【作者】 夏欧东；

【导师】 吕志平；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2013， 博士

【摘要】 研究背景：辨证论治是中医学的特色和精髓,突出了个性化的中医思路。“证”是中医学的一个特有概念,是对疾病过程中所处一定阶段的病位、病因、病性以及病势所作的病理性概括。证是机体内因和环境外因综合作用的机体反应状态,随着病程的发展而不断发生变化。证是对疾病当前本质所作的结论。因此,证的研究是中医学现代化研究的核心内容。近年来,随着基因组测序工作的完成,人们研究的重点已经从基因转移到蛋白质功能方面,蛋白质在疾病的发生发展中承担着重要的角色,这使得蛋白质组学在人类疾病的研究中有着极为重要的价值。蛋白质组学是一门在整体水平上高通量、大规模地研究蛋白质组成及其动态变化的活动规律,从而得到疾病、生理生化过程中的整体情况的新兴学科。蛋白质组学从一个机体或一个组织,一个细胞等不同层次“整体”的蛋白质活动的角度,来揭示和阐明证候形成和发展的基本规律。其理论和技术特点以及其整体、动态性的核心思想,与中医学的“整体观”和“辨证观”等理论体系有很大的相似性,这种研究思路与中医整体观和中药多靶点整合调节的特点不谋而合,具有明显的中医特色。肝郁证是由于肝的疏泄功能异常,疏泄不及而致气机郁滞所引起的情绪不宁、胸胁闷胀或易怒善哭为特征的一类临床病证,常见于临床多种疾病。临床流行病学调查显示,肝脏病证在五脏病证中占总体病证的40%,而肝郁证又是肝病证候的核心,也是中医肝病发病学的重要环节,因此肝郁证的本质研究一直是中西医结合领域研究的热点。近年来对肝郁证本质的研究颇多,已初步证实肝郁证具有现代病理生理学基础,并部分地阐释了中医理论的科学性,取得了阶段性成果：认为肝郁证和应激密切相关,并涉及机体神经、内分泌、循环、消化、免疫等多系统多器官的病理改变。近年来对应激相关的下丘脑-垂体-肾上腺皮质系统(Hypothalamic Pituitary Gonadal Axis,HPA)和蓝斑-交感-肾上腺髓质系统(locus coeruleus-sympathetic-adrenal medulla system,LC-NE)研究较多,我们课题组之前的研究表明肝郁证可引起蓝斑中酪氨酸羟化酶(tyrosine hydroxylasw,TH)、c-fos表达的上调,进一步阐明了蓝斑和肝郁证的相关性。肝郁证是中医经典病证,其方证研究也一直是其本质研究的一个重要方面。中医证候研究具有“以方测证,方证互参”的特点。一方面通过辨证的结果来确立相应的治法方药,一方面又根据方药的组成和功效来推测疾病的病机和症状。理论上讲,每一个方剂都有自己使用的病证,只有当其功效与疾病的病机相应时,方剂的效用才达到最佳。通过这一特征,我们可以通过建立预设的动物疾病模型,然后给予预设的方药,选择和疾病证候相关的差异指标,通过对用药后动物的观察和研究,确认该模型是否为预设的疾病模型。同时,在确定模型的情况下,选用特定方剂,通过观察差异指标,可以证实该方剂是否是治疗此病证的有效方剂。因此,本课题以蓝斑为切入点,通过蛋白质组学进行差异蛋白鉴定,并从中选取鉴定出的已知蛋白羧肽酶A抑制剂(Latexin/endogenous carboxypeptidase inhibitor,ECI),沉默信息调节基因2(silent information regulator2,Sirt2),转体基因蛋白(Transthyretin,TTR),并通过逍遥散进行反证,同时选用了西药阳性对照盐酸氟西汀(氟西汀是临床经典抗抑郁药,可有效改善闷闷不乐,兴趣减退等肝郁证症状),希望为蓝斑和肝郁证的相关性提供更进一步的证据,并对进一步研究肝郁证的本质打下一定的基础。研究目的：采用孤养加慢性不可预知温和刺激(chronic unexpected mild stress,CUMS)制备肝郁证大鼠模型,观察肝郁证大鼠在一般状态、行为学及从蛋白质的角度,对肝郁证模型大鼠蓝斑组织蛋白质组的差异表达进行比较分析并进行验证,从动物模型的角度找到肝郁证相关的蛋白质组群,从而为建立证候的微观辨证指标体系奠定基础,也为从临床疾病的角度对肝郁证相关蛋白质组的全面研究提供技术和理论支持。并通过比较逍遥散组和氟西汀组与模型组大鼠之间的蛋白表达差异,探讨逍遥散对肝郁证的作用机理,并以方测证,方证互参,为肝郁证本质的阐述提供客观依据。研究方法：本研究在肝郁证理论指导下建立孤养加CUMS肝郁证大鼠模型。并运用蛋白质组学研究中常用的二维凝胶电泳(two-dimensional gel eleetrophoresis,2-DE)分离方法,结合基质辅助激光解吸电飞行时间质谱技术(matrix assisted laser desorption time-of-flight mass spectrometry,MALDI-OF-MS),分析模型组大鼠蓝斑组织的蛋白质组和正常组大鼠之间的表达差异。1、孤养加CUMS肝郁证大鼠模型的建立分两次造模,第一次用于差异蛋白的检测和鉴定,第二次用于蛋白功能的验证。参照相关文献报道,从慢性心理应激的角度出发,将18只雄性SD大鼠随机分为正常组,模型组,逍遥散组,每组6只(第二次为72只,分组增加盐酸氟西汀组,每组18只)。除正常组外,其他各组大鼠均单笼饲养,并给予慢性不可预知温和刺激,每天2次,持续3周。逍遥散组在模型制备的基础上加用逍遥散干预,氟西汀组加用氟西汀干预,正常组不给任何干预,群养。2、蛋白质样本的提取和样本混合以液氮研磨蓝斑组织,低温裂解蓝斑细胞,提取蓝斑组织的蛋白质样本。采用BSA法对蛋白质样本进行定量。对组内的蓝斑组织,按等质量进行样本混合。3、蓝斑组织的2-DE分离在常规蛋白质组2-DE分离技术基础上,引入“盐桥”和低电压除盐方法,进一步优化蓝斑组织蛋白质组2-DE分离技术。对同类混合样本以组间配对的形式,同时进行2-DE分离,并重复3次。4、凝胶染色和图像分析对2-DE分离所获得的凝胶,采用新型的胶体考染技术,不仅可以提高凝胶染色的灵敏度,而且还能减少对后期质谱检测的干扰。凝胶经过染色和脱水后,用光密度透射扫描仪扫描而形成图像。用PDQuest7.0软件分析凝胶图像,获取每个蛋白质的表达量、等电点、分子量等信息。并将组间表达量相差2倍以上的蛋白质斑点设定为差异表达蛋白质,由软件自动生成分析结果,再加以人工验证。5、差异表达蛋白质斑点质谱鉴定和数据库确认用MALDI-TOF-MS对分析所获得的差异蛋白质斑点进行肽质量指纹(peptide mass figerint,PMF)的检测。首先,在凝胶上切取差异表达蛋白质斑点,再经水洗、脱色、还原与烷基化、酶解、萃取、点样和质谱分析等步骤获取每个蛋白质斑点的PMF。然后,利用Masoct软件搜索各大生物和蛋白质相关数据库,寻找与目的PMF相匹配的蛋白质,并查询该蛋白质的相关信息。6、运用化学发光免疫法检测各组大鼠血清中三碘甲状腺氨酸(T3)、甲状腺素(T4)和促甲状腺激素(TSH)含量。7、运用苏木精-伊红(hematoxylinandeosinstain,HE)染色,观察大鼠蓝斑的形态学变化。8、用免疫组化法(immunology and histology chemistry,IHC)检测蓝斑中Laxetin、 Sirt2和TTR的表达变化。用Imageproplus6.0软件进行分析。9、运用免疫印迹法(weastern blot, WB)检测Laxeti、Sirt2和TTR在蓝斑中的表达变化10、用实时荧光定量聚合酶链反应(real time-polymerase chain reaction,RT-PCR)技术检测Laxetin、Sirt2和TTR中mRNA在蓝斑中的表达情况。11、所有数据用spss13.0软件进行分析统计。实验结果均用平均值±标准差(X±S)表示,采用SPSS13.0软件统计包处理。体重的变化采用重复测量数据的方差分析,其余指标多样本均数比较采用单因素方差分析,方差齐性,多重比较采用LSD法检验,若方差不齐,采用Welch分析,多重比较采用Tamhane’s T2法检验,以P<0.05作为具有显著性差异。结果：1、成功制备了孤养加CUMS肝郁证大鼠模型。造模3周后,从一般情况来看,模型组大鼠开始表现为躁动,易激惹,随后逐渐安静,表现为倦怠少动,喜欢贴壁,反应迟钝,食欲减退,伴有毛色暗淡无光泽,松散,大便稀软等肝郁证表现。逍遥散组和氟西汀组上述反应明显低于模型组,且两药物组间无显著性差异(P>0.05)。而正常对照组未见异常行为状态。各组间体重增长存在显著差异(P=0.017),模型组生长较缓慢,逍遥散组和氟西汀组服药后体重增长缓慢的情况得到改善,且与模型组有显著差异(P=0.000)。糖水消耗实验提示各组间糖水偏好存在显著性差异((P=0.000),模型组的糖水偏好度明显小于其他组。旷场实验发现模型组大鼠的直立次数和爬格数均显著低于正常组(P=0.000),逍遥散组和氟西汀组则显著优于模型组,但和正常组比较仍有差异(P=0.000)。说明孤养加CUMS确能造成大鼠的肝郁证,能有效的模拟临床中肝郁证兴趣丧失、快感缺乏等症状。2、获得了高质量的蓝斑组织蛋白质组2-DE凝胶电泳图像。经2-DE分离和对凝胶图像的软件分析,发现了21个蛋白质斑点在三组间(正常组,模型组,逍遥散组)有差异表达。3、差异蛋白质斑点的质谱检测：出峰率达100%,经过对PMF的分析和数据库检测,确定肝郁证模型大鼠中表达具有显著性差异的蓝斑组织蛋白质17个。4、血清中T3和T4含量在模型组中最高,逍遥散组和氟西汀组次之,正常组中含量最低(P=0.000),TSH含量则在各组大鼠间的差异均无统计学意义(P=0.740)。5、HE染色显示各组大鼠蓝斑无明显的形态学改变。6、IHC检测发现模型组蓝斑Laxetin、Sirt2和TTR的阳性神经元数目均小于正常组,染色强度减弱,平均光密度值也低于正常组。逍遥散组和氟西汀组则可以改善这种情况。7、WB检测模型组Laxetin、Sirt2和TTR的表达量均低于正常组,逍遥散组和氟西汀组则较模型组表达增加。8、运用RT-PCR技术检测Laxetin、Sirt2和TTR的mRNA在LC中的表达,发现与正常组比较,模型组表达量均减少,逍遥散组和氟西汀组则较模型组升高。结论：对肝郁证孤养加CUMS动物模型进行了蓝斑的蛋白质组的研究,通过分析模型大鼠蓝斑组织蛋白质组的差异表达,获得和肝郁证动物模型密切相关的差异蛋白21个,其中17个可以获得数据库的匹配信息,确定蛋白质名称。我们在这17个蛋白质中选取了Laxetin、Sirt2和TTR这三种蛋白进行进一步的验证。这3种蛋白双向电泳结果提示Latexin和TTR在模型组表达下降,Sirt2在模型组表达上升。我们又采用IHC、WB、RT-PCR三种实验技术同时证明在肝郁证中Laxetin、Sirt2和TTR蛋白表达下降,且逍遥散和氟西汀可以改善这一情况,提示这三种蛋白参与了肝郁证的发病过程,且逍遥散和氟西汀均对其有效,疗效无显著差异。同时提示双向电泳鉴定出的蛋白表达情况只是前期的一种提示,并不一定是100%匹配的,这样我们后面采用三种不同技术验证3种蛋白的表达增加了结果的可信度。该结果为进一步研究肝郁证的分子机制提供了理论基础,也使肝郁证的实质研究有了进一步的发展。但关于这三种蛋白质和肝郁证之间的直接关系尚未明确验证和阐释,有待进一步研究。更多还原

【Abstract】 Research background:Syndrome differentiation and treatment are the characteristic and the essence of the Traditional Chinese Medicine.It emphasizes the personalized thinking of TCM."Syndrome" is a unique concept of TCM, it refers to the pathological generalization of the location, etiology, nature and the progression of diseases.Syndrome is a state of our body caused by internal and external factors, it is changing with the development of disease.Syndrome is conclusion about the current essence of the disease.So, the study of nature of disease is the central content of the modernization of TCM.In recent years,with the draft of the human genome has been completed,the focus of research is moving beyond genomics to proteomics.Protein has been playing an increasingly important role in the development of disease.This makes Proteomics quite valuable in the study of human diseases.As a new subject,proteins research the composition and of protein and its dynamic variety from the whole to grasp the whole situation of the physiological and biochemical process in the disease.Proteomics is an new research field that focuses on a whole set of proteins in a cell or a tissue of an organism.lt explains and clarifies the basic rules of the formation and development of Syndrome.It has overall and dynamic characters,and this is its core idea and it has its own theory and technology traits.From these points,Proteomics is very similar to the TCM theoretical system of the overall concept and individualization.This kind of research idea coincides with the characters of TCM.So this idea has obvious characters of Chinese medicine science.Liver depression syndrome is one of the common diseases which is caused by the dysfunction of liver and with the characters of depression,choking sensation in chest and irritable.Clinical epidemiology survey showed that liver diseases in the the viscera account for40%of the overall disease.Liver depression syndrome is the core syndrome of the liver disease and play an important role in the pathogenesis of the liver disease.Therefore,the nature of liver depression syndrome is always the hot topic in the area of Combine traditional Chinese and western medicine.There have been more and more researches about nature of the liver depression syndrome in recent years.Some preliminary research suggests that liver depression syndrome has modern pathophysiology basis and partly explain the scientificalness of TCM theory.We have achieved some stage achievements.Some researches indicate that Liver depression syndrome is likely to result from regulating function disorder of high nervous centre induced by negative psychic stress in which pathological changes of multi-system including nerve,endocrine,circulation,digestion,immunity,feeling and motion etc are involved.In recent years,the study of HPA and LC-NE axis has been developed rapidly.We seminar previously discovered liver depression syndrome can cause the up regulation of TH and c-fos in LC.This proved the relationship between LC and liver depression syndrome.Liver depression syndrome is one of the classic syndromes of liver disease of TCM.The research about its prescription and syndrome is always the most important aspect.The TCM Syndrome research has two feartures.One is to establish corresponding treatment through the result of syndrome differentiation,and the other is to infer the mechanism and symptoms of the disease from the composition and effect of the prescription.In theory,every prescription has its own applicable syndromes and only when the effect of prescription is appropriate for the mechanism of the disease,effect of the prescription can achieve optimal.Through this characteristic,we can establish the pre-determined animal disease models and then interfere them with the pre-determined prescription.After that, we select some abnormal indicators related to the disease and reaearch the animal models that are interfered to estimate whether the disease models are our pre-determined or not.At the same time,in the circumstance that the validation of the animal disease model is good,we observe and study some abnormal indicators and make sure the prescription we select is effect for this disease.Therefore, we hope to provide further evidence for the correlation of liver depression syndrome and LC and lay a solid foundation for the further study of the nature of liver depression syndrome by research into LC according to the methods of proteomics.Meanwhile, we selected some proteins and used xiaoyaosan to disprove it.Fluoxtine was selected as the positive-controlled medicine for its classic anti-depression effect.Objective:To set up the rat model of liver depression syndrome using the method of separation and chronic unpredictable mild stress to find the differentia of general condition and behaviorististics and from the point of proteomics,find the differentially expressed protein in the rat model of liver depressiom.And select the protein which we are interested in to verify.To discover the proteins related to liver depression syndrome from the reaearch of rat model and lay the foundation for the microcosmic differentiation indicatoers system of syndrome.At the same time, to provide technical and theoretical support for the comprehensive investigation on the related proteins from the point of clinical diseases.To explain the essence of liver depression syndrome and to study the clinical effect and mechanism of xiaoyaosan treating the it by comparing the differential expression proteins in the group of xiaoyaosan,fluoxetine and the model group.Methods:This study established the rat model of liver depression syndrome by the method of separation and chronic unpredictable mild stress(CUMS).We used two dimensional gel electrophoresis(2-DE)to sepatate the LC proteins,combining with matrix assisted laser desorption time-of-flight mass spectrometry(MALDI-OF-MS),to analyse the differential expression proteins in rat LC between the model group and the normal group.1. Established the rat model of liver depression syndrome by the method of separation and CUMS.We established two times of the rat model.The first time is for detection and identification to the differential proteins, the second time is for validation.We used the method of separation and CUMS according to the related literature reports. Eighteen female SD rats with SPF level and were into three groups according to a completely random method.Those were normal control group,liver depression syndrome model group,xiaoyaosan group and fluoxetine group.In each group, there were six rats.(in the second time,there were72rats and were randomly divided into four groups.) Except normal group,rats of other groups were feed separately and were induced by a group of CUMS2times every day for three weeks.Xiaoyaosan group was combined with the interferon of xioayaosan on the basis of the model rats and fluoxetine group with fluoxetine intervention.At the same time, the normal group was feed freely and collectively.2. The extraction of protein sample and sample mix.Grinded the LC organization using the liqid nitrogen and lysed the LC cells at a low tempreture.We extracted proteins from LC and made protein quantification by the method of BCA.And mixed the LC regnization in the same group according to the weight.3.2-DE separation of LC.We introduced the methods of salt brige and low voltage desalination on the basis of conventional separation technology of proteins to future optimize this technology.The samples were pair-matched.We made the2-DE separation for all the groups and repeated for three times.4. Gel staining and image analysis.A new kind of colloidal dye technology was used on the gel we obtained from the2-DE separation.On the one hand,this method can improve the sensitivity of gel staining,on the other hand,it can reduce the interference in the later mass-spectrometric detection.After dyeing and dehydration,the gels were scanned by optical density transmission scanners and the images were got. We used the software of PDquest7.0to analyze the images of gels and got the quantity of each protein expression,soelectric point and the molecular weight of proteins and so on.The protein spot whose expression has more than two times difference was defined to differentially-expressed protein,the results were automatically generated by the software and then were verified manually.5. The mass spectrometry identification and database confirm of differentially expressed protein spots.To the differentially expressed protein spots which we received,we applied MALDI-TOF-MS to make the detection of peptide mass figerint(PMF).We cut out the protein spots first and then got the PMF of every protein spot by the steps of water-washing, decolorizing, reduction and alkylation, digestion, extraction, sample and mass spectrometry.After these, we searched major biological and protein-related database by the software of Mascot, to find the proteins which are matched with the PMF we have got and know about the related information.6. CLIA to test T3, T4and TSH levels of serum in different groups.7. HE stain to observe morphological changes of LC.8. Immunohistochemical method to detect the expression changes of the protein we choose in LC and analyze the results by Imageproplus6.0software.9. Western blot method to detect the expression changes of the protein we choose in LC.10. RT-PCR technology to detect the expression changes of total RNA of the gene we choose in LC.11. Statistical analysis.The experimental results are expressed by x±s. SPSS13.0software was applied for it.ANOVA of repeated measures was applied for the weight changes.For the other indicators, the mean comparison of multiple samples used One Way ANOVA to analyse.When the variance was homogeneous, Least-significant difference (LSD) was applied for multiple comparison and when the variance was heteroscedastic, Tamhane’s T2was applied for multiple comparison and the value of P≤0.05would mean significant difference.Results:1. We made the rat model of liver depression syndrome succeedly.During the3weeks we interfered them by CUMS procedures,the model rats first were easily angery and then displayed the typical appearance such as low activity,slow reaction,reduces food and drink.Rats from xiaoyaosan group and fluoxtein group also showed some above abnormalities,but they were not obvious.There were no difference between the two medicine groups.Rats in normal group lived well.The rats’weight increase was significantly different between groups(P=0.017).It showed rats in model group grow slowest,while rats in xiaoyaosan group and fluoxetine group grow more than them.Fluid consumption test reavealed that the model group lose appetite for sweet water after3weeks.Statistics showed that there were differences between the three groups(P=0.000).The open field test reavealed that the erect times and travelling grids both reduced in model group and had statistical differences(P=0.000),xiaoyaosan group and fluoxetine group were better than it,but still have a gap with the normal group(P=0.000).The results showed that separation and CUMS could eatablish the model of liver depression syndrome succeedly and simulate clinical symptoms of liver depression syndrome such as interest loss and anhedonia.2.2-DE was used to separate the whole proteins of LC and the ideal2-DE maps were obtained.Tweenty one differential protein spots were checked out in the three groups(normal, model and xiaoyaosan group).3. We found seventeen differential proteins by the mass spectrometry identification and database confirm of differentially expressed protein spots.4. T3and T4levels in serum was highest in the model group and lowest in the normal group, the levels in xiaoyaosan group and fluoxetine group were between the two groups (P=0.000).TSH levels of serum in all the groups were not statistically significant (P=0.740).5. HE staining showed no obvious morphological change of locus coeruleus in all of groups. 6. Expression results of Immunohistochemical detection of LC showed that the number and mean density of Laxetin,Sirt2and TTR positive cells in model group were lower than others(P<0.05).7. Expression results of Weatern Blot showed that the expression of Laxetin, Sirt2and TTR were all lower than others(P<0.05).8. The expression of LaxetinmRNA, Sirt2mRNA and TTRmRNA by RT-PCR was observed that all were reduced in model group while xiaoyaosan group and fluoxetine group were higher than it.Conclusion:We eatablished the rat model of liver depression syndrome by separation and CUMS and researched the LC proteomics.21differential proteins related to liver depression syndrome were obtained,among them17proteins could be matched with the information in database to determine their names.Then,we selected the proteins of Latexin,Sirt2and TTR from the17proteins to further verified.Among the3proteins,2-DE results suggested that the expression of Latexin and TTR were reduced in model group and Sirt2was up-regulated.Then IHC,WB and RT-PCR technologys were used to prove that the expression of Laxetin,Sirt2and TTR was reduced in liver depression syndrome and xiaoyaosan,fluoxetine could improve this condition. The results revealed the three proteins involved in the process of liver depression syndrome.Xiaoyaosan and fluoxetine play the same role in it and have no differences. This result suggests that the expression of proteins identified by2-DE is just a prompt and is not necessarily a100%exact match.So we used three other methods to further verify the expression of the three proteins to increase the credibility.The results provided theoretical basis for the further research of the molecular mechanisms of liver depression syndrome, and make the essence study of liver depression syndrome have a further development.But the direct relationship between the three proteins and liver depression syndrome has not yet validated and elaborated. This remains to be further research.更多还原