【摘要】：目前現(xiàn)代醫(yī)學(xué)對原發(fā)性高血壓的發(fā)病原因及發(fā)病機(jī)制的研究還不能夠作出明確闡述,多數(shù)認(rèn)為其是多種因素相互作用導(dǎo)致的以患者動脈血壓升高為特征的心血管綜合征,其常隨著病程的發(fā)展而伴有一系列心血管系統(tǒng)的損害改變,嚴(yán)重威脅人類的生命健康�，F(xiàn)代醫(yī)學(xué)針對本病已研發(fā)并改良出多種一線抗高血壓治療西藥,具有較好降壓效應(yīng)及一定的心血管系統(tǒng)保護(hù)作用,在較大程度上控制了本病病程的不良發(fā)展及降低并發(fā)癥的發(fā)生,但目前臨床常用的控制高血壓西藥治療仍存在一定的缺陷,如對患者頭部昏沉、頭暈、失眠等自覺癥狀改善程度不甚理想；某些治療藥物仍具有相對明顯的副作用,如對患者糖耐量、脂質(zhì)代謝的不良影響、可引起外周水腫,甚至有研究還認(rèn)為部分高血壓患者的性功能障礙可能與利尿劑等藥物有關(guān),以上缺陷均對患者日常生活質(zhì)量造成不良影響。并且多數(shù)本病患者需要長期甚至終身服用控制血壓藥物,這一筆長期的醫(yī)療支出,對于患者個人、家庭以及社會而言均是長期的經(jīng)濟(jì)負(fù)擔(dān),尤其在我國部分經(jīng)濟(jì)欠發(fā)達(dá)的農(nóng)村甚至三、四線城市的醫(yī)保資金支出幅度尚未能與一線城市相比擬,故而這部分地區(qū)的患者及其家庭仍將承擔(dān)較多的支出費(fèi)用,相應(yīng)則更是明顯地增加了其家庭經(jīng)濟(jì)負(fù)擔(dān)。本病西醫(yī)病名雖在中醫(yī)學(xué)古代文獻(xiàn)無明確記載,但現(xiàn)代中醫(yī)學(xué)研究根據(jù)本病的臨床癥狀及其證候表現(xiàn),多將其歸屬于傳統(tǒng)中醫(yī)學(xué)的“眩暈”、“肝風(fēng)”、“頭痛”等范疇進(jìn)行辯證治療。針灸療法作為中醫(yī)療法一重要組成部分,從歷代針灸文獻(xiàn)及現(xiàn)代學(xué)者開展的多項針灸降壓療效觀察研究均可看出,針灸降壓具有良好的降壓效應(yīng),且突出表現(xiàn)在短期降壓效應(yīng)尤其對收縮壓的良性調(diào)節(jié)方面,以及對患者臨床不適癥狀的明顯改善和毒副作用明顯低于西藥降壓藥物治療,并且價格低廉、操作簡便,是值得大區(qū)域推廣應(yīng)用。但目前對針灸降壓機(jī)制尚無法明確闡述,在一定程度上限制了在更大范圍乃至全球范圍的推廣,故而對針灸降壓機(jī)制的研究顯得異常重要。針灸治療以經(jīng)絡(luò)理論為理論基礎(chǔ),人體經(jīng)絡(luò)內(nèi)連臟腑,外絡(luò)肢節(jié),將人體連為一有機(jī)整體,故而針灸治療效應(yīng)常是人體多靶點共同作用的結(jié)果,這對其治病機(jī)制、尤其組穴處方的針灸效應(yīng)機(jī)制的闡述增加了許多難度。根據(jù)經(jīng)穴-腦相關(guān)假說,針灸的治病必須先經(jīng)過腦作為信號樞紐進(jìn)行調(diào)整和整合之后才能作用到生物體的各個靶點,進(jìn)而起治療效應(yīng)。故而針灸降壓機(jī)制的中樞及外周神經(jīng)、體液因子調(diào)節(jié)機(jī)制應(yīng)該為互相作用的關(guān)系,各機(jī)制間不可截然分開,由于目前研究結(jié)果對各部分機(jī)制的相互關(guān)系尚未能很好的闡述清楚,所以本課題研究仍是以中樞機(jī)制為研究目標(biāo)。本課題前期實驗研究結(jié)果表明針刺SHR大鼠太沖穴雖然未能使血壓下降至正常水平,與模型組大鼠血壓情況相比仍具有良好的降壓效應(yīng),但實驗中選取的針灸學(xué)理論認(rèn)為對眩暈、頭痛等癥狀有改善作用的曲泉、沖陽針刺治療均未發(fā)現(xiàn)有良好的降壓效應(yīng),再次驗證了腧穴的治療效應(yīng)具有特異性。同時,目前研究普遍認(rèn)為延髓里存在最低級最基本的心血管調(diào)節(jié)中樞,也含有許多在中樞神經(jīng)系統(tǒng)上、下行投射聯(lián)系通路中不可缺少的神經(jīng)核團(tuán),均認(rèn)為其與血壓變化調(diào)節(jié)密切相關(guān)。目前對針刺能否影響延髓進(jìn)而出現(xiàn)相關(guān)調(diào)節(jié)效應(yīng)以及針刺調(diào)控高血壓的中樞靶點機(jī)制中蛋白質(zhì)相關(guān)的具體途徑尚未能明確闡述,故而本課題選取與血壓調(diào)節(jié)密切相關(guān)的延髓為研究對象,研究實驗中所取經(jīng)穴產(chǎn)生差異性降壓效應(yīng)的中樞機(jī)制,進(jìn)而探討經(jīng)穴效應(yīng)特異性的中樞機(jī)制。目的：在課題組前期研究所發(fā)現(xiàn)太沖、曲泉、沖陽及非穴針刺降壓具有差異性、穴位效應(yīng)具有特異性的基礎(chǔ)上,繼續(xù)選取以上腧穴進(jìn)行針刺干預(yù),應(yīng)用質(zhì)譜分析及蛋白組學(xué)技術(shù),研究同樣具有改善眩暈、頭痛癥狀但治療效應(yīng)具有特異性的太沖、曲泉、沖陽針刺干預(yù)后各相應(yīng)SHR大鼠延髓的蛋白表達(dá)差異情況,為闡述針刺降壓效應(yīng)中樞機(jī)制及經(jīng)穴效應(yīng)特異性的中樞機(jī)制研究提供一定的理論線索。方法：選用SPF級9周齡雄性自發(fā)性高血壓大鼠55只,隨機(jī)分為太沖組、曲泉組、沖陽組、非穴組和模型組,每組11只。選擇9周齡正常雄性Wistar大鼠8只作為正常組。太沖組、沖陽組、曲泉組和非經(jīng)穴組分別針刺雙側(cè)太沖、沖陽、曲泉和非穴各5min。針刺時使用襪套套住大鼠頭部進(jìn)行固定。在大鼠清醒狀態(tài)下進(jìn)針,行捻轉(zhuǎn)手法,捻轉(zhuǎn)頻率80±10次/min,捻轉(zhuǎn)角度180°±30°。先左后右,兩側(cè)各針刺2.5min,共5min。上午十二時前完成針刺,每天一次,連續(xù)七天。正常組、模型組大鼠以同樣方法抓握固定5min,不予針刺。第七天針刺結(jié)束后將各組大鼠端頭取腦并分離延髓組織,然后經(jīng)研磨組織、提取蛋白、二維凝膠電泳分離蛋白、掃描圖像、圖像分析等步驟獲得各組差異蛋白點,再用質(zhì)譜儀分析蛋白并從MASCOT數(shù)據(jù)庫中檢索、鑒定出各組的差異蛋白。結(jié)果：(1)用Imagemaste 2D 5.0軟件對獲取的二維凝膠電泳圖進(jìn)行分析,以模型組作為參考膠與其他各組結(jié)果進(jìn)行比較,各組大鼠相應(yīng)的延髓表達(dá)差異蛋白點結(jié)果如下：①太沖組大鼠表達(dá)上調(diào)的差異蛋白點有15個,下調(diào)的差異蛋白點有11個；②沖陽組大鼠表達(dá)上調(diào)的差異蛋白點有34個,下調(diào)的差異蛋白點有28個；③曲泉組大鼠表達(dá)上調(diào)的差異蛋白點有14個,下調(diào)的差異蛋白點有13個；④非穴組大鼠表達(dá)上調(diào)的差異蛋白點有33個,下調(diào)的差異蛋白點有27個。⑤正常組大鼠表達(dá)上調(diào)的差異蛋白點有28個,下調(diào)的差異蛋白點有13個;(2)全部實驗大鼠的差異蛋白質(zhì)點鑒定檢查結(jié)局：各實驗組共得出216個差異蛋白點,選取表達(dá)差異明顯并剔除重復(fù)點后進(jìn)行蛋白檢索鑒定,各組檢索結(jié)果共得出37個蛋白,具體如下：①太沖組表達(dá)上調(diào)的有6個：Protein LOC100911959:對細(xì)胞的生長還有發(fā)育、細(xì)胞周期及信號轉(zhuǎn)導(dǎo)起調(diào)節(jié)作用；Transitional endoplasmic reticulum ATPase:與細(xì)胞凋亡、生長以及DNA的修復(fù)相關(guān),在一些腫瘤中也是高表達(dá)并和腫瘤分型、分期、轉(zhuǎn)歸有關(guān)；MICOS complex subunit Mic60 (Fragment):參加調(diào)節(jié)線粒體,調(diào)節(jié)蛋白轉(zhuǎn)運(yùn)加工效率；Dihydropyrimidinase-related protein 3:參加軸突延長及神經(jīng)元極化形成,參與微管的動態(tài)調(diào)節(jié)；Cathepsin B:參與溶酶體降解蛋白質(zhì)的過程和免疫反應(yīng)抗原的加工、激素的活化；Peroxiredoxin-2:紅細(xì)胞中主要的過氧化氫清除者,保護(hù)紅細(xì)胞及紅細(xì)胞膜。②沖陽組上調(diào)的有4個：AP-2 complex subunit beta:載脂蛋白亞基,血漿脂蛋白組成部分,參與脂類代謝；Endoplasmin;Triosephosphate isomerase:糖酵解過程中重要的異構(gòu)酶,影響能量的有效生成；失調(diào)可導(dǎo)致免疫系統(tǒng)失調(diào)的多系統(tǒng)疾��；ATPsynthase subunit d, mitochondrial。下調(diào)的蛋白有6個：Succinyl-CoA ligase subunit beta; Mannose-6-phosphate isomerase; Protein Serpinb6:蛋白酶抑制因子;Eukaryotic translation initiation factor 2 subunit 1:參與真核細(xì)胞中的蛋白質(zhì)的翻譯,和腫瘤的發(fā)生發(fā)展有很大關(guān)系；Anamorsin; Protein Plcxd3,參與脂類代謝。③曲泉組上調(diào)的有4個：Septin-11:參與細(xì)胞胞質(zhì)分裂、細(xì)胞內(nèi)物質(zhì)轉(zhuǎn)運(yùn)、凋亡等細(xì)胞生理過程,控制胚胎在發(fā)育過程中相關(guān)的細(xì)胞的遷移；Glutamine synthetase:參與清理體內(nèi)游離銨離子的過程,對機(jī)體起保護(hù)作用；Heat shock cognate 71 kDa protein:維持細(xì)胞的生存和功能,提高細(xì)胞的抵抗力,協(xié)同免疫作用；SAPdomain-containing ribonucleoprotein.-參與細(xì)胞生長、代謝及癌變的重要轉(zhuǎn)錄或翻譯控制。下調(diào)的蛋白有4個：Lamin-B1:影響細(xì)胞增殖、遷移,對氧化應(yīng)激和信號通路發(fā)揮影響,在一些癌組織里面呈高表達(dá)；cAMP-dependent protein kinase type II-alpha regulatory subunit:調(diào)節(jié)蛋白質(zhì)磷酸化；Secernin-1:調(diào)節(jié)細(xì)胞胞吐作用,在一些腫瘤中呈高表達(dá)；Septin 8 (Predicted):參與細(xì)胞內(nèi)功能調(diào)節(jié),與神經(jīng)遞質(zhì)的傳遞與神經(jīng)功能障礙等疾病直接相關(guān)。④非穴組上調(diào)的有3個：Heat shock 70 kDa protein 4:參與熱激響應(yīng),可能有心肌保護(hù)作用；Alanine--tRNA ligase, cytoplasmic:參與蛋白質(zhì)生物合成過程；Dihydropyrimidinase-related protein。下調(diào)的有2個：Hsp90 co-chaperone Cdc37:參與腫瘤生理與病理的多個過程；Guanine nucleotide-binding protein G(o) subunit alpha:參與離子通道及信號轉(zhuǎn)導(dǎo)調(diào)節(jié),一些腫瘤的發(fā)生可能和它的紊亂有很大關(guān)系。⑤模型組對比于正常大鼠,其表達(dá)上調(diào)的蛋白有2個：Glycine--tRNA ligase (Fragment):催化氨基酸與tRNA結(jié)合的特異性酶,參加了基因的表達(dá)控制,可以抑制細(xì)胞凋亡；Spermatid-associated protein:和不孕不育有關(guān)。表達(dá)下調(diào)的有6個：Protein disulfide-isomerase A3:參與細(xì)胞的信號轉(zhuǎn)導(dǎo),促進(jìn)糖蛋白折疊質(zhì)量控制在內(nèi)質(zhì)網(wǎng)中；Fructose-bisphosphate aldolase C:和一些腫瘤細(xì)胞中起誘導(dǎo)作用的糖代謝酶有關(guān)系；Biliverdin reductase A:參加了細(xì)胞信號通路的磷酸化的調(diào)節(jié),影響了代謝的相關(guān)蛋白、凋亡的分子細(xì)胞周期等相關(guān)蛋白、信號通路的蛋白,免疫分子還有氧化應(yīng)激因子等的基因表達(dá)；Inositol monophosphatase 1:參加了磷脂酰肌醇信號的傳遞過程；Phosphoribosyl transferase domain containing 1:和殺傷腫瘤細(xì)胞功能相關(guān)。結(jié)論：1.SHR大鼠相較于wistar大鼠其延髓中參與胰島素代謝及能量代謝的部分相關(guān)蛋白出現(xiàn)表達(dá)差異,結(jié)合其他學(xué)者相關(guān)研究推測血壓變化可能與能量代謝異常有相關(guān)性；SHR大鼠可能比正常大鼠更易患免疫功能缺陷相關(guān)疾病。2.針刺太沖穴的降壓效應(yīng)中樞機(jī)制可能與延髓中參與能量代謝部分相關(guān)蛋白密切相關(guān)；針刺沖陽穴可導(dǎo)致延髓中參與影響脂類代謝部分相關(guān)蛋白表達(dá)差異,與其具有胃經(jīng)調(diào)節(jié)生物體水谷代謝的循經(jīng)治療效應(yīng)相吻合；針刺曲泉穴可導(dǎo)致延髓中參與影響免疫反應(yīng)的部分蛋白表達(dá)差異,與其作為作為肝經(jīng)合穴可直達(dá)調(diào)理肝臟進(jìn)而調(diào)氣機(jī)、理肝血而調(diào)和人體陰陽相吻合。以上各經(jīng)穴針刺作用效應(yīng)是多靶點多渠道一起作用的結(jié)果,為針刺太沖穴降壓效應(yīng)中樞機(jī)制及經(jīng)穴效應(yīng)特異性研究提供一定研究參考。3.針刺非穴對大鼠延髓蛋白表達(dá)的影響其作用靶點相對來說較為簡單且無明顯規(guī)律性。4.經(jīng)穴治療效應(yīng)及其相應(yīng)中樞作用機(jī)制具有特異性。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R245

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