【作者】 马朝群；

【导师】 许芝银；

【作者基本信息】 南京中医药大学 ， 中医外科学， 2009， 博士

【摘要】 研究背景:乳腺增生病是乳腺小叶在成熟期或周期变化中生理性增生与复旧不全造成的乳腺正常结构紊乱,病理表现为上皮增生,纤维组织增生及部分以淋巴细胞为主的炎性细胞浸润,统称为乳腺结构不良。在各种乳腺疾病中,乳腺增生病的发病率占乳房疾病的75%,约占育龄妇女的40%。发病年龄集中于20～50岁,45～50岁达到高峰,50岁以后发病率急骤下降。流行病学资料表明乳腺癌的发生与乳腺增生性疾病有密切关系,特别是乳腺不典型增生。国内外专家认为,此病癌变率占10～20%,发生乳癌的危险比健康妇女高1.4～2.5倍。正常乳腺的上皮细胞向恶性转化,是在各种内外因素作用下发生的一个渐进过程,大约需15～35年。在这漫长的数十年中,通过人为的积极干预,如改善环境、行为干预、精神疏导、药物治疗等,对患者的免疫机能和内分泌功能起良性调节作用,有可能将病变逆转,从而减少和防止乳腺癌的发生。因此防治本病有着重要的临床意义和广阔的应用前景。本病属于中医“乳癖”的范畴。中医学认为肾虚肝郁、冲任失调为发病之本,气滞痰凝血瘀为发病之标,病位在肝脾肾,病性属本虚标实。西医认为其形成是因内分泌失调,雌激素增多或雌孕激素比例失调及乳腺组织中雌激素受体增多所致。在治疗方面,西医多采用内分泌治疗和手术疗法,但存在着服药期间疗效好、停药后反跳明显、短期用药可干扰人体激素间的平衡、手术会对周围腺体组织产生刺激等不足。目前以辨证论治为基础的中医药在治疗乳腺良性增生疾病方面具有明显的疗效和综合优势。具体主要有辨证论治及中成药、单味药治疗。但目前还存在着不少问题,如辨证分型没有统一的标准,部分研究缺乏对照组等。本文运用中医药三种不同治法即疏肝理气、调摄冲任和补肾疏肝法分别治疗乳腺增生型大鼠,观察其疗效并探讨作用机理,为此病的治疗提供一个新的思路和易于推广、经济实用、行之有效的方法。方法:通过肌肉注射苯甲酸雌二醇和黄体酮的方法,复制出大鼠乳腺增生模型,将60只大鼠随机分组,每组10只。予以药物干预,分别进行镇痛实验,比较各组的镇痛效果;观察大鼠的行为学指标,对大鼠的血浆性激素水平:雌二醇(E2)、孕酮(P)、垂体泌乳素(PRL)、促黄体生成激素(LH)、促卵泡成熟激素(FSH)含量进行测定;在实验过程中对大鼠的乳头高度及直径进行测量,了解各组药物对大鼠乳腺充血和水肿的减轻程度,并在光镜下对大鼠的乳腺组织进行病理学观察,对乳腺小叶的腺泡数量,腺上皮增生、腺泡分泌现象,导管上皮增生程度等进行分析,比较各组药物对大鼠乳腺增生的干预程度;并采用免疫组化和原位杂交法检测大鼠乳腺组织中ER、PR、P53和端粒酶的表达水平。结果:补肾疏肝法对乳腺增生型小鼠具有较好的非特异性镇痛作用,且明显优于单纯疏肝理气或单纯调摄冲任法,亦优于乳癖消和三苯氧胺(P＜0.05)。此法能改善大鼠的“竖毛、皮毛欠光、脱毛、倦怠、弓背、怕冷、反应迟钝、易激惹、急躁”等肾虚肝郁证候群,具有统计学意义(P＜0.05)。实验表明,三苯氧胺组、乳癖消组以及补肾疏肝组(以下称:混合组)在乳房直径方面均能得到有效改善(P＜0.01),而疏肝理气组和调摄冲任组则无显著性变化(P＞0.05),说明混合组效果优于单纯的调摄冲任组和疏肝理气组;而在降低乳头高度方面,各治疗组虽然相较于空白对照组有明显降低(P＜0.05),但治疗组之间并无明显性差异。对大鼠的乳腺组织进行形态学观察,结果表明疏肝理气与调摄冲任法合用可较好地对抗外源性雌、孕激素引起的实验性大鼠乳腺增生。调节大鼠体内性激素代谢水平方面,混合组以及三苯氧胺组对于降低乳腺增生大鼠血清E2水平有极显著性效果(P＜0.01),同时,单纯疏肝理气组也能有效降低乳腺增生大鼠血清E2水平且具有统计学意义(P＜0.05);疏肝理气组、调摄冲任组、混合组与空白对照组比较能显著升高乳腺增生大鼠血清P水平(P＜0.05),其中后两者具有极显著性差异(P＜0.01);在PRL与LH水平方面,各治疗组与空白对照组之间比较并无显著性差异(P＞0.05);另外,调摄冲任组与空白对照组相比能降低FSH含量(P＜0.05),而混合组和三苯氧胺组能显著性降低其含量(P＜0.01)。当大鼠乳腺组织形态表现为明显增生时,乳腺内ER、PR表达水平也随之显著升高,混合组在抑制大鼠乳腺组织增生的同时,乳腺ER、PR表达水平随之降低,且明显低于空白模型对照组(P＜0.01)。我们采用免疫组化方法测定治疗前后突变型P53蛋白的表达,结果表明,大鼠乳腺增生时乳腺组织P53染色灰度明显低于正常大鼠及混合组者,而后二者之间的染色灰度值则相近,说明乳腺增生大鼠乳腺组织P53蛋白是明显增加的,疏肝补肾中药能抑制P53蛋白的产生、释放,从而对乳腺组织起到保护作用(P＜0.01)。对于大鼠乳腺组织内端粒酶的测定,我们发现除乳癖消组外,其余各组与空白模型组对照均能降低端粒酶的阳性表达率(P＜0.05),且三苯氧胺组和混合组效果明显,优于单纯疏肝理气组和调摄冲任组(P＜0.01)。结论:补肾疏肝法对乳腺增生型大鼠具有显著的镇痛效果,并能一定程度上改善其肾虚肝郁症状,较好地对抗外源性雌、孕激素引起的实验性大鼠乳腺增生,调节大鼠体内性激素代谢水平,改善乳腺组织内ER、PR、P53及端粒酶的表达释放,从而对乳腺增生具有较好的治疗效果。更多还原

【Abstract】 Background: Hyperplasia of Mammary Gland (HMG) refers to disordered mammary structure due to physiological hyperplasia or incomplete renewing of lobular mammary gland during the maturing period or with periodical change, pathologically presented by epithelial hyperplasia, fibrous hyperplasia or lymphocyte predominated inflammatory cell infiltration, generally entitled as mammary dysplasia. Among variety of mammary gland diseases, the incidence of HMG reaches 75%, it accounts for 40% diseases of women of childbearing age. The disease mainly affect women of age 20 through 50, which reaches a peak at age 45 to 50; and declines sharply after 50. Epidemiological data indicates that breast cancer is closely related to proliferative breast diseases, in particular mammary atypical hyperplasia. Domestic and overseas professionals consider that it’s carcinomatous change rate accounts for 10～20%, the risk of the occurrence of breast cancer in women with HMG is 1.4 to 2.5 times higher than in healthy women. The transforming of normal mammary epithelial cells to malignant condition is a gradual process, around 15 to 35 years, owing to various internal and external factors. During this long transforming period of several decades, active interventions, such as improvement in the environment, behavior intervention, mental ease, medication treatment, etc. all can have positive regulatory effect on the immune function and endocrine function; it may reverse the disease process to reduce or to prevent the occurrence of breast cancer finally. Therefore, prevention and treatment of this disease has important clinical significance and broad application prospects. This disease belongs to the scope of Chinese Medicine "Ru Pi". Traditional Chinese Medicine (TCM) believes that kidney deficiency and liver Qi stagnation, disordered Qi and blood in Chong and Ren meridians are the root cause (Ben), Qi stagnation, accumulation of sputum, and blood stasis are symptoms and signs (Biao), diseased location is liver, spleen and kidney, it is characterized by Ben deficiency and Biao excess. Western medicine considers HMG is caused by endocrine disorders, estrogen level rising, disordered ratio of estrogen and progesterone, or an increase in estrogen receptor in mammary tissue. In terms of the treatment, western medicine often adopts the endocrine therapy or surgical therapy, however, there are some inadequacies, such as the efficacy can be good when taking medicine, while there is rebound phenomenon after drug withdrawal, short-term medication will interfere with hormone balance as well, the operation can be a stimulating factor to the mammary tissue around. Currently, TCM treatment, based on syndrome differentiation, has evident effect characterized by comprehensive advantages for proliferative benign mammary diseases, including syndrome differentiation treatment, formulated herbal medicine treatment and single herb treatment. Nevertheless, there are still some problems, there is no uniformed standard for syndrome type differentiation, some studies do not have control group, etc. In this paper, three different TCM treatments, i.e. Soothing Liver Qi, Regulating Chong and Ren Meridian, Tonifying Kidney and Soothing Liver Qi, are applied to treat rat with MGH, the effect was observed, the acting mechanism was explored. The study result has provided a new way of thinking for the treatment of this disease, which is effective, cost-effective, can be widely applied.Methods: Through intramuscular injection of estradiol benzoate and progesterone, rat model of MGH were produced. 60 rats were randomized into six groups with 10 in each group, all receive drug intervention. The analgesia experiment were conducted to compare the analgesic effect of different interventions; behavior indicators were observed, plasma sex hormone levels were detected including estradiol (E2), progesterone (P), pituitary prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH); measure the height of the nipple on rats, to detect the degree of congestion and edema of mammary tissue; observe pathological signs through light microscope, focusing on the number of breast lobules of acinar, adenomatosis, acinar secretion phenomenon, and the ductal hyperplasia degree, to compare the effect of different interventions on mammary gland hyperplasia; detect ER, PR, P53 and the expression level of telomerase in rat’s mammary tissue by immunohistochemistry method or by in situ hybridization method.Results: Treatment method of Soothing Liver Qi and Regulating Chong and Ren Meridian shows non-specific analgesic effect on mice model, better than either the treatment method of Soothing Liver Qi, or method of Regulating Chong and Ren Meridian, it is also better than Ru Pi Xiao group or tamoxifen group, all have statistical significance (P <0.05). This method can improve syndromes of kidney deficiency and liver Qi stagnation, i.e., pilo-erection, less fur-ray, lipsotrichi, lassitude, roach back, aversion to cold, unresponsive, irritable, fidget, with statistical significance (P < 0.05). Experiments also show that breast diameter significantly change in Tamoxifen group, or Ru Pi Xiao group, or Tonifying Kidney and Soothing Liver Qi group (referred as, mixed group, in following sections) (P <0.01), while there is no significant change for breast diameter in the group of Soothing Liver Qi, or Regulating Chong and Ren Meridian group (P> 0.05), this experiment indicates that the mixed group is superior to Soothing Liver Qi group or Regulating Chong and Ren Meridian group. With respect to decreasing nipple height, there is no apparent differences among the treatment groups, though there is significance when compared with blank control group (P <0.05). Morphological observation of mammary tissue of rats indicated that, group of applying treatment method of both Soothing Liver Qi and Regulating Chong and Ren Meridian has better effect against rat MGH induced by exogenous estrogen and progesterone. Regarding adjust the level of sex hormone, mixed group, tamoxifen group have a significant effect in reducing serum E2 level (P <0.01), meanwhile, Soothing Liver Qi group can effectively reduce the serum E2 level which is statistical significant (P <0.05); Soothing Liver Qi group, Regulating Chong and Ren Meridian group, mixed group significantly increase serum P level when compared with blank control group (P <0.05), further, there is a significant difference between the latter two groups (P <0.01); for the level of PRL and LH, there is no significant difference between treatment groups and control group (P> 0.05); besides, regulating Chong and Ren Meridian group can reduce FSH concentration compared with control group (P <0.05), the mixed group and the tamoxifen group can significantly reduce it’s concentration (P <0.01). When mammary gland tissue manifested obvious hyperplasia morphologically, mammary ER, PR expression levels significantly increase. The mixed group shows effect of inhibiting mammary tissue hyperplasia, and decreasing ER, PR level, there is statistical significant when compared with blank model control group (P <0.01). Immunohistochemistry method was used to determine mutant P53 protein expression before and after the treatment, the result shows, P53 in mammary tissue of rat with HMG is much lower than that in normal rat and in mixed group, and the staining gray values between the latter two groups are similar, these signs indicate that P53 protein is increased in the mammary tissue of rat with HMG, herbs of Soothing Liver Qi and Tonifying Kidney can inhibit the production and release of P53 protein which has a protective effect on mammary tissue as a result (P <0.01). Testing of telomerase in rat mammary tissue reveals that, aparting from group Ru Pi Xiao, the rest groups and the blank model group, all can decrease the expression of telomerase (P <0.05), further, it is more obvious in tamoxifen group and in the mixed group, which is better than Soothing Liver Qi group and than Regulating Chong and Ren Meridian group (P <0.01).Conclusion: Treatment method of Tonofying kidney and Soothing Liver Qi has significant analgesic effect on rat with HMG, it can improve the symptoms of Kidney deficiency and Liver Qi stagnation to a certain extent, the treatment method has inhibiting effect on rat mammary gland hyperplasia induced by exogenous estrogen and progesterone, it can regulate the sex hormone metabolism in rats, ameliorate the expression and releasing of ER, PR, P53 in mammary tissue, as a result, has therapeutic effect on HMG更多还原