针刺太冲穴对SHR大鼠延髓蛋白差异性表达影响的研究

发布时间：2018-08-23 14:47

【摘要】：目前现代医学对原发性高血压的发病原因及发病机制的研究还不能够作出明确阐述,多数认为其是多种因素相互作用导致的以患者动脉血压升高为特征的心血管综合征,其常随着病程的发展而伴有一系列心血管系统的损害改变,严重威胁人类的生命健康。现代医学针对本病已研发并改良出多种一线抗高血压治疗西药,具有较好降压效应及一定的心血管系统保护作用,在较大程度上控制了本病病程的不良发展及降低并发症的发生,但目前临床常用的控制高血压西药治疗仍存在一定的缺陷,如对患者头部昏沉、头晕、失眠等自觉症状改善程度不甚理想；某些治疗药物仍具有相对明显的副作用,如对患者糖耐量、脂质代谢的不良影响、可引起外周水肿,甚至有研究还认为部分高血压患者的性功能障碍可能与利尿剂等药物有关,以上缺陷均对患者日常生活质量造成不良影响。并且多数本病患者需要长期甚至终身服用控制血压药物,这一笔长期的医疗支出,对于患者个人、家庭以及社会而言均是长期的经济负担,尤其在我国部分经济欠发达的农村甚至三、四线城市的医保资金支出幅度尚未能与一线城市相比拟,故而这部分地区的患者及其家庭仍将承担较多的支出费用,相应则更是明显地增加了其家庭经济负担。本病西医病名虽在中医学古代文献无明确记载,但现代中医学研究根据本病的临床症状及其证候表现,多将其归属于传统中医学的“眩晕”、“肝风”、“头痛”等范畴进行辩证治疗。针灸疗法作为中医疗法一重要组成部分,从历代针灸文献及现代学者开展的多项针灸降压疗效观察研究均可看出,针灸降压具有良好的降压效应,且突出表现在短期降压效应尤其对收缩压的良性调节方面,以及对患者临床不适症状的明显改善和毒副作用明显低于西药降压药物治疗,并且价格低廉、操作简便,是值得大区域推广应用。但目前对针灸降压机制尚无法明确阐述,在一定程度上限制了在更大范围乃至全球范围的推广,故而对针灸降压机制的研究显得异常重要。针灸治疗以经络理论为理论基础,人体经络内连脏腑,外络肢节,将人体连为一有机整体,故而针灸治疗效应常是人体多靶点共同作用的结果,这对其治病机制、尤其组穴处方的针灸效应机制的阐述增加了许多难度。根据经穴-脑相关假说,针灸的治病必须先经过脑作为信号枢纽进行调整和整合之后才能作用到生物体的各个靶点,进而起治疗效应。故而针灸降压机制的中枢及外周神经、体液因子调节机制应该为互相作用的关系,各机制间不可截然分开,由于目前研究结果对各部分机制的相互关系尚未能很好的阐述清楚,所以本课题研究仍是以中枢机制为研究目标。本课题前期实验研究结果表明针刺SHR大鼠太冲穴虽然未能使血压下降至正常水平,与模型组大鼠血压情况相比仍具有良好的降压效应,但实验中选取的针灸学理论认为对眩晕、头痛等症状有改善作用的曲泉、冲阳针刺治疗均未发现有良好的降压效应,再次验证了腧穴的治疗效应具有特异性。同时,目前研究普遍认为延髓里存在最低级最基本的心血管调节中枢,也含有许多在中枢神经系统上、下行投射联系通路中不可缺少的神经核团,均认为其与血压变化调节密切相关。目前对针刺能否影响延髓进而出现相关调节效应以及针刺调控高血压的中枢靶点机制中蛋白质相关的具体途径尚未能明确阐述,故而本课题选取与血压调节密切相关的延髓为研究对象,研究实验中所取经穴产生差异性降压效应的中枢机制,进而探讨经穴效应特异性的中枢机制。目的：在课题组前期研究所发现太冲、曲泉、冲阳及非穴针刺降压具有差异性、穴位效应具有特异性的基础上,继续选取以上腧穴进行针刺干预,应用质谱分析及蛋白组学技术,研究同样具有改善眩晕、头痛症状但治疗效应具有特异性的太冲、曲泉、冲阳针刺干预后各相应SHR大鼠延髓的蛋白表达差异情况,为阐述针刺降压效应中枢机制及经穴效应特异性的中枢机制研究提供一定的理论线索。方法：选用SPF级9周龄雄性自发性高血压大鼠55只,随机分为太冲组、曲泉组、冲阳组、非穴组和模型组,每组11只。选择9周龄正常雄性Wistar大鼠8只作为正常组。太冲组、冲阳组、曲泉组和非经穴组分别针刺双侧太冲、冲阳、曲泉和非穴各5min。针刺时使用袜套套住大鼠头部进行固定。在大鼠清醒状态下进针,行捻转手法,捻转频率80±10次/min,捻转角度180°±30°。先左后右,两侧各针刺2.5min,共5min。上午十二时前完成针刺,每天一次,连续七天。正常组、模型组大鼠以同样方法抓握固定5min,不予针刺。第七天针刺结束后将各组大鼠端头取脑并分离延髓组织,然后经研磨组织、提取蛋白、二维凝胶电泳分离蛋白、扫描图像、图像分析等步骤获得各组差异蛋白点,再用质谱仪分析蛋白并从MASCOT数据库中检索、鉴定出各组的差异蛋白。结果：(1)用Imagemaste 2D 5.0软件对获取的二维凝胶电泳图进行分析,以模型组作为参考胶与其他各组结果进行比较,各组大鼠相应的延髓表达差异蛋白点结果如下：①太冲组大鼠表达上调的差异蛋白点有15个,下调的差异蛋白点有11个；②冲阳组大鼠表达上调的差异蛋白点有34个,下调的差异蛋白点有28个；③曲泉组大鼠表达上调的差异蛋白点有14个,下调的差异蛋白点有13个；④非穴组大鼠表达上调的差异蛋白点有33个,下调的差异蛋白点有27个。⑤正常组大鼠表达上调的差异蛋白点有28个,下调的差异蛋白点有13个;(2)全部实验大鼠的差异蛋白质点鉴定检查结局：各实验组共得出216个差异蛋白点,选取表达差异明显并剔除重复点后进行蛋白检索鉴定,各组检索结果共得出37个蛋白,具体如下：①太冲组表达上调的有6个：Protein LOC100911959:对细胞的生长还有发育、细胞周期及信号转导起调节作用；Transitional endoplasmic reticulum ATPase:与细胞凋亡、生长以及DNA的修复相关,在一些肿瘤中也是高表达并和肿瘤分型、分期、转归有关；MICOS complex subunit Mic60 (Fragment):参加调节线粒体,调节蛋白转运加工效率；Dihydropyrimidinase-related protein 3:参加轴突延长及神经元极化形成,参与微管的动态调节；Cathepsin B:参与溶酶体降解蛋白质的过程和免疫反应抗原的加工、激素的活化；Peroxiredoxin-2:红细胞中主要的过氧化氢清除者,保护红细胞及红细胞膜。②冲阳组上调的有4个：AP-2 complex subunit beta:载脂蛋白亚基,血浆脂蛋白组成部分,参与脂类代谢；Endoplasmin;Triosephosphate isomerase:糖酵解过程中重要的异构酶,影响能量的有效生成；失调可导致免疫系统失调的多系统疾病；ATPsynthase subunit d, mitochondrial。下调的蛋白有6个：Succinyl-CoA ligase subunit beta; Mannose-6-phosphate isomerase; Protein Serpinb6:蛋白酶抑制因子;Eukaryotic translation initiation factor 2 subunit 1:参与真核细胞中的蛋白质的翻译,和肿瘤的发生发展有很大关系；Anamorsin; Protein Plcxd3,参与脂类代谢。③曲泉组上调的有4个：Septin-11:参与细胞胞质分裂、细胞内物质转运、凋亡等细胞生理过程,控制胚胎在发育过程中相关的细胞的迁移；Glutamine synthetase:参与清理体内游离铵离子的过程,对机体起保护作用；Heat shock cognate 71 kDa protein:维持细胞的生存和功能,提高细胞的抵抗力,协同免疫作用；SAPdomain-containing ribonucleoprotein.-参与细胞生长、代谢及癌变的重要转录或翻译控制。下调的蛋白有4个：Lamin-B1:影响细胞增殖、迁移,对氧化应激和信号通路发挥影响,在一些癌组织里面呈高表达；cAMP-dependent protein kinase type II-alpha regulatory subunit:调节蛋白质磷酸化；Secernin-1:调节细胞胞吐作用,在一些肿瘤中呈高表达；Septin 8 (Predicted):参与细胞内功能调节,与神经递质的传递与神经功能障碍等疾病直接相关。④非穴组上调的有3个：Heat shock 70 kDa protein 4:参与热激响应,可能有心肌保护作用；Alanine--tRNA ligase, cytoplasmic:参与蛋白质生物合成过程；Dihydropyrimidinase-related protein。下调的有2个：Hsp90 co-chaperone Cdc37:参与肿瘤生理与病理的多个过程；Guanine nucleotide-binding protein G(o) subunit alpha:参与离子通道及信号转导调节,一些肿瘤的发生可能和它的紊乱有很大关系。⑤模型组对比于正常大鼠,其表达上调的蛋白有2个：Glycine--tRNA ligase (Fragment):催化氨基酸与tRNA结合的特异性酶,参加了基因的表达控制,可以抑制细胞凋亡；Spermatid-associated protein:和不孕不育有关。表达下调的有6个：Protein disulfide-isomerase A3:参与细胞的信号转导,促进糖蛋白折叠质量控制在内质网中；Fructose-bisphosphate aldolase C:和一些肿瘤细胞中起诱导作用的糖代谢酶有关系；Biliverdin reductase A:参加了细胞信号通路的磷酸化的调节,影响了代谢的相关蛋白、凋亡的分子细胞周期等相关蛋白、信号通路的蛋白,免疫分子还有氧化应激因子等的基因表达；Inositol monophosphatase 1:参加了磷脂酰肌醇信号的传递过程；Phosphoribosyl transferase domain containing 1:和杀伤肿瘤细胞功能相关。结论：1.SHR大鼠相较于wistar大鼠其延髓中参与胰岛素代谢及能量代谢的部分相关蛋白出现表达差异,结合其他学者相关研究推测血压变化可能与能量代谢异常有相关性；SHR大鼠可能比正常大鼠更易患免疫功能缺陷相关疾病。2.针刺太冲穴的降压效应中枢机制可能与延髓中参与能量代谢部分相关蛋白密切相关；针刺冲阳穴可导致延髓中参与影响脂类代谢部分相关蛋白表达差异,与其具有胃经调节生物体水谷代谢的循经治疗效应相吻合；针刺曲泉穴可导致延髓中参与影响免疫反应的部分蛋白表达差异,与其作为作为肝经合穴可直达调理肝脏进而调气机、理肝血而调和人体阴阳相吻合。以上各经穴针刺作用效应是多靶点多渠道一起作用的结果,为针刺太冲穴降压效应中枢机制及经穴效应特异性研究提供一定研究参考。3.针刺非穴对大鼠延髓蛋白表达的影响其作用靶点相对来说较为简单且无明显规律性。4.经穴治疗效应及其相应中枢作用机制具有特异性。
[Abstract]:At present, the etiology and pathogenesis of essential hypertension can not be clearly explained in modern medicine. Most of them believe that it is a cardiovascular syndrome characterized by elevated arterial blood pressure caused by the interaction of multiple factors. It often accompanies a series of cardiovascular system damage changes with the development of the course of the disease. Modern medicine has developed and improved a variety of first-line antihypertensive western medicine for this disease, which has better antihypertensive effect and certain cardiovascular system protection, to a large extent, to control the adverse development of the disease and reduce the incidence of complications, but currently commonly used in clinical control of hypertension. There are still some defects in the treatment of Western medicine, such as unconscious symptoms such as dizziness, dizziness, insomnia and so on. Some drugs still have relatively obvious side effects, such as adverse effects on glucose tolerance and lipid metabolism of patients, can cause peripheral edema, and even some studies believe that sexual function of hypertension patients. Obstacles may be related to drugs such as diuretics, which have a negative impact on the quality of daily life of patients, and most patients need long-term or even lifelong use of blood pressure control drugs. This long-term medical expenditure is a long-term financial burden for patients, families and society, especially in China. The expenditure of medical insurance funds in the underdeveloped rural areas and even in the third and fourth tier cities has not been comparable to that in the first tier cities, so the patients and their families in this part of the country will still bear more expenditure expenses, and the corresponding economic burden of their families will be obviously increased. However, according to the clinical symptoms and symptoms of the disease, modern Chinese medicine studies usually classify it as "dizziness", "liver wind" and "headache" in traditional Chinese medicine for dialectical treatment. The study shows that acupuncture and moxibustion have good antihypertensive effect, especially in short-term antihypertensive effect on the benign regulation of systolic blood pressure, as well as the obvious improvement of clinical discomfort symptoms and side effects of patients are significantly lower than those of Western antihypertensive drugs, and the price is low, the operation is simple, it is worthy of wide regional application. But at present, the mechanism of acupuncture and moxibustion can not be clearly explained, to a certain extent, limiting the promotion in a wider range and even the global scope, so the study of acupuncture and moxibustion mechanism is very important. The therapeutic effect of moxibustion is often the result of multi-target interaction of the human body, which makes it difficult to explain the therapeutic mechanism, especially the acupuncture effect mechanism of acupoint prescription. Therefore, the central and peripheral nerves of acupuncture and moxibustion hypotensive mechanism, humoral factors regulation mechanism should be interactive relationship, each mechanism can not be completely separated, because the current research results on the mechanism of the relationship between the various parts of the mechanism has not been well explained, so this topic is still to study the central mechanism. Previous experimental results showed that acupuncture at Taichong acupoint of SHR rats could not reduce blood pressure to normal level, and had a good effect on reducing blood pressure compared with the model group rats, but the acupuncture theory selected in the experiment suggested that Ququan could improve the symptoms of dizziness and headache, and Chongyang acupuncture treatment was not found. At the same time, it is generally believed that the medulla oblongata contains the lowest and most basic cardiovascular regulatory center, as well as many nuclei indispensable to the downward projection pathway of the central nervous system. At present, it is not clear whether acupuncture can affect the medulla oblongata and then produce related regulatory effects and the specific pathways related to protein in the mechanism of acupuncture regulating hypertension central target. Therefore, this topic selected the medulla oblongata which is closely related to blood pressure regulation as the research object to study the differential hypotension of the points selected in the experiment. Objective: To investigate the central mechanism of acupoint effect and its specificity. The difference of protein expression in the medulla oblongata of SHR rats after acupuncture intervention by Taichong, Ququan and Chongyang was also found to improve the symptoms of dizziness and headache, but the therapeutic effect was specific. Fifty-five male spontaneously hypertensive rats were randomly divided into Taichong group, Ququan group, Chongyang group, non-acupoint group and model group with 11 rats in each group.Eight normal male Wistar rats aged 9 weeks were selected as normal group. The head of the rats was fixed. The rats in the normal group were treated with the same method for 5 minutes without acupuncture. On the seventh day, the rats in the model group were treated with the same method for 5 minutes without acupuncture. After acupuncture, the brain of rats in each group was taken out and the medulla oblongata was separated, and then the proteins were extracted from the ground tissues, separated by two-dimensional gel electrophoresis, scanned images and image analysis. The proteins in each group were analyzed by mass spectrometry and retrieved from MASCOT database. Results: (1) Ima was used to identify the proteins in each group. Gemaste 2D 5.0 software was used to analyze the obtained two-dimensional gel electrophoresis map. The results were compared with those of other groups using model group as reference gel. The results of the corresponding differential protein spots in the medulla oblongata of rats in each group were as follows: 1. There were 15 up-regulated and 11 down-regulated differential protein spots in Taichong group; 2. There were large differences in Chongyang group. There were 34 differential protein spots up-regulated and 28 down-regulated in mice, 14 up-regulated and 13 down-regulated in Ququan group, 33 up-regulated and 27 down-regulated in non-acupoint group. There were 28 protein spots and 13 down-regulated protein spots; (2) The results of differential protein spot identification in all experimental rats: 216 different protein spots were obtained in each experimental group, and 37 proteins were obtained in each group after distinct expression differences and repetitive points were removed. Protein LOC100911959: regulates cell growth and development, cell cycle and signal transduction; Transitional endoplasmic reticulum ATPase: is associated with apoptosis, growth and DNA repair; is also highly expressed in some tumors and is associated with tumor typing, staging, and prognosis; T Mic60 (Fragment): participate in the regulation of mitochondria and protein transport processing efficiency; Dihydropyrimidinase-related protein 3: participate in axon elongation and neuronal polarization formation, participate in the dynamic regulation of microtubules; Cathepsin B: participate in lysosome protein degradation process and immune reaction antigen processing, hormone activation; Peroxiredoxin-related protein 3: participate in the process of neuronal polarization and microtubule dynamic regulation; Cathepsin B: participate in lysosome protein degradation process 2: The main scavenger of hydrogen peroxide in red blood cells, protecting red blood cells and erythrocyte membranes. ATP synthase subunit d, mitochondrial. down-regulated six proteins: Succinyl-CoA ligase subunit beta; Mannose-6-phosphate isomerase; Protein Serpinb6: protease inhibitor; Eukaryotic translation initiation factor 2 subunit 1: participating in eukaryotic fineness Anamorsin; Protein Plcxd3, involved in lipid metabolism. 3. Four up-regulated proteins in Ququan group: Septin-11: involved in cytokinesis, intracellular substance transport, apoptosis and other cellular physiological processes, controlling the cell migration during embryonic development; Synthetase: Involves in the process of clearing free ammonium ions in the body and plays a protective role in the body; Heat shock cognate 71 kDa protein: maintains cell survival and function, improves cell resistance, synergistic immunity; SAP domain-containing ribonucleoprotein. - Involves in the important transcription or translation control of cell growth, metabolism and canceration. Lamin-B1: affects cell proliferation, migration, oxidative stress and signaling pathways, and is overexpressed in some cancerous tissues; cAMP-dependent protein kinase type II-alpha regulatory subunit: regulates protein phosphorylation; Secernin-1: regulates cell exocytosis, and is overexpressed in some cancers; Predicted: involved in the regulation of cellular function, and directly related to neurotransmitter transmission and neurological dysfunction and other diseases. Two of the opyrimidinase-related proteins were down-regulated: Hsp90 co-chaperone Cdc37: involved in many physiological and pathological processes of tumors; Guanine nucleotide-binding protein G(o) subunit alpha: involved in ion channels and signal transduction regulation, and some tumors may be associated with its disorders. _The model group was compared with the normal group. In rats, there are two up-regulated proteins: Glycine-tRNA ligase (Fragment): a specific enzyme that catalyzes the binding of amino acids to tRNA, participates in gene expression control and inhibits cell apoptosis; Spermatid-associated protein: associated with infertility and infertility. Signal transduction promotes folding quality control of glycoproteins in the endoplasmic reticulum; Fructose-bisphosphate aldolase C: is related to the inducible glycometabolism enzymes in some tumor cells; Biliverdin reductase A: participates in the regulation of phosphorylation of cell signaling pathways, affecting metabolic related proteins, and the molecular cell cycle of apoptosis. Inositol monophosphatase 1: participated in phosphatidylinositol signal transduction; Phosphoribosyl transfer domain containing 1: correlated with the function of killing tumor cells. CONCLUSION: 1. SHR rats in the medulla oblongata compared with Wistar rats. Some related proteins involved in insulin metabolism and energy metabolism were expressed differently, and combined with other related studies, it was speculated that the changes of blood pressure may be related to abnormal energy metabolism; SHR rats may be more susceptible to immunodeficiency-related diseases than normal rats. 2. The central mechanism of hypotensive effect of acupuncture at Taichong point may be related to the medulla oblongata. Acupuncture of Chongyang acupoint can lead to some related proteins in medulla oblongata involved in affecting lipid metabolism.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

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