| Breast cancer, the incidence of which increased year by year, is one of the most common malignant tumors in women. In view of the fact that breast cancer has now become common disease threatening women’s physical and mental health, along with the people enhance the awareness breast cancer and improve the quality of life, its prevention has become the emphasis and hotspot research in the field.Normal breast ductal epithelial cells change into cancer cells is a multi-stages and multi steps process. At present, the majority of scholars believe that the occurrence of breast cancer undergoes normal epithelial, usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), carcinoma in situ (CIS) and invasive breast cancer (IBC) of the continuous change process, In the current view, premalignant lesions and its previous stages can be reversed, therefore, copy this continuous change process, clarify its pathogenesis and pathological changes is the most effective way for timely and effective measures to prevent and control of breast cancer. In the course of the research, a series of clinical patients’samples cannot be obtained with the continuous process of pathological changes of the same individual. The animal model is a powerful tool to achieve this goal that not only simple, convenient and easy to operate, but also can simulate patients internal environment in the maximum degree. In present, the animal tumor model methods can be divided into spontaneous, portability and induced model. Compared to the other two methods, induced model method is using carcinogenic factors contact directly or indirectly with animal, which can guarantee the premise of the original in physiological, anatomical features, simulation the development disease of internal environment in the greatest extent and reproduce the whole process of breast cancer. In presentment, breast cancer animal models and mammary gland hyperplasia animal models have been relatively mature, but the premalignant lesions of breast disease animal model is difficult to accurately grasp the experimental end point, too early or too late are both lead this experiment failure. Therefore, seldom report about successful premalignant breast disease animal.At present, early menarche, late menopause, long menstrual period, have been recognized as risk factors of breast cancer. Clinically, tamoxifen and aromatase inhibitors for breast cancer endocrine therapy drugs have been widely used, implies that estrogen play a crucial role in in the development of breast cancer. But how estrogen carcinogenesis in vivo, whether the specific molecular mechanisms and similar to the human body in animal models, there is no clear and unified understanding.This experiment used the 28 day old female SD rats and adopts the DMBA gavage (15mg/kg, first,55th,96th days respectively to intervene) combined with estrogen (0.5mg/kg/d) and progesterone (4 mg/kg/d) sequential administration (every 5 days for a delivery cycle) method, application of visual inspection, palpation morphological observation assisted with ultrasound, magnetic resonance imaging and the final pathology diagnosis of premalignant lesions of experimental endpoint determination, successfully induce premalignant breast disease rat model. On the base of this section, we used ELISA and immunohistochemistry technique, to detect the expression of 2-hydroxyl-estradiol (2-OH-E2),4-hydroxyl-estradiol (4-OH-E2), 16a-hydroxyl-estrone (16a-OH-E1), ERa, ERp and PI3K-85a in the normal breast tissue, premalignant lesions and breast cancer tissue which reserved in the process of this model, to study estrogen carcinogenesis mechanism and evaluate furtherly utility value in this model.Section I The Establishment and Evaluation of Premalignant breast disease in Rat ModelBackground:The development of breast cancer from UDH to ADH to CIS, and eventually to IBC, is a series of sequential steps, these lesions may coexist in the same individual breast tissues. Shaaban et al use epidemiological research and summarized that, if does not impose any intervention measures, the relative risk of UDH, ADH, DCIS eventually develop for IBC relative risk (RR) is 1.5-2,4-5 and 8-10 respectively. ADH and DCIS occur before IBC, called the premalignant lesions.In the devolvement of breast cancer, premalignant lesions as one important link, is a relatively long process, moreover, intervention at this stage can present or even completely reversed the development of breast cancer. The establishment of premalignant breast disease of animal model can simulate patients’complicated pathophysiologic state in the greatest extent and provide an experimental platform for discussion the mechanism of breast cancer and two grade prevention of breast cancer.As early as in 1961, Huggins et al found, DMBA and N-methyl-N-nitrosourea (MNU) as carcinogens in rats, specifically induce rat mammary tumor. Breast is a hormone sensitive organ, estrogen and progesterone cyclically effect on the breast can make the gland hyperplasia and involution occurs regularly. Elevated hormone levels not only increase the incidence of tumors but also can make the tumor occurrence in advance. We therefore hypothesized that application of mammary specific carcinogen MNU or DMBA combined with estrogen and progesterone, control the drug dose and action cycle appropriately, may appear the stage of breast premalignant cancer longer.The difficult of premalignant lesion animal model is to determine the endpoint of premalignant disease in this model. Pathology as the gold standard for diagnosis of tumor, can accurately judge the nature of the tumor, but in this diagnosis, the animal must killed or biopsy of suspicious lesions after anesthesia and may interrupt the natural evolution in a certain extent of disease. This method is restricted in the whole process of establish animal model. So, it is imperative to form simple, reasonable and convenient indicators to help make sure the end of premalignant lesions in noninvasive conditions.Ultrasound examination has been widely used in clinical in screening and aided diagnosis of benign and malignant breast tumors because of its timeliness, convenience, noninvasive economy and can be repeatedly short-term repeated observation. In ultrasonic inspection, two dimensional ultrasound as currently the most commonly used technique in breast diseases diagnoses, can display the malignant tumor characteristics clearly such as:irregular shape, spiculation, calcification, aspect ratio, posterior echo attenuation and lateral acoustic shadow. Breast Magnetic Resonance Imaging (MRI) because of its good soft tissue resolution, non-invasive, no radiation and multi direction, multi parameters, imaging sequence, and is not affected by breast soft tissue density, become an important supplementary method of auxiliary examination of breast diseases. Including plain scan, dynamic enhanced imaging and diffusion weighted imaging and other imaging methods. Plain scan only can through different signal intensity and morphological features to diagnosis of breast disease. Dynamic enhanced scanning displayed the lesion site and its surrounding characteristics of morphology and hemodynamics of relevant information more clearly by injecting contrast, after multi phase scanning, drawing dynamic enhancement curve, has great supporting role in differential diagnosis of breast tumor in nature.In summary, we believe that the model of premalignant breast disease in rats, the evaluation index is currently no unified, refer to the corresponding clinical indicators, combined with the specific circumstances of our laboratory, pseudo gross morphology, imaging and pathology three aspects to evaluate this model.Objective:This study perform 4 animal experiments, from the type of carcinogen carcinogen, route of administration, dosage of estrogen and progesterone, cycle, respectively, in order to explore the best induced tat model method that can prolonged the window phase in premalignant lesions, and most similar to human disease progress, to provide animal platform for the prevention of breast cancer. In the process of modeling, ultrasound, MRI imaging, combined with morphological observation, reference to gold standard in the diagnosis of the final pathology, to evaluate and determine the end point of premalignant lesions of breast disease animal model of end point.Materials and methods and results:1. Application of carcinogens and estrogen and progesterone to establish the rat model of premalignant breast disease. Through four animals experiments, identified carcinogens and estrogen and progesterone route of administration, dosage and administration times.(1). Experiment one:the selection of carcinogenic agents, DMBA or MNU. Eight SD rats were randomly divided into seven groups, were as follows intervention:group one,6 rats, blank control group; group two,12 rats, DMBA100mg/kg intragastric; group three,10 rats, DMBA100mg/kg intragastric+estrogen and progesterone 5 days a cycle; group four,12 rats, DMBA100mg/kg intragastric+estrogen and progesterone 30 days a cycle; group five,10 rats, MNU50mg/kg intragastric; group six,10 rats, MNU50mg/kg intragastric+estrogen and progesterone 5 days a cycle;group seven, 10 rats, MNU50mg/kg intragastric+estradiol and progesterone for 30 days in a cycle.Result:Before the end of each experiment, DMBA groups (groups two, three and four) total mortality was 38.23% caused by toxic carcinogenic agent, MNU groups (group five, six and seven) total mortality was 43.33% caused by carcinogenic agent toxicity; in a total of 60 days from 120th to 180th day began of the experiment, the number of premalignant breast disease in DMBA each groups were 5 (62.50%),3 (50%),4 (57%) separately. At the end of experiment, all survival rats in the DMBA groups were observed IBC, which mainly appears in 150th-200th days from the begin of experiment. In all MNU groups were not found premalignant breast disease except only one rat in group seven. In MNU groups, the number of rats eventually appears IBC were 4,5, and 5. MNU groups have so low detection premalignant breast disease rate and high mortality, indicating that MNU is more toxic and have a narrow window phase of premalignant breast disease, which cannot easily be detected. In the follow-up study of selection, we decided to use DMBA as a premalignant breast disease induced agent.(2). Experiment two:the administration of carcinogen DMBA, intragastric or intraperitoneal injection. SD rats were divided into six groups randomly, each group were 5 rats, were as follows intervention:group one, DMBA60mg/kg intraperitoneal injection+estradiol and progesterone 5 days a cycle; group two, DMBA50mg/kg intraperitoneal injected+estradiol and progesterone 5 days a cycle; group three, DMBA100mg/kg intragastric+estrogen and progesterone 5 days a cycle; group four, DMBA60mg/kg intragastric+estrogen and progesterone 5 days a cycle; group five, DMBA50mg/kg intragastric+estrogen and progesterone 5 days a cycle; group six, DMBA40mg/kg intragastric+estradiol and progesterone for 5 days a cycle.Result:All of the rats in intraperitoneal administration groups were death in the first-19th days of the experiment began, a large number of pale bloody ascites were found by autopsy, the size of liver and kidney increases, confirmed that acute hepatic and renal necrosis by pathology. DMBA gavage groups also have some degrees of toxicity of death, but because of the number rats in each group less, each group had no statistical significance. Intraperitoneal injection of DMBA has high toxicity and mortality, in subsequent experiments, we continue to use DMBA intragastric administration mode.(3). Experiment three:the choice of estrogen and progesterone administration cycle,5 days or 30 days. SD rats were randomly divided into four groups, were as follows intervention:group one,6 rats, control group; group two,15 rats, DMBA 100mg/kg intragastric+estradiol and progesterone 30 days a cycle; group three,15 rats, DMBA100mg/kg intragastric+estradiol and progesterone 5 days a cycle; group four,15 rats, only single drug DMBA100mg/kg gavage.Result:We increased the number rats in each group in this experiment, DMBA monotherapy, DMBA combined with estrogen and progesterone 5 days a cycle, DMBA combined with estrogen and progesterone 30 days a cycle were compared, the results showed that in the DMBA combined with estrogen and progesterone 5 days a cycle group, premalignant cancer detection rate (55.60%) was higher than that of group two (40%) and group four (37.50%), suggesting that estrogen and progesterone 5 days a cycle can help stabilize premalignant breast disease of rats. After 30 days at the intervention of carcinogen, the rats still have higher death,5 (33.33%),6 (40%),7 (46.67) rats were dead in group two, three and four.(4). Experiment four:the dosage and time selection of carcinogen DMBA.50 rats were randomly divided into 5 groups,10 rats in each group, this experiment use low dosage several times to control, the dose of carcinogen DMBA was from lOmg/kg to 30mg/kg, each group with increasing 5mg/kg, respectively, the rats were conducted in experiment first,55th,96th day of the three time points DMBA gavage, starting from the second day, all rats were in accordance with estrogen and progesterone 5 days a cycle intervention. Group one, DMBA10mg/kg; group two, DMBA15mg/kg; group three, DMBA20mg/kg; group four, DMBA25mg/kg; group five, DMBA30 mg/kg.Result:This study found except for the last group with 50% mortality, all of the other groups’death rate was lower than 20%. From the carcinogen intervention of 121th day to 200th day, in 79 days, with a total of 19 rats were first found premalignant breast from all of the rats, of which group two were the highest-7 (87.50%), and the time of first appeared premalignant breast disease were from 121th to 181th days of 60 days in group two. Window phase in group three was the highest-51 days, followed by group two of 38 days, the group five is the shortest-8 days.2. Evaluation indexes in establish rat model of precancerous breast disease. All of the above experiment rats were underwent morphological observation and palpation at the second day after the carcinogen intervention. After 10 weeks the carcinogen intervention, use ultrasound twice a week, if ultrasound showed a clear mass, then underwent tumor resection (including tumor and surrounding a small amount of normal gland) for pathological diagnosis under general anesthesia, if ultrasound examination showed suspicious lesions, then the MRI auxiliary diagnosis were needed, tumor resection for pathological diagnosis under general anesthesia if MRI showed suspicious lesions.200th days after carcinogen intervention, all rats were kill to death, take out the breast tissue and suspicious site to make pathological diagnosis.(1). The gross morphology, including inspection and palpation. Inspection:the abdomen has uplift or not, papilla and surrounding skin color, size of the nipple; palpation contents:the texture of abdominal subcutaneous breast pad, swelling material. Recorded the tumor’s size, shape, texture, edge, activity etc. once tumor was discovered.(2). Ultrasound observation included:boundary, the edge of the tumor, internal echo, form, whether fine punctate hyperechoic calcifications, there is posterior echo attenuation or not, blood flow signals of Adler semi quantitative grading, internal arteries blood flow index.(3). MRI observation included:ordinary scan and enhanced scan, abdominal subcutaneous tissue signal intensity, there is abnormal enhancement or not, dynamic enhancement curve type hemodynamic performance.(4). Pathological observation:all organizations were stained with HE, immunohistochemistry was used to examine the molecular breast myoepithelial marker P63 for further diagnosis if there is some uncertain diagnosis whit HE.Results:1. In the general shape, the performance for the breast skin ruddy, uplift, nipple increase; palpation subcutaneous breast pad thickening, soft, masses shape are often irregular, the surface is unsmooth and boundary is unclear.2. Ultrasound display, tumor has unclear and irregular boundary, shape irregular, can be expressed as uneven or having hornlike projections, low internal echo, sometimes clearly visible echogenic calcification, a small part of a posterior echo attenuation, semi quantitative blood flow signals are often Adler grade Ⅰ-Ⅱ, internal arteries blood flow index is RI> 0.7. Magnetic resonance T1W1 showed low or intermediate signal, abnormal enhancement, mostly punctate foci or mass lesions, ductal carcinoma in situ were catheter like or segment enhancement in enhancement scanning. Dynamic enhanced hemodynamic curve showed outflow type or platform.3. Pathological observation, by HE staining and P63 immunohistochemical diagnosis, premalignant breast disease divided into the following several pathology types:atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS).Conclusion:1. DMBA has more weak toxicity than MNU, in the process of premalignant breast disease modeling, can increase the probability of premalignant breast disease in rat model.2. DMBA gavage more suitable for the route of administration, fractionated low dose administered, security molding effect and effectively reduce the mortality of experimental rats.3. Estrogen and progesterone by 5 days a cycle mode of administration stimulated subcutaneous abdominal glands hyperplasia in rats and adapted the inherent physiological cycle in rats, can appear hyperplasia involution change regularity, create the appropriate body within the environment, extent the process of premalignant breast disease relatively in the process of modeling.4. Ultrasound showed inhomogeneous low echo, focal enhancement of blood flow; dynamic enhancement time signal intensity curves in MRI performance showed flow type or platform type, which can be as index of premalignant breast disease in vivo dynamic observation.5. DMBA (15mg/kg first,55th,96th day respectively by gavage) combined with estrogen (0.5mg/kg/d intraperitoneal injection) and progesterone (4 mg/kg/d intraperitoneal injection) sequential administration (every 5 days for a delivery cycle) can be successfully established stable animal model of premalignant breast disease.Section Ⅱ The Mechanism of Premalignant breast disease in Rat Model Background:Background:Application of the carcinogen DMBA combined with estrogen and progesterone 5 days a cycle have been confirmed a successfully method to establish rat model of premalignant breast disease in our first section. At the same time, we also found that estrogen and progesterone intervention with the same physiological cycle in rats (5 days a cycle) is more suitable to establish the model of precancerous lesions of rats. The same dose administered of DMBA, different estrogen and progesterone intervention (5 days and 30 days for a cycle), the final appearance of premalignant breast disease is in different rates. Therefore, DMBA simply only as initiating factors for tumor development, cyclical changes in vivo estrogen and progesterone can provide the microenvironment for tumor occurrence and development and affected the occurrence and development of tumor speed in a certain extent.Estrogen and progesterone for 5 days in a cycle of sequential administration synergistic effects with endogenous hormone on the breast, which promote breast ductal and acinar growth regularly and hyperplasia of mammary gland involution on the organization, may make the stagnation in the atypical hyperplasia period longer. This study use 28 days rats as our experimental model, which endogenous hormone levels in rats were lower and has not formed the normal physiological cycle yet, through injection of exogenous hormones (5 day cycle) help rats reproductive system and endocrine system to mature, the exogenous hormone and endogenous hormone secretion in rats with formation of synchronization effect, synchronization to promote breast tissue hyperplasia development, make breast more synchronous appear continuous process include normal hyperplasia, atypical hyperplasia and carcinoma, so as to conducive to judge and control the endpoint of experiment correctly.Estrogen, as an important endocrine hormone in women, plays an important role in the reproductive system, sports system and cardiovascular system etc.. So far, although the reason of breast cancer is not clear and unified understanding, but at present the evidence clearly confirmed that estrogen plays a positive role in promoting and development of breast cancer. As early as in 1998 October, the US Food and Drug Administration (FDA) formally approved that estrogen antagonist-tamoxifen can be used to prevent women who have a high risk for breast cancer. In the early 1990s, represented by tamoxifen in breast cancer endocrine chemical prevention experiment carried out gradually, ten years follow-up data suggest that tamoxifen can reduce the rate of recurrence and metastasis of postoperative breast cancer patients significantly.The present study showed that estrogen in the body through estrogen receptor (ER) signal transduction mechanism and estrogen metabolism two ways to produce carcinogenic effects. Estrogen combined to its receptor, induced receptor dimerization reaction and combinated the downstream target gene, activated MAPK, PI3K, JNK pathway, stimulateed the breast cells to proliferate continually, which increasing the chance of errors during DNA replication, resulting in gene mutation, leading to breast cancer. Among them, the PI3K pathway as a classical pathway in the model, plays an important role in the inducing cell differentiation, proliferation, invasion and metastasis.In the metabolic pathways, estrogen mainly through hydroxylation reaction, generate 2-OH-E2 and 2-hydroxyl-estrone (2-OH-E1),4-OH-E2 and 4-hydroxyl-estrone and (4-OH-E1),16α-OH-E2 and 16 a-hydroxy-estrone (16a-OH-E1). These compounds, 4-OH-E2 and 16α-OH-E1 have carcinogenic effect, while 2-hydroxyl can generate anticancer drugs 2-methoxy-estradiol and 2-methoxy-estrone under the action of catechu phenol-o-methyl transferase (COMT). So,2-hydroxy and 16 a-hydroxyl, 2-hydroxyl and and 4-hydroxyl ratio, determine breast cell cancerization proportion, the one who has higher metabolism ratio of 16 a-hydroxyl and 4-hydroxy estrogen, the one has higher risk of breast cancer than that of the high proportion of estrogen metabolism through 2-hydroxyl.SD rats’estrogen structure are very similar to people’s, and have the same physiological function, estrogen receptors are also divided into A-F 6 districts and have the same structure and function in every district between people and SD rats, most of the breast tumors are hormone dependent tumors. So, we speculate that estrogen in rats may also have the similar carcinogenesis with people. This study use ELISA and immunohistochemistry in normal breast tissue, premalignant tissues and breast cancer tissues from which has been successfully established rat model of premalignant breast disease, detected the key index of estrogen pathways in the analysis and comparison, discuss estrogen action mechanism in this model, evaluation of its practical value.Objective:To explore the mechanism of estrogen in the model of premalignant breast disease that has been successfully establishedMaterials and methods:1. Applicate ELISA technique to detect the levels of 2-OH-E2,4-OH-E2 and 16a-OH-E[in normal breast tissue, premalignant lesions and breast carcinoma.2. Using immunohistochemistry technique to detect the levels of ERa, ERβ and PI3K-P85a in normal breast tissue, premalignant lesions and breast carcinoma.3. Statistical analysis:Statistical analysis was conducted using SPSS20.0 professional analysis software. ELISA data use MasterPlexReaderFit professional analysis software.Results:1.2-OH-E2,4-OH-E2 and 16a-OH-E1 content were gradually increased in the rat breast normal tissue, premalignant lesions and breast cancer tissues, especially for the 4-OH-E2 and 16α-OH-E1 content increased more obviously (P<0.001).2. The ratio of 4-OH-E2 and 2-OH-E2 has the highest level in the premalignant breast tissue and have a significant difference in normal tissue (P=0.002), breast cancer tissue (P=0.030).3. Ratio of 16a-OH-E1 and 2-OH-E2 were increased gradually in the rat breast normal tissue, premalignant lesions and breast cancer tissues, there is significant difference only in normal breast tissue and premalignant lesions to breast cancer tissues (P< 0.001).4. The expression range and total score of ERa gradually increased in the breast normal tissue, premalignant lesions and breast cancer tissues, there is si gnificant difference in normal breast tissue to premalignant lesions (scale:P< 0.001; total score:P=0.003) and no statistically significant differences in prem alignant lesions to breast cancer tissues. There is no statistically significant diff erence in intensity in the three groups.5. The expression range of ERβ gradually decreased in the breast normal tissue, premalignant lesions and breast cancer tissues, there is significant difference in normal breast tissue to premalignant lesions (P=0.008), no statistically significant differences in intensity. There is statistically significant differences in the total score between normal breast tissues and breast cancer (P=0.006).6. The expression range and intensity of PI3K-P85α in rat breast normal tissue, premalignant lesions and breast cancer tissues were gradually increasing. Range, intensity and total score between every group had statistical difference (P<0.001)Conclusion:1. In the model of premalignant breast disease, estrogen metabolism mainly through 4-OH-E2 and 16α-OH-E1 pathway.2. In the model of premalignant breast disease, estrogen binding ERα mainly as estrogen receptor way, starting downstream of ERα, upregulating expression of PI3K-P85.3. There are specific changes in the expression of ERα and the ration of 4-OH-E2 and 2-OH-E2 in normal breast tissue to premalignant breast disease tissues, which can be used as an auxiliary index in the diagnosis of premalignant breast tumors.4. The effects of estrogen pathways to breast cancer are similar between human and rat. This model can simulate the human body environment carcinogenic process in the greatest degree, which has higher use value. |
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