外燥小鼠模型的建立与评价

发布时间：2018-04-26 22:18

本文选题：外燥 + 小鼠模型　；参考：《湖北中医药大学》2011年博士论文

【摘要】：中医证候动物模型是在中医学整体观念及辨证施治思想指导下,运用脏象学说和病因病机理论,把人类疾病原型的某些特征在动物身上模拟复制,是中医学人体证候的具体再现,是开展中医证候及中药药理研究和新药开发的一个重要途径。燥,为自然界六气之一,是秋之主气。“岁金太过”或“岁木不及”,则燥气可化为致病因素,称为燥邪,因其来源于外,故常称为外燥。外燥是中医病因学说的重要组成部分,但对外燥理论与实验的研究均较少。本课题在前期研究的基础之上,拟通过总结中医动物模型以及中医证候动物模型的研究方法,结合外燥的病机特点,探讨建立外燥动物模型的途径,并通过实验研究观察外燥动物模型的病理变化规律,对外燥动物模型进行评价,为建立稳定的、可靠的、可重复的外燥小鼠动物模型提供依据。一、理论研究 (一)外燥小鼠模型造模因素的选择中医动物模型复制的诸多方法虽然各自有显著的优点,但是均有明显的不足之处。课题组在导师张六通教授的指导下,回顾了半个世纪以来中医证候动物模型的研究历程,认为中医证候动物模型的研究应该从三个方面出发：优化动物模型的造模因素；建立规范的动物模型评价方法；选择具有特征性的评价指标。外燥动物模型应该在外燥相关理论的指导下,复制符合外燥病机和证候的动物模型。外燥的流行责之于“岁金太过”或“岁木不及”,其产生原因与我国的地理结构和气候特点相关,我国的气候存在湿→干转换时间短于干→湿转换时间的特征,所以秋之燥气容易化为燥邪。模拟秋季气候变化的方法建立外燥小鼠模型,既可较好地体现外燥的变化规律,也可避免以极端干燥地区湿度条件为基础的研究结果对动物模型稳定性和可重复性的影响。 (二)外燥小鼠模型评价方法的探讨外燥的病理变化有两个主要特点：燥性干涩,易伤津液；燥易伤肺,影响肺的宣发肃降功能,可见口鼻干燥,咽干口渴,干咳少痰,或痰液胶黏难咯等症状。现代研究发现卫气的防御作用与肺部免疫效应密切相关,研究显示卫气的运行节律与免疫节律高度一致,并且卫气虚的患者IgG,IgA,IgE的含量有明显变化。外燥动物模型支气管肺泡灌洗液中IgA、IgG和SP-A含量的变化可以较全面地反映呼吸道不同部位免疫能力的改变,可以反映外燥动物模型肺气卫外功能变化的规律。气道粘膜由细胞层和粘液层组成。气道粘液层有两部分：凝胶层和溶胶层。气道粘液层的变化,可在一定程度上反映吸入空气对气道津液代谢影响的规律。气道Muc5ac mRNA及其蛋白的表达可在一定程度上反映气道凝胶层的变化,而气道AQP5 mRNA及其蛋白的表达可提示气道溶胶层的变化,通过观察气道Muc5ac和AQP5 mRNA及其蛋白表达的变化可揭示肺津液代谢的规律。二、实验研究 (一)动物及分组 SPF级BALB/c小鼠70只,体重18±1g,雌雄各半。由湖北省实验动物研究中心提供,许可证号：SCXK(鄂)2008-2005。实验动物在实验室常规饲养7天后随机分为7组：正常对照组(C组)、温燥模型Ⅰ组(WⅠ组)、温燥模型Ⅱ组(WⅡ组)、常燥模型Ⅰ组(QⅠ组)、常燥模型Ⅱ组(QⅡ组)、凉燥模型Ⅰ组(LⅠ组)、凉燥模型Ⅱ组(LⅡ组),置人工气候箱内进行造模。C、WⅠ、QⅠ、LⅠ组小鼠于第7日上午, wⅡ、QⅡ、LⅡ组小鼠于第13日上午按实验要求取材,取材前4小时禁水禁食。 (二)指标检测 1.小鼠一般情况观察观察小鼠的神态、活动情况、皮毛色泽及有无脱落,饮食状况及粪便物理性状变化。 2.小鼠肺组织结构观察采用苏木素伊红(HE)染色法。 3.支气管肺泡灌洗液(BALF)中IgG、IgA和SP-A蛋白含量的检测。采用定量ELISA试剂盒检测。 4.肺组织Muc5ac mRNA和AQP5 mRNA表达的测定采用SYBR Green I荧光嵌合法进行实时定量荧光PCR检测。 5.肺组织Muc5ac蛋白表达的测定采用免疫组织化学染色法检测。 6.肺组织AQP5蛋白表达的测定, 采用蛋白免疫印迹(Western blotting)检测。 (三)统计学分析采用SPSS15.0统计软件对所得数据进行处理分析。实验数据均以x±s表示,两组均数间比较采用t检验,多组均数间两两比较采用方差分析,P0.05为差异有统计学意义。三、结果本课题外燥小鼠模型的复制条件,既体现了秋季气候变化的过程,又体现了秋季气候变化的特征,较好地体现外燥的致病特点。 “邪之所凑,其气必虚”(《素问·评热病论》),外燥导致各组外燥小鼠模型的正气受到不同程度的损伤,尤其是肺气的宣发功能。肺气的宣发功能改变主要表现为肺气卫外和肺气宣发津液两方面外燥小鼠模型BALF中IgA、IgG和SP-A含量的变化体现了肺气卫外功能的变化。实验结果显示,与IgG含量的变化不同,虽然温燥、凉燥和常温燥小鼠模型BALF中IgA、SP-A含量的变化存在一定的差异,但是各组IgA、SP-A含量变化的趋势是一致的。外燥小鼠模型气道AQP5 mRNA及其蛋白表达的下降,提示外燥导致气道内津液含量下降,同时也导致了津液不能外达气道。而气道Muc5ac mRNA及其蛋白表达的增加,可能是一种保护性反应,以防止气道水液进一步的散失,也可能是外燥导致的一种病理变化。Muc5ac表达增加所反映的凝胶层的增厚与AQP5表达降低所代表的溶胶层的变薄,均可降低气道粘膜纤毛的转运率,导致气道防御能力下降,即肺气卫外能力降低。外燥小鼠模型出现毛色枯燥、饮水量增加,粪便略显干燥等症状,也是外燥之“燥胜则干”的表现。咳嗽的发生一方面是由于气道内痰液的刺激,另一方面也可能由气道反应性增强所致。外燥小鼠模型气道AQP5的降低和Muc5ac表达的增加,不仅可导致气道反应性增加,而且可导致了气道粘膜纤毛转运率的下降,使气道内痰液形成成为可能,并且提示形成的痰液性质较为粘稠。综上所述,外燥小鼠模型不仅体现了外燥形成的病机,而且具有外燥的基本症状和体征,是研究外燥发病机理较好的平台。四、结论本课题成功地建立了外燥小鼠模型,该模型接近人类外感燥邪的发病机制,较好地体现了外燥导致肺气输布津液的功能下降,体内津液不能外达,体表津液无以为继而发生干燥的病机,并且可以推断模型存在外燥之干咳少痰,或痰液胶黏难咯等症状。 1.外燥小鼠模型的造模因素模拟早秋,中秋和晚秋气候特点建立外燥小鼠模型的条件如下：早秋(温燥)：温度22士2℃,相对湿度33士2%；中秋(常燥)：温度15℃士2℃,相对湿度33士2%；晚秋(凉燥)：温度8士2℃,相对湿度33士2%；人工外燥环境风速设定为2.5m/s。 2.外燥小鼠模型的评价 (1)外燥小鼠模型毛色枯燥,偶见耸毛、蜷缩等现象,饮水量有所增加,粪便略显干燥。 (2)外燥小鼠模型气道粘膜皱襞增多,杯状细胞增生明显,气道粘液厚度均呈现下降趋势。 (3)外燥小鼠模型气道BALF中IgA含量增加,SP-A的含量增加。 (4)外燥小鼠模型气道AQP5 mRNA及其蛋白表达下降,Muc5ac mRNA及其蛋白表达增加。
[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.

【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R-332

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