本文選題：柴胡疏肝散 + 雌鼠�。� 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:"方證相關(guān)"蘊(yùn)含方藥與病證之間存在某種對(duì)應(yīng)的規(guī)律,中醫(yī)方劑的研究應(yīng)考慮到證候的選擇性。中醫(yī)證候通常具有跨系統(tǒng)和動(dòng)態(tài)演變的特點(diǎn),證候模型復(fù)制中需要注意跨系統(tǒng)多指標(biāo)的綜合評(píng)價(jià)。男女有異,生理有別,相同證候在不同性別患者身上存有差異,但目前基于性別差異的證候模型的系統(tǒng)探查及評(píng)價(jià)還很少見。本課題以女性肝郁證為切入點(diǎn),側(cè)重從性腺軸的角度,對(duì)束縛肝郁雌鼠涉及神經(jīng)-內(nèi)分泌的現(xiàn)代內(nèi)涵進(jìn)行探查;同時(shí)基于方證相關(guān)的理論,選擇針對(duì)肝郁證的代表方柴胡疏肝散,觀察該方對(duì)雌性肝郁模型大鼠的調(diào)節(jié)作用,為疏肝-肝郁證關(guān)聯(lián)的現(xiàn)代生物學(xué)內(nèi)涵提供理解。論文包括文獻(xiàn)綜述和實(shí)驗(yàn)研究?jī)蓚�(gè)部分,其中文獻(xiàn)綜述主要圍繞中醫(yī)肝臟與生殖系統(tǒng)、肝郁證與女性生殖疾病的關(guān)系進(jìn)行相關(guān)文獻(xiàn)的搜集與整理,提出女性肝郁證神經(jīng)內(nèi)分泌病生理的概念。實(shí)驗(yàn)研究探查了束縛肝郁模型雌鼠的中醫(yī)證候和神經(jīng)內(nèi)分泌功能狀態(tài)及柴胡疏肝散的相關(guān)作用。方法:大鼠隨機(jī)分為正常對(duì)照組、肝郁模型組(以下簡(jiǎn)稱模型組)、柴胡疏肝散大劑量組(柴大組)、柴胡疏肝散小劑量組(柴小組),每組10只。后三組按慢性應(yīng)激束縛法造模連續(xù)4周,正常對(duì)照組不予處理。造模第2周末(造模第14天),柴大組和柴小組分別按相當(dāng)于藥材1.26g/kg和0.31g/kg的劑量(相當(dāng)于人用量的2和0.5倍)給予柴胡疏肝散灌胃,每日1次,連續(xù)2周;模型組和正常對(duì)照組大鼠給予等量蒸餾水。于造模第27天,對(duì)各組大鼠陰道涂片進(jìn)行觀察,擇動(dòng)情前期/動(dòng)情間期(實(shí)驗(yàn)第28-30天),禁食禁水12h,腹腔麻醉,經(jīng)腹主動(dòng)脈取血,分離血清與血漿;斷頭后低溫下迅速取下丘腦、垂體、雙側(cè)卵巢等組織,分別于10%中性福爾馬林中固定和液氮中速凍后置于-80℃冰箱保存,樣本待測(cè)。觀測(cè)指標(biāo):1)動(dòng)情周期,陰道涂片觀測(cè)造模前和給藥前后的大鼠動(dòng)情周期;2)記錄造模期間大鼠的體重和外觀行為(行為狀態(tài)、活躍程度、情緒反應(yīng)、睡眠狀態(tài)、飲食狀態(tài)、皮膚毛發(fā)),實(shí)驗(yàn)第2周末和第4周末做糖水偏嗜實(shí)驗(yàn),4周末做曠場(chǎng)實(shí)驗(yàn);3)放免法和酶聯(lián)法測(cè)定下丘腦-垂體-腎上腺/甲狀腺/性腺軸的相關(guān)指標(biāo)。各組數(shù)據(jù)以x±s表示,采用SPSS 20.0軟件處理,組間比較采用單因素方差分析(one-way ANOVA),用SNK法檢驗(yàn);非正態(tài)分布資料采用非參數(shù)統(tǒng)計(jì)分析。計(jì)數(shù)資料采用百分率表示,組間差異使用Fisher精確檢驗(yàn)。所有圖表均用GraphPad Prism 6 軟件制作。結(jié)果:(1)證候?qū)W指標(biāo):①一般外觀行為的變化:與正常組比較,模型大鼠造模第一周開始出現(xiàn)多動(dòng)興奮、煩躁易怒、大便干結(jié),2周后逐漸出現(xiàn)扎堆少動(dòng),行動(dòng)遲緩,飲食減少,大便干燥,毛發(fā)不榮;第2-4周的體重均見不同程度的降低(p0.05或p0.01)。第2周和第4周糖水偏嗜實(shí)驗(yàn)中糖水偏嗜度分別增加和降低(p0.01);第4周曠場(chǎng)實(shí)驗(yàn)中的中央?yún)^(qū)停留時(shí)間、穿格次數(shù)、直立次數(shù)均降低(p0.01)。與模型組比較,柴大組大鼠活動(dòng)狀態(tài)、情緒睡眠、飲食大便趨于適度;第3-4周體重均見明顯升高(p0.05或p0.01)。第4周糖水偏嗜度增加(p0.01),曠場(chǎng)實(shí)驗(yàn)中的中央?yún)^(qū)停留時(shí)間、穿格次數(shù)、直立次數(shù)均見增加(p0.01);柴小組大鼠的上述指標(biāo)無明顯變化(p0.05)。與柴小組比較,柴大組大鼠第4周上述指標(biāo)均有顯著差異(p0.05或p0.01)。②實(shí)驗(yàn)室關(guān)聯(lián)指標(biāo)的變化:與正常組比較,模型組大鼠血中5-HT、DA、NE明顯降低,E和β-EP明顯升高(p0.01或p0.05)。與模型組比較,柴大組大鼠血中5-HT和NE明顯升高,E和β-EP明顯降低(p0.01或p0.05);柴小組大鼠血中DA明顯升高(p0.01)。與柴小組比較,柴大組大鼠血中5-HT和NE顯著升高、E顯著降低(p0.01或p0.05)。(2)下丘腦-垂體-腎上腺皮質(zhì)軸指標(biāo):與正常組比較,模型組大鼠下丘腦CRH、血清ACTH、CORT均顯著升高(p0.01)。與模型組比較,柴大組大鼠下丘腦CRH、血清ACTH、CORT均顯著降低(p0.01);柴小組大鼠CRH、ACTH明顯降低(p0.01或p0.05),CORT呈降低趨勢(shì)p0.05)。與柴小組比較,柴大組大鼠下丘腦CRH、血清CORT均顯著降低(p0.01或p0.05)。(3)下丘腦-垂體-甲狀腺軸指標(biāo):與正常組比較,模型組大鼠下丘腦TRH、血中T3、T4升高,TSH降低,差異均有統(tǒng)計(jì)學(xué)意義(p0.01或p0.05)。與模型組比較,柴大組大鼠下丘腦TRH、血中T3、T4降低,TSH升高,差異均有統(tǒng)計(jì)學(xué)意義(p0.01);柴小組大鼠下丘腦TRH、血中T3、T4降低,TSH升高,僅T4差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。與柴小組比較,柴大組大鼠下丘腦TRH、血中T3、T4降低,TSH升高,差異均有統(tǒng)計(jì)學(xué)意義(p0.01或p0.05)。(4)下丘腦-垂體-性腺軸指標(biāo):①動(dòng)情周期的變化:實(shí)驗(yàn)期間,正常組大鼠動(dòng)情周期穩(wěn)定,模型組大鼠的動(dòng)情周期紊亂,呈現(xiàn)"前-中-后-間"延長(zhǎng)或滯遲,實(shí)驗(yàn)第4周大鼠動(dòng)情周期紊亂率為100%。與模型組比較,柴大組和柴小組大鼠動(dòng)情周期紊亂率分別為20%和80%,其中柴大組紊亂率顯著降低(p0.01)。與柴小組比較,柴大組大鼠動(dòng)情周期紊亂率明顯下降p0.05)。②下丘腦-垂體-性腺軸激素的變化:與正常組比較,模型組大鼠血中GnRH、FSH、LH、PRL、P、T、E2均顯著升高p0.01或p0.05)。與模型組比較,柴大組大鼠血中GnRH、FSH、LH、PRL、P、T均顯著降低(p0.01),E2有降低趨勢(shì);柴小組大鼠血中FSH、LH、PRL、P、E2均顯著降低(p0.01),GnRH、T僅有降低趨勢(shì)。與柴小組比較,柴大組大鼠血中GnRH和FSH降低顯著(p0.01),其余指標(biāo)差異無統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:慢性束縛法建立的雌性肝郁模型大鼠出現(xiàn)與中醫(yī)證候相關(guān)的外觀行為和實(shí)驗(yàn)室指標(biāo)的變化,并伴有腎上腺皮質(zhì)軸、甲狀腺軸、性腺軸的功能亢進(jìn)及動(dòng)情周期紊亂,提示女性肝郁證存在神經(jīng)-內(nèi)分泌多系統(tǒng)的功能失調(diào)。中醫(yī)肝郁證的治療代表方-柴胡疏肝散對(duì)該雌性肝郁模型大鼠的中醫(yī)證候和上述神經(jīng)內(nèi)分泌系統(tǒng)相關(guān)指標(biāo)均有良好的改善作用,大劑量作用明顯優(yōu)于小劑量。該研究結(jié)果為認(rèn)識(shí)女性肝郁證的現(xiàn)代病理生理內(nèi)涵和中醫(yī)方證相關(guān)(疏肝方-肝郁證關(guān)聯(lián))及柴胡疏肝散的臨床應(yīng)用提供了一定的實(shí)驗(yàn)證據(jù)。
[Abstract]:Objective: "there are some corresponding rules between the formula" contains prescriptions and syndromes of traditional Chinese medicine formula, consideration should be given to the selective syndrome. TCM syndrome usually has the characteristics of cross system and dynamic evolution of syndrome model replication should pay attention to in the cross system of multi index comprehensive evaluation. Men and women are different. Don't have the same physiological syndrome, there are some differences in the different gender of patients, but the current system of exploration and evaluation of syndrome model based on gender difference is still very rare. The female liver depression syndrome as the starting point, mainly from the gonadal axis angle, to detect the connotation of the modern female rat liver bound to neuroendocrine at the same time; formula based on the theory of choice for the party on behalf of Ganyu Chaihushugansan, regulated by the side of the female Ganyu model rats, to provide an understanding of modern biology - liver stagnation of the liver Qi connection of culvert. The paper includes two parts: literature review and experimental research, the literature review mainly focuses on the Chinese medicine liver and reproductive system, collect and collate relevant literatures on the relationship between liver stagnation syndrome and female genital diseases, female Ganyu concept of neuroendocrine disease physiology. Experimental study on exploration of the shackles of Ganyu model female rats of TCM syndrome and nerve endocrine function and Chaihushugansan related. Methods: the rats were randomly divided into normal control group, depression model group (hereinafter referred to as the model group), CHSGS group of large dose (Chai Dazu), CHSGS group of small dose (Chai Xiaozu), 10 rats in each group. The three groups according to the chronic stress binding method modeling for 4 consecutive weeks, normal control group were not treated. Modeling the second week (fourteenth day of modeling), Chai Da group and group Chai respectively according to the equivalent of 1.26g/kg and 0.31g/kg medicine dose (equivalent to the amount of 2 and 0.5 people Times) give CHSGS gavage, 1 times daily for 2 weeks; the model group and normal control group rats were given distilled water. On the twenty-seventh day of modeling, the rats vaginal smears were observed, choose proestrus / diestrus (the 28-30 day), fasting 12h, abdominal cavity anesthesia, abdominal aortic blood, serum and plasma separation; after decapitation under low temperature rapidly from the hypothalamus, pituitary, ovary and other tissues, respectively, to the preservation of frozen in liquid nitrogen fixation and placed in a -80 C refrigerator 10% neutral formalin in the sample. Observation index: 1) the estrous cycle, vaginal smear observation made and before the model before and after Administration of the estrous cycle of rats; 2) to record the weight and appearance and behavior during the modeling rats (behavior, active, emotional reactions, sleep, diet, skin and hair), experiment second and fourth weeks for sucrose preference test, 4 weekend to do field test; 3 ) related indexes of hypothalamic pituitary adrenal / thyroid / gonadal axis radioimmunoassay and ELISA method. All the data were expressed by X + s, using SPSS 20 software, compared with the single factor analysis of variance (one-way ANOVA), with SNK test method; non normal distribution data collected for statistical analysis non parametric. Count data expressed by the percentage difference between the two groups using the Fisher exact test. All charts are used GraphPad Prism 6 software. Results: (1) syndrome index: the change of general appearance behavior: compared with the normal group, model rats in the first week with excitement, irritability after 2 weeks, dry stool, gradually appear to get together to move less, slow, eating less, dry stool, hair glory; different degrees of reduction were seen in article 2-4 week weight (P0.05 or P0.01) for second weeks and fourth weeks. Sucrose preference test in sucrose preference degree were increased 鍜岄檷浣，

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