本文選題：外燥 + 小鼠模型��； 參考：《湖北中醫(yī)藥大學(xué)》2011年博士論文

【摘要】：中醫(yī)證候動(dòng)物模型是在中醫(yī)學(xué)整體觀念及辨證施治思想指導(dǎo)下,運(yùn)用臟象學(xué)說和病因病機(jī)理論,把人類疾病原型的某些特征在動(dòng)物身上模擬復(fù)制,是中醫(yī)學(xué)人體證候的具體再現(xiàn),是開展中醫(yī)證候及中藥藥理研究和新藥開發(fā)的一個(gè)重要途徑。 燥,為自然界六氣之一,是秋之主氣�！皻q金太過”或“歲木不及”,則燥氣可化為致病因素,稱為燥邪,因其來源于外,故常稱為外燥。外燥是中醫(yī)病因?qū)W說的重要組成部分,但對外燥理論與實(shí)驗(yàn)的研究均較少。本課題在前期研究的基礎(chǔ)之上,擬通過總結(jié)中醫(yī)動(dòng)物模型以及中醫(yī)證候動(dòng)物模型的研究方法,結(jié)合外燥的病機(jī)特點(diǎn),探討建立外燥動(dòng)物模型的途徑,并通過實(shí)驗(yàn)研究觀察外燥動(dòng)物模型的病理變化規(guī)律,對外燥動(dòng)物模型進(jìn)行評價(jià),為建立穩(wěn)定的、可靠的、可重復(fù)的外燥小鼠動(dòng)物模型提供依據(jù)。 一、理論研究 (一)外燥小鼠模型造模因素的選擇 中醫(yī)動(dòng)物模型復(fù)制的諸多方法雖然各自有顯著的優(yōu)點(diǎn),但是均有明顯的不足之處。課題組在導(dǎo)師張六通教授的指導(dǎo)下,回顧了半個(gè)世紀(jì)以來中醫(yī)證候動(dòng)物模型的研究歷程,認(rèn)為中醫(yī)證候動(dòng)物模型的研究應(yīng)該從三個(gè)方面出發(fā)：優(yōu)化動(dòng)物模型的造模因素；建立規(guī)范的動(dòng)物模型評價(jià)方法；選擇具有特征性的評價(jià)指標(biāo)。外燥動(dòng)物模型應(yīng)該在外燥相關(guān)理論的指導(dǎo)下,復(fù)制符合外燥病機(jī)和證候的動(dòng)物模型。 外燥的流行責(zé)之于“歲金太過”或“歲木不及”,其產(chǎn)生原因與我國的地理結(jié)構(gòu)和氣候特點(diǎn)相關(guān),我國的氣候存在濕→干轉(zhuǎn)換時(shí)間短于干→濕轉(zhuǎn)換時(shí)間的特征,所以秋之燥氣容易化為燥邪。模擬秋季氣候變化的方法建立外燥小鼠模型,既可較好地體現(xiàn)外燥的變化規(guī)律,也可避免以極端干燥地區(qū)濕度條件為基礎(chǔ)的研究結(jié)果對動(dòng)物模型穩(wěn)定性和可重復(fù)性的影響。 (二)外燥小鼠模型評價(jià)方法的探討 外燥的病理變化有兩個(gè)主要特點(diǎn)：燥性干澀,易傷津液；燥易傷肺,影響肺的宣發(fā)肅降功能,可見口鼻干燥,咽干口渴,干咳少痰,或痰液膠黏難咯等癥狀。 現(xiàn)代研究發(fā)現(xiàn)衛(wèi)氣的防御作用與肺部免疫效應(yīng)密切相關(guān),研究顯示衛(wèi)氣的運(yùn)行節(jié)律與免疫節(jié)律高度一致,并且衛(wèi)氣虛的患者IgG,IgA,IgE的含量有明顯變化。外燥動(dòng)物模型支氣管肺泡灌洗液中IgA、IgG和SP-A含量的變化可以較全面地反映呼吸道不同部位免疫能力的改變,可以反映外燥動(dòng)物模型肺氣衛(wèi)外功能變化的規(guī)律。 氣道粘膜由細(xì)胞層和粘液層組成。氣道粘液層有兩部分：凝膠層和溶膠層。氣道粘液層的變化,可在一定程度上反映吸入空氣對氣道津液代謝影響的規(guī)律。氣道Muc5ac mRNA及其蛋白的表達(dá)可在一定程度上反映氣道凝膠層的變化,而氣道AQP5 mRNA及其蛋白的表達(dá)可提示氣道溶膠層的變化,通過觀察氣道Muc5ac和AQP5 mRNA及其蛋白表達(dá)的變化可揭示肺津液代謝的規(guī)律。 二、實(shí)驗(yàn)研究 (一)動(dòng)物及分組 SPF級BALB/c小鼠70只,體重18±1g,雌雄各半。由湖北省實(shí)驗(yàn)動(dòng)物研究中心提供,許可證號：SCXK(鄂)2008-2005。 實(shí)驗(yàn)動(dòng)物在實(shí)驗(yàn)室常規(guī)飼養(yǎng)7天后隨機(jī)分為7組：正常對照組(C組)、溫燥模型Ⅰ組(WⅠ組)、溫燥模型Ⅱ組(WⅡ組)、常燥模型Ⅰ組(QⅠ組)、常燥模型Ⅱ組(QⅡ組)、涼燥模型Ⅰ組(LⅠ組)、涼燥模型Ⅱ組(LⅡ組),置人工氣候箱內(nèi)進(jìn)行造模。C、WⅠ、QⅠ、LⅠ組小鼠于第7日上午, wⅡ、QⅡ、LⅡ組小鼠于第13日上午按實(shí)驗(yàn)要求取材,取材前4小時(shí)禁水禁食。 (二)指標(biāo)檢測 1.小鼠一般情況觀察 觀察小鼠的神態(tài)、活動(dòng)情況、皮毛色澤及有無脫落,飲食狀況及糞便物理性狀變化。 2.小鼠肺組織結(jié)構(gòu)觀察 采用蘇木素伊紅(HE)染色法。 3.支氣管肺泡灌洗液(BALF)中IgG、IgA和SP-A蛋白含量的檢測。 采用定量ELISA試劑盒檢測。 4.肺組織Muc5ac mRNA和AQP5 mRNA表達(dá)的測定 采用SYBR Green I熒光嵌合法進(jìn)行實(shí)時(shí)定量熒光PCR檢測。 5.肺組織Muc5ac蛋白表達(dá)的測定 采用免疫組織化學(xué)染色法檢測。 6.肺組織AQP5蛋白表達(dá)的測定, 采用蛋白免疫印跡(Western blotting)檢測。 (三)統(tǒng)計(jì)學(xué)分析 采用SPSS15.0統(tǒng)計(jì)軟件對所得數(shù)據(jù)進(jìn)行處理分析。實(shí)驗(yàn)數(shù)據(jù)均以x±s表示,兩組均數(shù)間比較采用t檢驗(yàn),多組均數(shù)間兩兩比較采用方差分析,P0.05為差異有統(tǒng)計(jì)學(xué)意義。 三、結(jié)果 本課題外燥小鼠模型的復(fù)制條件,既體現(xiàn)了秋季氣候變化的過程,又體現(xiàn)了秋季氣候變化的特征,較好地體現(xiàn)外燥的致病特點(diǎn)。 “邪之所湊,其氣必虛”(《素問·評熱病論》),外燥導(dǎo)致各組外燥小鼠模型的正氣受到不同程度的損傷,尤其是肺氣的宣發(fā)功能。肺氣的宣發(fā)功能改變主要表現(xiàn)為肺氣衛(wèi)外和肺氣宣發(fā)津液兩方面 外燥小鼠模型BALF中IgA、IgG和SP-A含量的變化體現(xiàn)了肺氣衛(wèi)外功能的變化。實(shí)驗(yàn)結(jié)果顯示,與IgG含量的變化不同,雖然溫燥、涼燥和常溫燥小鼠模型BALF中IgA、SP-A含量的變化存在一定的差異,但是各組IgA、SP-A含量變化的趨勢是一致的。 外燥小鼠模型氣道AQP5 mRNA及其蛋白表達(dá)的下降,提示外燥導(dǎo)致氣道內(nèi)津液含量下降,同時(shí)也導(dǎo)致了津液不能外達(dá)氣道。而氣道Muc5ac mRNA及其蛋白表達(dá)的增加,可能是一種保護(hù)性反應(yīng),以防止氣道水液進(jìn)一步的散失,也可能是外燥導(dǎo)致的一種病理變化。Muc5ac表達(dá)增加所反映的凝膠層的增厚與AQP5表達(dá)降低所代表的溶膠層的變薄,均可降低氣道粘膜纖毛的轉(zhuǎn)運(yùn)率,導(dǎo)致氣道防御能力下降,即肺氣衛(wèi)外能力降低。外燥小鼠模型出現(xiàn)毛色枯燥、飲水量增加,糞便略顯干燥等癥狀,也是外燥之“燥勝則干”的表現(xiàn)。 咳嗽的發(fā)生一方面是由于氣道內(nèi)痰液的刺激,另一方面也可能由氣道反應(yīng)性增強(qiáng)所致。外燥小鼠模型氣道AQP5的降低和Muc5ac表達(dá)的增加,不僅可導(dǎo)致氣道反應(yīng)性增加,而且可導(dǎo)致了氣道粘膜纖毛轉(zhuǎn)運(yùn)率的下降,使氣道內(nèi)痰液形成成為可能,并且提示形成的痰液性質(zhì)較為粘稠。 綜上所述,外燥小鼠模型不僅體現(xiàn)了外燥形成的病機(jī),而且具有外燥的基本癥狀和體征,是研究外燥發(fā)病機(jī)理較好的平臺。 四、結(jié)論 本課題成功地建立了外燥小鼠模型,該模型接近人類外感燥邪的發(fā)病機(jī)制,較好地體現(xiàn)了外燥導(dǎo)致肺氣輸布津液的功能下降,體內(nèi)津液不能外達(dá),體表津液無以為繼而發(fā)生干燥的病機(jī),并且可以推斷模型存在外燥之干咳少痰,或痰液膠黏難咯等癥狀。 1.外燥小鼠模型的造模因素 模擬早秋,中秋和晚秋氣候特點(diǎn)建立外燥小鼠模型的條件如下：早秋(溫燥)：溫度22士2℃,相對濕度33士2%；中秋(常燥)：溫度15℃士2℃,相對濕度33士2%；晚秋(涼燥)：溫度8士2℃,相對濕度33士2%；人工外燥環(huán)境風(fēng)速設(shè)定為2.5m/s。 2.外燥小鼠模型的評價(jià) (1)外燥小鼠模型毛色枯燥,偶見聳毛、蜷縮等現(xiàn)象,飲水量有所增加,糞便略顯干燥。 (2)外燥小鼠模型氣道粘膜皺襞增多,杯狀細(xì)胞增生明顯,氣道粘液厚度均呈現(xiàn)下降趨勢。 (3)外燥小鼠模型氣道BALF中IgA含量增加,SP-A的含量增加。 (4)外燥小鼠模型氣道AQP5 mRNA及其蛋白表達(dá)下降,Muc5ac mRNA及其蛋白表達(dá)增加。
[Abstract]:Under the guidance of the overall concept of traditional Chinese medicine and the theory of syndrome differentiation and treatment, the TCM syndrome model uses the theory of visceral image and the theory of etiology and pathogenesis to simulate and replicate some characteristics of the prototype of human disease on the animal. It is a concrete reproduction of the syndromes of the Chinese medicine. It is an important part of the research of TCM syndromes and pharmacology and the development of new drugs. Way.
Dryness, one of the six natural gases in nature, is the main gas of autumn. "Age gold too" or "old wood is inferior", dryness gas can be used as a pathogenic factor, which is called dryness. Because of its origin, it is often called external dryness. External dryness is an important part of the theory of etiological factors in traditional Chinese medicine, but the study on the theory of external dryness and testing is less. This topic is based on the basis of earlier research. On the basis of summarizing the research methods of the animal model of traditional Chinese medicine and the animal model of TCM syndrome, combining with the characteristics of the external dryness, the way to establish the model of the external dryness animal is discussed, and the pathological changes of the animal model of the dryness animal are observed by the experimental study, and the model of the dryness animal is evaluated, in order to establish a stable, reliable and repeatable external dryness. The animal model of mice provides the basis.
First, theoretical research
(1) the selection of model factors for the model of external dryness mice
Many methods of animal model reproduction of traditional Chinese medicine have obvious advantages, but they have obvious shortcomings. Under the guidance of Professor Zhang Liutong, the subject group reviewed the research course of TCM syndrome animal model in the last half century, and thought that the research of TCM syndrome animal model should start from three aspects: optimizing the animal. The modeling factors of the model, the establishment of a standardized animal model evaluation method, and the selection of the characteristic evaluation index. The animal model of the external dryness animal should be guided by the theory of external dryness and the animal model which conforms to the pathogenesis and syndrome of the external dryness.
The cause of the epidemics of the external dryness is the "age gold too" or "the age of the old wood", the cause of which is related to the geographical structure and climate characteristics of our country. The climate of our country has the characteristics of wet to dry conversion time shorter than dry to wet conversion time, so the dry gas of autumn is easily turned into dry evil. It can not only reflect the changing rules of the external dryness, but also avoid the effect of the research results based on the humidity conditions in extreme dry areas on the stability and repeatability of animal models.
(two) a study on the evaluation method of the model of external dryness mice
The pathological changes of external dryness have two main characteristics: dry dry, easy to hurt the body fluid; dryness is easy to hurt the lung. It affects the function of the lung. It can be seen that the mouth and nose are dry, the dry mouth is thirsty, dry cough is less phlegm, or the mucus of sputum is difficult to cough and so on.
Modern research has found that the defense function of the health gas is closely related to the immune effect of the lung. The study shows that the rhythm of the operation is highly consistent with the immune rhythm, and the content of IgG, IgA and IgE in the patients with Qi deficiency can be significantly changed. The changes in the content of IgA, IgG and SP-A in the bronchoalveolar lavage fluid of the animal model of the external dryness animal can reflect the total respiration in a more comprehensive way. The change of immune function in different parts of the road can reflect the change of the function of the lung qi outside the dry animal model.
The airway mucous membrane consists of the cell layer and the mucus layer. There are two parts of the airway mucus layer: the gel layer and the sol layer. The changes in the airway mucus layer can reflect the effect of the inhaled air on the metabolism of the airway body fluid to a certain extent. The expression of the airway Muc5ac mRNA and its protein can reflect the changes in the airway gelatin layer to a certain extent, and the airway AQP5 The expression of mRNA and its protein can indicate the changes in the airway sol layer. By observing the changes in the expression of Muc5ac and AQP5 mRNA in the airway, the regulation of the metabolism of lung fluid can be revealed.
Two, experimental research
(I) animals and groups
There were 70 SPF BALB/c mice weighing 18 + 1g, each half of male and female. Provided by the Hubei provincial laboratory animal research center, the license number is SCXK (E) 2008-2005..
The experimental animals were randomly divided into 7 groups: normal control group (group C), temperature dryness model I group (group W I), temperature dryness model II Group (group W II), normal dryness model I (group Q I), normal dryness model II Group (group Q II), cold dryness model I (group L I), cold dryness model II Group (L group), set model.C in artificial climate box, W I, Q I, L. On the morning of the seventh day, the mice in group w II, Q II and L II were sampled according to the experimental requirements on the morning of the morning of the thirteenth day and fasted for 4 hours before sampling.
(two) index detection
General observation of 1. mice
The state of mind, activity, fur color and abscission, diet and changes in physical characteristics of feces were observed.
Observation on the structure of lung tissue of 2. mice
The hematoxylin (HE) staining method was used.
3. detection of IgG, IgA and SP-A protein levels in bronchoalveolar lavage fluid (BALF).
Quantitative ELISA kit was used to test it.
Determination of expression of Muc5ac mRNA and AQP5 mRNA in 4. lung tissues
SYBR Green I fluorescence embedding method was used for real-time quantitative PCR detection.
Determination of the expression of Muc5ac protein in 5. lung tissues
The immuno histochemical staining was used to detect it.
6. determination of AQP5 protein expression in lung tissue,
Protein immunoblotting (Western blotting) was used to detect it.
(three) statistical analysis
The data were processed and analyzed with SPSS15.0 statistical software. The experimental data were expressed in X + s. The two groups were compared with the t test, and 22 of the multiple groups were compared with the analysis of variance. P0.05 was statistically significant.
Three, the result
The replicating condition of the dryness mouse model not only embodies the process of climate change in autumn, but also embodies the characteristics of climate change in autumn, which can better reflect the pathogenic characteristics of external dryness.
The spirit of the external dryness model of the mice is damaged in different degrees, especially the function of the lung qi. The change of the function of lung qi is mainly manifested in two aspects: Lung Qi and lung qi.
The changes in the IgA, IgG and SP-A content in the BALF of the dryness mice model showed that the changes in the function of lung qi were different. The experimental results showed that the changes of the content of IgG were different from that of IgG, although there was a certain difference in the content of IgA and SP-A in the model BALF of cold dryness and normal temperature dryness, but the trend of IgA and SP-A content in each group was the same.
The decrease in the expression of AQP5 mRNA and its protein in the airway of the dryness mouse model suggests that the content of the body fluid in the airway decreases and the body fluid can not reach the airway. The increase in the expression of Muc5ac mRNA and its protein in the airway may be a protective reaction to prevent the further loss of the airway water and may also be caused by external dryness. The thickening of the gelatin layer and the thinning of the sol layer represented by the increase of.Muc5ac expression in the pathological changes could reduce the transport rate of the mucociliary body of the airway and decrease the ability of the airway defense, that is, the ability to decrease the lung qi and the outside and the air. The mice model of the external dryness was dry, the amount of drinking water increased, and the excrement was slightly dry, and so on. It is also the expression of "dry win is dry".
The occurrence of coughing is due to the stimulation of the sputum in the airway and the enhancement of the airway responsiveness. The decrease of the airway AQP5 and the increase of the expression of Muc5ac in the model of external dryness can not only lead to an increase in airway responsiveness, but also lead to a decrease in the ciliary transport rate of the airway mucosa and the formation of sputum in the airway. Yes, and the forms of the sputum are more viscous.
In summary, the model of dryness mice not only embodies the pathogenesis of dryness, but also has the basic symptoms and signs of external dryness. It is a good platform for the study of the pathogenesis of dryness.
Four. Conclusion
This model has successfully established a model of external dryness mice. This model is close to the pathogenesis of human exogenous pathogenic dryness. The model can better reflect the decline of the function of the body fluid, the body fluid in the body can not be reached, the body surface of body fluid is dry and the body surface is dry, and it can be inferred that the model exists dry cough, less sputum, or sputum glue. Symptoms such as sticky difficulty.
Modeling factors of 1. dryness mice
Early autumn, mid autumn and late autumn climate characteristics of the establishment of the conditions of the dryness mice as follows: early autumn (Wen Zao): temperature 22 2 degrees, relative humidity 33 2%; Mid Autumn Festival (Chang Zao): temperature 15 C 2, relative humidity 33, 2%; late autumn (cold): temperature 8 2, humidity 33 2%; artificial wind environment wind speed set to 2.5m/s.
Evaluation of 2. dryness mice model
(1) the dry mouse model is dull in hair, occasionally shrugged and curled. The amount of drinking water has increased and the stool is slightly drier.
(2) in the external dry mouse model, the mucosal fold increased, the goblet cell proliferation was obvious, and the airway mucus thickness showed a downward trend.
(3) the IgA content in the airway BALF increased and the SP-A content increased in the dry mouse model.
(4) the expression of AQP5 mRNA and protein in airway was decreased, and the expression of Muc5ac mRNA and its protein increased in the dry mouse model.

【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R-332

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