組增加更為明顯(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組中以3d組的bcl-2基因表達(dá)量最高,其次為24h組,最后為7d組(p0.05)。(3)bcl-2 m RNA/bax m RNA:運(yùn)動(dòng)訓(xùn)練組bcl-2 m RNA/bax m RNA比值均較對(duì)照組與模型組明顯增加(p0.05);模型組與對(duì)照組的bcl-2 m RNA/bax m RNA比值無(wú)明顯差異(p0.05);3個(gè)運(yùn)動(dòng)訓(xùn)練組中以3d組bcl-2 m RNA/bax m RNA比值最高,其次為24h組,再次為7d組(p0.05)。結(jié)論:1、運(yùn)動(dòng)訓(xùn)練能顯著改善腦出血模型大鼠的認(rèn)知功能,且在生命體征平穩(wěn),無(wú)嚴(yán)重并發(fā)癥,神經(jīng)功能障礙癥狀沒(méi)有繼續(xù)進(jìn)展的情況下,腦出血后3天開(kāi)始介入運(yùn)動(dòng)訓(xùn)練對(duì)大鼠認(rèn)知功能的改善最有益;2、運(yùn)動(dòng)訓(xùn)練改善腦出血模型大鼠認(rèn)知功能的機(jī)制可能與促進(jìn)腦出血模型大鼠海馬CA3區(qū)BDNF蛋白、IL-10蛋白及Bcl-2 m RNA的表達(dá),抑制Bax m RNA表達(dá),抑制海馬CA3區(qū)神經(jīng)元凋亡有關(guān)。
[Abstract]:Objective: To explore the effects of different time window intervention training on cognitive function and hippocampal plasticity in experimental cerebral hemorrhage rats, and to provide basis and guidance for clinical treatment. Methods: 1, modeling and intervention methods: injection of type I collagenase and hepatin sodium into the right caudate nucleus of rats to establish experimental cerebral hemorrhage model in the control group. The same part of the rat was injected into the same volume of physiological saline instead of collagenase and heparin sodium. The improved neural function defect score (Modified Neurological Severity Score, m NSS) was made after the model, and the positron emission computed tomography / computer tomography (Positron Emission Tomography/Computed Tomography PET/CT) was performed in 12h after the operation. Examination, score more than 8.5 points and PET/CT examination confirmed the existence of hematoma was a successful model, and the exercise training group was divided into 24 hours (24h) group, 3 days (3D) group, 7 days (7D) group according to the time of beginning intervention exercise training. After the model success, 24h, 3D, 7d began to exercise training, the total training time was 3 weeks, the control group and the model group did not exercise. Training.2, index observation: (1) after the training, Y- labyrinth test was carried out to rats in each group to evaluate the learning ability of rats and one time passive avoidance response test in order to evaluate the retention ability of the memory. (2) the pathological changes in the hemorrhage area of cerebral hemorrhage model rats were observed by HE staining method; (3) the immunohistochemical method was used to detect the brain out of the rats. The protein expression of endogenous neurotrophic factor (Brain derived neurotrophic factor, BDNF) and interleukin -10 (Interleukin 10, IL-10) in the hippocampal CA3 region of the right brain tissue of the rat's right blood model was expressed by the mean photosensitivity (Optical density, OD), and (4) the hippocampus of the right brain tissue of the rat model of cerebral hemorrhage was detected by the RT-PCR method. The gene expression of region Bax and Bcl-2 expressed.3 with gray value. Statistical analysis: the data processing of this experiment was used to analyze the IBM SPSS Statitistics 17 software, and the average number + standard deviation (? X + s) used in the experimental data was expressed, the comparison of the detection indexes between the rats in each group, the comparison of the average optical density value of the BDNF, and the Bax M The comparison of Cl-2 m RNA/bax m RNA grayscale ratio was compared with single factor analysis of variance (one-way ANOVA). The difference was statistically significant with P0.05. Results: 1, learning and memory ability test: the score of discrimination learning ability of the exercise training group was lower than that of the model group, 24h group, 3D group, 7d group met the standard of learning. The number of 74.25 + 10.10,66.25 + 9.67,77.50 + 10.88 in the model group was 104.92 + 12.25 (P0.05). There was no significant difference between the group 24h and the 7d group in the exercise training group (P0.05), but all were significantly higher than that of the 3D group (P0.05), and the memory retention of the exercise training group. All the 24h, 3D and 7d groups were significantly better than the model group. The latency period (step-through latency, STL) was 272.7s, 283.1s, 242.3s, and STL was 132.8s (P0.05) after the electric shock of the model group, and there was no significant difference in the memory retention ability of the exercise group and the exercise group, but all were significantly higher than the memory retention ability of the group. Score (P0.05).2, brain tissue morphology observation: in the control group, the brain tissue of the control group did not have hematoma in the right caudate nucleus, and the successful rat model showed that there was an irregular bleeding area in the right caudate nucleus, and the swelling of the brain tissue around the hematoma was obvious on the 1 day after the operation, and the hematoma was basically absorbed on the 28 day after the operation, leaving one of the basic absorption of the hematoma after the operation. The cystoid lacunae and the swelling of the brain tissue around the capsule obviously subsided. The hematoma absorption in the exercise training group was slightly faster than the model group.3, HE staining: the model group and the exercise training group had a typical pathological change compared with the control group. The brain tissue structure of the right caudate nucleus in the control group was uniform, the staining was all clear, the cytoplasm was rich, and the nucleus was in the middle. No obvious glial cell proliferation and neovascularization were found in the model group. The hematoma in the lesion area of the model group was incomplete, the surrounding tissue was loose, the nonhomogeneous distribution, the inhomogeneous and uneven vacuoles were arranged, with a small amount of glial cell proliferation and neovascularization. Compared with the model group, the hematoma absorption in the 24h exercise training group was over. There was a slight improvement in the degree of looseness in the surrounding tissue. The vacuoles of different sizes were still visible, accompanied by obvious glial cell proliferation and neovascularization. Compared with the 24h exercise training group, the hematoma in the 3D group and the 7d group was absorbed completely, the degree of porosity in the surrounding tissue was obviously improved, the vacuoles decreased obviously, the neovascularization and the degree of glial cell proliferation were basic. The expression of.4, BDNF and IL-10 protein: (1) the expression of BDNF protein in the hippocampus CA3 area of the right brain tissue of the control group was very little. The expression of BDNF protein in the model group and the exercise training group was significantly higher than that in the control group (P0.05), and the expression of BDNF protein in the 3 exercise groups was significantly higher than that in the model group (P0.05); the 3 exercise training groups, 3D group and 24 were in 3 exercise training groups. There was no significant difference in the expression of BDNF protein in group H (P0.05), but obviously higher than that in group 7d (P0.05). (2) the expression of IL-10 protein in the hippocampus CA3 region of the right brain tissue of the control group was very few. The expression of IL-10 protein in the model group and the exercise training group was significantly higher than that in the control group (P0.05), and the expression of IL-10 protein in the 3 exercise group was more than that of the model group. Xian Zenggao (P0.05); the expression of IL-10 protein in group 3D was the highest in 3 exercise groups, but there was no significant difference in the expression of IL-10 protein between group 24h and 7d group (P0.05); 5, Bax m RNA, bcl-2 m,: (1) the expression of gene expression in the hippocampus of the right large brain tissue of the control group was very few, model group and exercise training The expression of Bax gene in the practice group was significantly higher than that in the control group (P0.05); the expression of Bax gene in the model group was more obvious than that in the 3 exercise group (P0.05); the highest expression of Bax gene in the 7d group was the most obvious in the 3 exercise training groups (P0.05), and the Bax gene expression of 24h and 3D groups was not significantly different (P0.05). (2) the expression of Bcl-2 The expression of bcl-2 gene in the hippocampal CA3 region of the right brain tissue of the rats was lower, and the expression of bcl-2 gene in the model group and the exercise training group increased (P0.05), and the bcl-2 gene expression in the 3 exercise groups was more obvious than that in the model group (P0.05); the bcl-2 gene expression in the 3D group was the highest in the 3 exercise group, followed by the 24h group. At last, 7d group (P0.05). (3) Bcl-2 m RNA/bax m RNA ratio in Bcl-2 m RNA/bax m RNA: sports training group increased significantly than that of the control group and the model group, and the ratio of the model group and the control group was not significant. 7d group (P0.05). Conclusion: 1, exercise training can significantly improve the cognitive function of cerebral hemorrhage model rats, and in the condition of stable life signs, no serious complications, and the symptoms of nerve dysfunction do not continue to progress, 3 days after cerebral hemorrhage, intervention exercise training is the most beneficial to improve the recognition function of rats; 2, exercise training to improve cerebral hemorrhage model. The mechanism of cognitive function of type rats may be related to promoting the expression of BDNF protein, IL-10 protein and Bcl-2 m RNA, inhibiting the RNA expression of Bax m and inhibiting the apoptosis of hippocampal CA3 region neurons in the hippocampus CA3 area of cerebral hemorrhage model rats.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R743.34

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