【作者】 熊新贵；

【导师】 梁清华；

【作者基本信息】 中南大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:1.复制具有病证结合特点的高血压肝阳上亢证与出血性中风肝阳化风证及缺血性中风肝阳化风证动物模型。2.研究肝阳上亢证与肝阳化风证垂体、肾上腺、甲状腺及脾淋巴细胞蛋白质的表达规律,从神经内分泌-免疫系统的蛋白质表达水平探讨二证本质内涵,为肝阳上亢证与肝阳化风证开辟新的研究领域。3.寻找肝阳上亢证与肝阳化风证相同与差异表达的蛋白,揭示同病异证和异病同证的本质内涵。方法:1.高血压肝阳上亢证动物模型的复制:75只SD大鼠随机分为正常组15只(自由饮水),高血压肝阳上亢证组60只,采用附子汤灌胃(附子汤的剂量为20 ml/kg·d),同时喂1.5%高渗盐水的方法,共6周,6周后从激惹程度、结膜充血情况、血压测定及旋转时间等方面进行模型评价。2.出血性中风肝阳化风证动物模型的复制:在成功复制高血压肝阳上亢证模型基础上,采用Ⅶ胶原酶脑内定位注射诱导大鼠脑出血复制成出血性中风肝阳化风证模型,从行为学与形态学观察评价模型成功与否。3.缺血性中风肝阳化风证动物模型的复制:在成功复制高血压肝阳上亢证模型基础上,用改良线栓法造成大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO),复制成缺血性中风肝阳化风证模型,从行为学与TTC染色评价模型成功与否。4.蛋白质组学检测及基因、蛋白质水平验证:应用双向凝胶电泳技术(two-dimensional gel electrophoresis,2DE)分别分离各组垂体、肾上腺、甲状腺及脾淋巴细胞蛋白质,改良的考马斯亮蓝染色使凝胶中的蛋白质点可视化;扫描凝胶成像,Pdquest图像分析软件进行图像分析并识别差异表达的蛋白质与共同表达的蛋白质;应用基质辅助激光解吸电离飞行时间质谱技术(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS)得到相应的肽质量指纹图谱(peptide mass fingerprint,PMF),搜索数据库进行鉴定,运用RT-PCR与western blotting方法从基因与蛋白质两方面验证差异表达蛋白。结果:1.动物模型的复制:60只SD大鼠成功复制成高血压肝阳上亢证模型48只,模型成功率为80%;模型成功后随机分为高血压肝阳上亢组14只,出血性中风肝阳化风组17只(实验中死亡2只)和缺血性中风肝阳化风组17只(实验中死亡3只)。2.动物模型的评价:①高血压肝阳上亢证组大鼠出现不同程度的性情变化,表现为烦躁、易激惹、互相打斗,部分大鼠双眼结合膜颜色加深变红,与正常组比较差异有显著性(P＜0.05);与正常组相比旋转时间明显缩短(P＜0.05);随造模时间延长,血压逐步上升,至第6周达到高峰,与正常组比较差异有显著性(P＜0.01);②造模术后12h出血性肝阳化风证组与缺血性肝阳化风证组大部份出现了结膜充血,且其易激惹程度进一步加重,表现为提尾时尖叫、惊跳,甚至咬人或同笼大鼠频繁打斗,与正常组、肝阳上亢组比较差异有显著性意义(P＜0.05);旋转时间较术前更加缩短,且血压进一步上升,与正常组、肝阳上亢证组比较差异有显著性(均P＜0.05);脑出血术后神经功能缺损评分＞2分的为13只;脑缺血术后神经功能缺损评分＞2分的为12只;出血性中风肝阳化风证HE染色示脑内形成直径3mm左右血肿,光镜下见出血灶内充满变性红细胞、大量中性粒细胞和吞噬细胞浸润,无正常结构,血肿周围神经细胞排列紊乱,缺血性中风肝阳化风证TTC染色示缺血区脑组织颜色苍白,其余部分染色为均匀红色,缺血的大鼠脑缺血体积百分比为(15-45%),正常组全脑染色为均匀红色。3.垂体、肾上腺、甲状腺组织及脾淋巴细胞双向凝胶电泳图谱的建立和图象分析:在相同条件下分别对垂体、肾上腺、甲状腺及脾淋巴细胞总蛋白质各进行了3次双向凝胶电泳分离,考染显色后得到肯景清晰、分辨率高、重复性好的2-DE图谱各3块,其总蛋白质的分布模式非常相似,使用PDQuest软件对凝胶图谱进行图像分析结果显示,垂体组织的2-DE图谱的蛋白质点约1100±32个;肾上腺的2-DE图谱的蛋白质点约800±56个;甲状腺的2-DE图谱的蛋白质点约500±17个;脾淋巴细胞的2-DE图谱约的蛋白质点约1200±24个,大多数蛋白质点在位置和丰度上是一致的,主要分布在pH4-8和Mr20-60kD的范围内。4.选取四组中差异表达和共同表达的蛋白质点作进一步的质谱鉴定,从匹配的蛋白质斑点中随机选取了61个分辨较清楚的点进行MALDI-TOF-MS分析,共有46个蛋白质点得到鉴定,根据NCBI、MSDB数据库中提供的的信息,这些差异蛋白质的功能涉及抗氧化应激、代谢相关酶、蛋白质合成与降解、细胞骨架相关蛋白、分子伴侣、出凝血相关等,其中类固醇激素合成急性调控蛋白(steroidogenicacute regulatory protein,STAR)、泛素C末端水解酶L1(Ubiquitincarboxyl-terminal hydrolase isozyme L1,UCH-L1)、二氢嘧啶酶相关蛋白2(Dihydropyrimidinase-related protein 2,DRP-2)、硫氧还蛋白过氧化物酶(Peroxiredoxin,PRDX)等可能与肝阳上亢证与肝阳化风证神经内分泌-免疫网络的相互作用有关。5.差异蛋白验证结果:(1)RT-PCR结果显示:STARmRNA、DRP-2mRNA及PRDX2mRNA在高血压肝阳上亢证、出血性中风肝阳化风证及缺血性中风肝阳化风证组表达均较正常组明显增强(p＜0.01),而在出血性中风肝阳化风证及缺血性中风肝阳化风证组表达又较高血压肝阳上亢证组表达增强(P＜0.05),出血性中风肝阳化风证及缺血性中风肝阳化风证两组间比较比较无明显差异(P＞0.05);UCH-L1mRNA在高血压肝阳上亢证、出血性中风肝阳化风证及缺血性中风肝阳化风证组表达均较正常组明显减弱(p＜0.01),而在出血性中风肝阳化风证及缺血性中风肝阳化风证组表达又较高血压肝阳上亢证组表达明显减弱(P＜0.05),出血性中风肝阳化风证及缺血性中风肝阳化风证两组间比较无明显差异(P＞0.05),且STARmRNA、DRP-2mRNA、PRDX2mRNA及UCH-L1mRNA在垂体与。肾上腺中的表达水平一致。(2)Western blotting结果显示:STAR在高血压肝阳上亢证、出血性中风肝阳化风证及缺血性中风肝阳化风证组表达均较正常组明显增强(p＜0.01),而在出血性中风肝阳化风证及缺血性中风肝阳化风证组表达又较高血压肝阳上亢证组表达增强(P＜0.05),出血性中风肝阳化风证及缺血性中风肝阳化风证组两组间比较无明显差异(P＞0.05);UCH-L1在高血压肝阳上亢证、出血性中风肝阳化风证及缺血性中风肝阳化风证组表达均较正常组明显减弱(p＜0.01),而在出血性中风肝阳化风证及缺血性中风肝阳化风证组表达又较高血压肝阳上亢证组表达明显减弱(P＜0.01),出血性中风肝阳化风证及缺血性中风肝阳化风证组两组间比较无明显差异(P＞0.05),结果与蛋白质组学及RT-PCR研究的结果一致。结论:1.成功复制了高血压肝阳上亢证、出血性中风肝阳化风证及缺血性中风肝阳化风证动物模型,具有病证结合的特点。2.肝阳上亢证与肝阳化风证的形成过程是一个多蛋白质参与的复杂病理过程,且神经内分泌系统与免疫系统的多个蛋白质表达发生了明显的改变,提示二证神经内分泌-免疫网络出现了明显的紊乱。3.肝阳上亢证与肝阳化风证既有相同的蛋白质表达又有差异的蛋白质表达,提示二证既有相同的物质基础又有不同的本质内涵,为肝阳上亢证与肝阳化风证本质研究提供了科学依据。更多还原

【Abstract】 Objective:(1)To establish the model of hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome which have the characteristic of combined Disease and Syndrome.(2)To investigate the protein profile in the rat pituitary,adrenal gland, thyroid gland and splenic lymphocyte using proteomics techniques, and to elucidate the nature of Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome from the view of the neuroendocrine-immune system,which can be helpful to provide new field for approaching the nature of the two Syndromes.(3)To elucidate the essence of the "different syndrome complexes in the same disease" and "the same syndrome complex in different diseases" by analyzing the undifferential and differential expression proteins between Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome.Methods:(1)Randomly selected 15 rats from 70 male SD rats as normal control group,and the remnant 60 rats as hypertensive with Liver-Yang Hyperactivity Syndrome which was made through pouring "Aconite Decoction" and drinking 1.5%hypertonic saline for six weeks. Observing the degree of irritation and conjunctiva hyperemia and measuring the blood pressure and rotation time after six weeks.(2)The model of hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome was made through injecting TypeⅦcollagenase into the right globus pallidus(GP) of rats by stereotaxic method on the base of the model of hypertensive with Liver-Yang Hyperactivity Syndrome.Then,the brain tissues were made sections,stained by HE method,to evaluate the histological changes.Furthermore,neuropathological profile was observed at the same time.(3)The model of cerebral ischemia with Liver-Yang Forming Wind Syndrome was made through occluding the middle cerebral artery on the base of the model of hypertensive with Liver-Yang Hyperactivity Syndrome.Then,the brain tissues were made sections,stained by TTC method,to evaluate the histological changes.Furthermore, neuropathological profile was observed at the same time.(4) The total proteins of pituitary,adrenal gland,thyroid gland and splenic lymphocyte in the rats were separated by two-dimensional gel electrophoresis(2-DE) respectively.The gels of 4 groups were stained by Coomassie brilliant blue,scanned by ImageScanner and analyzed in PDQuest software.The undifferential and differential expression protein spots were identified by peptide mass fingerprint(PMF) based on matrix -assisted laser desorption/ionization time of flight mass spectrometry(MALDI- TOF-MS) and database searching,then some of them were validated by RT-PCR and western blotting.Results:(1)Copying rats model:Forty-eight rats were reproduced into a successful model of hypertensive with Liver-Yang hyperactivity Syndrome among 60 rats,with the incidence of 80.0%approximately. The 48 rats were randomly divided into three groups:hypertensive with Liver-Yang Hyperactivity Syndrome group including 14 rats, hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome group including 17 rats and cerebral ischemia with Liver-Yang Forming Wind Syndrome group including 17 rats.(2)Evaluation of the models:①hypertensive with Liver-Yang hyperactivity Syndrome model group with high aggression and conjunctiva hyperemia and high blood pressure and shortened rotation time.Compared with the normal control group the difference was significant(P＜0.05).②Affer operation,the majority of the hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome rats showed up conjunctival congestion and the aggression degree further deepened.Compared with the normal control group and hypertensive with Liver-Yang hyperactivity Syndrome,the difference were significant(P＜0.05).The rotation time of hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group shortened and blood pressure heightened.Compared with the normal control group and hypertensive with Liver-Yang hyperactivity Syndrome,the difference were significant(P＜0.05).After injecting TypeⅦcollagenase,the neurological deficits score of 13 rats are more than 2 scores.After occluding the middle cerebral artery,the neurological deficits score of 12 rats are more than 2 score.Lymphocytes,other kinds of leukocytes, glial cells and lots of red blood cells were observed in the tissues of hematoma area after stained by HE in the hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome group. Under staining with 0.1%TTC,the infarcted tissues caused by middle cerebral artery occlusion(MCAO) were white in color on the injured side,while the normal brain tissues on the non-injured side and the sham-operated controls were pink in color in cerebral ischemia with Liver-Yang Forming Wind Syndrome groups.(3) Establishment of 2-DE maps in the rat pituitary,adrenal gland,thyroid gland and splenic lymphocyte and image analysis:Well-resolved and reproducible 2-DE maps of rat pituitary,adrenal gland,thyroid gland and splenic lymphocyte from normal,hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group were acquired.As compared with the control 2-DE maps,the gels of the pituitary displayed approximately 1100±32 protein spots,the 2-DE maps of the adrenal gland displayed 800±56 spots,the 2-DE maps of the thyroid gland displayed 500±17 spots,and the 2-DE gels of the spleen lympholeukocytes displayed 1200±24 spots.The location and abundance of the majority of protein spots are consistent,mainly distributed in the pH4-8 and Mr20-60kD range.(4) Select the undifferential and differential expression proteins for further analysis by MALDI-TOF-MS.46 spots more than 2 folds differential expression protein spots were identified.The proteins were divided into seven groups based on their functions using information obtained from the Swiss-Prot and NCBInr websites:antioxidant,metabolic enzymes,protein biosynthesis and degradation,structural proteins, chaperone,thromboxane enzyme,and so on.Steroidogenic acute regulatory protein,ubiquitin carboxyl-terminal hydrolase isozyme L1, dihydropyrimidinase-related protein 2,peroxiredoxin may concerned with the neuroendocrine-immune network in Liver-Yang hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome.(5)Viladation of differentially expressed protein:①The results of RT-PCR:STAR mRNA、DRP-2 mRNA and PRDX2 mRNA is stronger in hypertensive with Liver-Yang Hyperactivity Syndrome, hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal contral group(P＜0.01) and it is also stronger in hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05).UCH-L1 mRNA is weaker in hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal contral group(P＜0.05) and it is also weaker in hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05).The expression level of STAR mRNA、DRP-2 mRNA、UCH-L1 mRNA and PRDX2 mRNA in pituitary and adrenal gland are identical.②The results of Western blotting:STAR is stronger in hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal contral group(P＜0.01) and it is stronger in hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05).UCH-L1 mRNA is weaker in hypertensive with Liver-Yang Hyperactivity Syndrome, hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal control group(P＜0.05) and it is weaker in hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05). The results are consistent with the results of proteomics and RT-PCR. Conclusion:(1)Founded successfully animal models of hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome.The models have the characteristic of combined Disease and Syndrome.(2) The forming process of Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome is a complex pathological process which many proteins involved in.The neuroendocrine-immune system was obviously changed,which suggest that the neuroendocrine - immune network of the two syndromes are obvious disturbed.(3) There are undifferential and differential proteins between Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome which suggest that two syndrome have the same basic material and different nature connotations.更多还原