【作者】 王剑；

【导师】 陈群；

【作者基本信息】 广州中医药大学 ， 中医诊断学， 2006， 博士

【摘要】 阴虚证是中医学中一个重要的证型,是指因阴液亏耗,滋润濡养功能和制约阳热功能低下,而产生以燥、热、升、动为特征的病理状态。人体是一个复杂的整体,中医学的“阴”有着广泛的物质和功能基础,阴虚证广泛见于临床各科疾病,在不同的疾病,不同的年龄,存在不同的病理生理改变。因此,很难用一项或二项所谓“黄金指标”来特异地反映阴虚证的本质,这意味着必须多系统、多层次来进行阴虚证机理的探讨。 本研究结合课题组的研究方向,①通过连续4W以温燥、辛热、利水中药组成的耗阴复方予小鼠灌服制备阴虚证小鼠模型,观察动物模型皮肤中透明质酸及其调控因素的变化;②参照皮肤光老化模型,通过连续6W高强度中长波段紫外线UVA、UVB照射小鼠背部裸露区皮肤,模拟外感热邪消耗阴液病变,观察与皮肤病变密切相关的基质金属蛋白酶及其金属蛋白酶组织抑制因子的表达,及补阴方药对上述病变的作用;③体外培养小鼠皮肤成纤维细胞,观察耗阴复方血清对成纤维细胞合成糖胺多糖的影响。通过上述三方面与皮肤相关的阴虚模型病理变化侧重反映阴虚证滋润濡养功能低下相关的病理改变;④通过4W连续24h光照抑制幼鼠褪黑素分泌,制备性成熟期前肾阴虚大鼠模型,观察其神经、内分泌变化及补阴方药的干预作用;⑤运用去卵巢手术加灌服耗阴中药复方制备更年期肾阴虚大鼠模型,观察其神经内分泌的变化;⑥以基因芯片技术观察更年期肾阴虚模型大鼠下丘脑基因表达谱的变化。通过上述三方面研究侧重反映阴虚证在生长发育和衰老过程中相关的病理改变。综合整体、细胞、基因多个层次,研究多个阴虚证模型相关系统的病变,期望对阴虚证的现代研究内容有多所创新,并为相关疾病防治药物或保健品的开发提供一些思路和方法。 通过实验研究,发现①连续4W以耗阴中药复方灌服制备的阴虚证模型小鼠皮肤中透明质酸含量、血液中表皮生长因子含量同步下降,血液中皮质酮有升高趋势。经补阴方药治疗后,此种病变得以逆转,说明阴虚证病理改变与皮肤间质透明质酸的减少,及其促生长因素——表皮生长因子含量的减少,降解因素皮质酮含量的增多密切相关; ②连续6W高强度中长波段紫外线UVA、UVB照射可致小鼠裸露部位皮肤萎缩,基质金属蛋白酶1和基质金属蛋白酶3高表达,而金属蛋白酶组织抑制因子表达受抑,胶原降解;而同期补阴方药治疗可部分改善此种病变。从造模因素与中医学中外感热毒的相关性,及从补阴方药的疗效反推,此种动物模型反映了外感阳热邪气所致皮肤阴虚病理改变的部分机理;更多还原

【Abstract】 Syndrome of Yin-deficiency is an important syndrome in traditional Chinese medicine (TCM), it means a kind of pathological stasis with character of dryness, heat, and excitement, it was caused by consumption of Yin-fluid, thereby decreased function of moistening, nourishing and restricting Yang. Human body is a complex whole system. "Yin" in traditional Chinese medicine has widespread material and functional basis. Syndrome of Yin-deficiency is widely seen in diseases of various clinical branches, and show different pathophysiologic mechanism in different diseases or different ages. Therefore, it is very difficult to rely on one or two items of so-called "golden index" to demonstrate fully the essence of syndrome of Yin-deficiency, and the study on the mechanism of syndrome of Yin-deficiency should be carried on in multi-systems and multi-levels.According our study objection, we carried on some study: ①The mouse model of Yin-deficiency syndrome was prepared by administrated with decoction consist of herbs of warm-dryness, pungent-heat, and promoting-urination for 4W, then the changes of the hyaluronic acid and the regulative factor in skin of the animal model were observed;②Referring to the way to induce skin photoage, high dose middle to long wave band ultraviolet ray UVA、 UVB was used to illuminate the exposed skin in the back of mouse for 6W, to simulate the pathological change of exogenous heat evil consuming Yin-fluid, then the expression of matrix metalloproteinases(MMPs) and tissue inhibitor of metalloproteinases-1(TIMP-1) were observed, the balance between MMPs and TIMP is closely correlated with skin pathological change, hydroxyproline was taken as the index of degradation of skin matrix. The effect of nourishing-Yin prescription of Chinese medicinal materials was studied on the above pathological change;③ Through in vitro cell culture offibroblast from mouse skin, the effect of blood serum containing Chinese material medica of warm-dryness, pungent-heat, promoting-urination on fibroblast synthesizing glycosaminoglycan was observed. The above three aspects of skin-correlation model of Yin-deficiency syndrome stress on reflecting the related pathological changes of low function of moistening and nourishing in syndrome of Yin-deficiency;?The kidney-Yin deficiency rat model before Sexual maturity was prepared by continuously 24h illumination for 4W to suppress the secretion of melatonin of young mouse, the changes of nerve and endocrine, the effect of Chinese material medica formula of nourishing-Yin was observed;(5)The kidney-Yin-deficiency rat model in menopause was prepared by using ovary-excision plus adminstrated with Chinese material medica of warm-dryness, pungent-heat, promoting-urination, the change of its neuroendocrine were observed;?The gene expression spectrum change in hypothalamus of rat model was studied by gene chip technology. The above three aspects aim to demonstrate the pathological changes of Yin deficiency syndrome in the growth, development and aging process. In the whole, cell and gene levels, several models of Yin deficiency were used to study the special pathological changes, hoping to innovate and affluence the content of modern research of Yin-deficiency syndrome, and to provide some idea and methods for exploiting of medicine and health maintenance product against the above diseases.Through experimental study, we discovered:(Dln the mouse model of Yin- deficiency syndrome which is prepared by administrated for 4W with Chinese material medica compound formula consist of warm-dryness, pungent-heat, promoting-urination, the hyaluronic acid content in the skin and the epidermal growth factor content in the blood decrease in synchronization, the corticosterone content in the blood showed the tendency of ascension. After treated with nourishing-Yin Chinese material medica formula, this pathological change was restored, which showed there existed close correlation between the pathological change of syndrome of Yin-deficiency and the reduction of the hyaluronic acid in the skin, and the reduction of the growth factor — —the epidermal growth factor, and the increase of the corticosterone content which was the adjustment factor of degradation;?Continuously illumination by high strength middle-long wave band ultraviolet ray U\^ UVB for 6W may caused the atrophy of exposed skin ofthe mouse, high expression of the matrix metal proteinasel and the matrix metal proteinase3, meanwhile the decreased expression of tissue inhibitor of metalloproteinases-1, followed by collagen degeneration;this pathological change was partly restored by treatment with nourishing Yin Chinese material medica formula in synchronization with light illumination. By referring to the exogenous pathogenic heat in traditional Chinese medicine, and from the curative effect of nourishing-Yin Chinese material medica formula, we primarily assumed this kind of animal model reflected partial mechanisms of pathological changes of skin Yin deficiency caused by exogenous pathogenic heat;(3)The fibroblast cultured by blood serum of Yin-consuming Chinese material medica compound prescription, showed reduction of synthesis of glycosaminoglycan, and restore to the normal level after given with blood serum of nourishing-Yin Chinese material medica compound prescription. The changes of the synthesis of glycosaminoglycan in the cell model reflected the opposite effect between Yin-consuming and Yin-nourishing Chinese material medica.?Male rats at 21 d after birth were exposed to 24h continual light for four weeks to inhibit melatonin secretion, the aim is to produce the model of deficiency of kidney-Yin. Compared to the normal group, model group changed in many aspects, such as body weight, serum MT declined significantly, serum NE, E and T increased significantly, While thyroid hormone and corticosterone changed lightly. In the treatment groups, low or high dose prescription of nourishing Yin and decreasing fire were given in the period of 24h continual light expose, it showed body weight and serum MT were increased to normal level, serum NE, E and T were decreased to normal level or even lower than normal level. All the results demonstrate that hypo-secretion of melatonin and the leaded hyperfunction of sympathetic nerve and the axle of hypothalamus-pituitary-sex gland, have some internal correlations with the syndrome of kidney-Yin deficiency with hyperfuction of fire. Our study firstly reported that the model of sexual precocity can be prepared with continual light expose.?The rat model kidney-Yin deficiency in menopause was made by ovary-excision plus administration with Chinese material medica of_ warm-dryness, pungent-heat, promoting-urination. It was discovered, in Yin-deficiency group, the content of estrogen, FSH in blood were in higherlevel than those in model of ovary-excision, the swelling and hypertrophy of various endocrine cells in the pituitary gland was obvious, which indicated that the function of various endocrine cells compensatorily ascend in the basis of pathological changes, this phenomenon is similar to the "interior heat due to Yin deficiency" in traditional Chinese medicine;The compensatorily ascent pituitary gland over-secreted follicle stimulating hormone increases the expression of aromatizing enzyme in the organizations of fat, liver, brain and so on, which increases the transformation from androgen produced by adrenal gland to estradiol. Estradiol affects the uterus, which improve the endometrium atrophy degree better than that in ovary-excision model group. Corticosterone content in blood in the imitative Yin-deficiency model group is obviously higher than that in the ovary-excision model group, and is close to that in the sham operation group. The pathology examination of the adrenal cortex discovered there is slight proliferation phenomenon, relating to the phenomenon of swelling and hypertrophy of various endocrine cells in the pituitary gland ,indicated that after administrated with Yin-consuming Chinese material medica, the function of hypothalamic-pituitary-adrenocortical axis in the Yin-deficiency model group is in higher level than that in the ovary-excision model group, which is similar to the function ascension of the adrenal cortex in the syndrome of interior heat due to Yin deficiency in clinical research.?Using gene array, we observed the gene expression changes in the hypothalamus of rat of kidney-Yin deficiency model. Taking the two times difference of gene expression as the significant standard, it was found that many gene expression changes associated with the kidney deficiency, such as the atrophy of nerve fiber demonstrated by the decreased expression of the following gene, myelin protein, procollagen, chondroitin sulfate proteoglycan, decorin, and myosin,etc;the above gene are the component of the nerve myelin, endoneurium, neurotubule and neurofilament;the decreased of anti-injury of hypothalamus neuron demonstrated by the decreased expression of the following gene, insulin-like growth factor, insulin-like growth factor binding protein, bone morphogenetic protein, etc, all the above gene have the function of nourishing nerve and promoting neuron development, restoring neuron injury;the decreased gene expression of many solute carrier family indicated the hypofunction of transport of organic anion, organic cation, neurotransmitter and the succeed decrease of cell metabolism which was alsoindicated by the gene expression decrease of many enzyme, involving the biological process of sugar, lipid, protein, and nucleic acid.The gene expression, correlated closely with the pathological changes of YIN-deficiency, involve the decreased expression of heat shock protein 70kD, which indicated the decreased function of against hazard stressor;the increased expression of corticotropin releasing hormone, which represent the hyper functional compensation of hypothalamus-pitutary;the decreased 5-HT receptor, which reflect the increased content of 5-HT in the hypothalamus and the disord of autonomic nerve;the increased Y-aminobutyric acid receptor compared with no change of glumatic acid receptor, which indicated the balance of excitement and inhibition towards excitement in the hypothalamus. There are also many active compound in the hypothalamus shows expression difference among YIN-deficiency group and other groups, such as tachykinins, vasoactive intestinal polypeptide, vasodilatator phosphoprotein, neuromedin, etc, the roles of those compound in the pathological changes of YIN-deficiency need further study.Our study abundance the contents of YIN-deficiency syndrome in the following aspects:?A new model-making methods of YIN-deficiency syndrome was established by administrating decoction consist of YIN-consuming herbs. It was discovered that hyaluronic acid in skin has positive correlation with fluid, their function also resemble very much. Prescription of nourish YIN exert its effect through improving the content of epidermal growth factor;? A new cell model reflecting YIN deficiency changes was primarily studied by in vitro cell culture of fibroblast using serum from the rat administrated with YIN-consuming herbs, and the evaluation criterion of effect of nourish YIN was also studied;(3) The internal correlations between pathological changes of skin photoage and pathological changes of YIN-deficiency was explored, it was discovered that nourish YIN prescription exert its anti-skin photoage through improving content of TIMP-1;? The internal correlation between the functional discord caused by hyposecretion of melation in impuberal period, and the syndrome of kidney YIN deficiency was primarily studied. It was firstly reported that sexual precocity model can be made by continual light expose;? By combing the pathological changes and function discord of pituitary, it was firstly reported that there exist close relation between consumptive function increase and fire caused by deficiency of YIN;(6) The concordant effect of genistein with melatonin on modulating the function of gonad axis’ in menopause, which further demonstrated the internal correlation h/twgfin.melatonin and kidney YIN;(7) A great deal of biological information of gene expression in hypothalamus of rat model of kidney YIN deficiency was studied, by which many study conclusion on YIN deficiency were proved, meanwhile some new active compounds about YIN deficiency were provide as clue for further study.更多还原