The Research On Histopathological Alterations During Breast Carcinogenesis In A Rat Model Induced By DMBA And Estrogen-Progestogen Combinations

BackgroundBreast cancer is one of the most common tumors in women in the world, accounting for about 1/4 of new cancer incidence in women, and its mortality which accouting for 15% is in first place now, with the development of people’s living standard, its incidence is increasing year by year. In rencent years, more attentions have been paid on the prevention of breast cancer, the researchers had established various animal model of breast cancer and investigated the etiology and development mechanism of breast cancer, this would play an important role in prevention of breast cancer.The studies found that the development of breast cancer is a muti-step process, progressing sequentially from normal to usual hyperplasia, to atypical hyperplasia, to in situ, and invasive stages. At present, researchers showed that the stage of premalignant lesion and before of the stage could be reversed, herein, to copy this process and to definite the mechanism and histopathological changes would be helpful for seeking the effective methods to prevent and control the breast carcinoma. In view of the fact that the mammary gland is also a hormone-dependent organ, estrogen(E) play a main role of stimulating ductal growth and epithelial hyperplasia. The main role of progestogen(P) is to promote the development of breast lobules and acini, based on E stimulation of breast ductal development can make the breast to be fully developed. Meanwhile, the studies showed that 7,12-dimethylbenz(a)anthracene (DMBA) is a highly toxic chemical and frequently used to induce tumors in animal experiments, breast cancer was initiated by DMBA under hormonal conditions in which suitable levels were present. So far, although more and more breast cancer model was established, and provided the basement for the studies of development mechanism and therapy methods, the comprehensive and systematic research on histomorphological alternations of these models was very rare. In our study, we planed to induce breast carcinogenesis in rat by DMBA and estrogen-progetogen combination, and to observe the histopathological changes of rat mammary gland, to stain the diagnosis related marker P63, CK5/6, CK34 β E12 and breast cancer related protein E-Cadherin, PCNA, Bcl-2 and investigate the carcinogenesis of breast cancer, the study not only provide the histopathological basement for the further research on the breast cancer models, but also the theory proof for the pathogenesis and treatment of breast cancer.Part OneHistomorphological observations during carcinogenesis in rat mammary gland induced by DMBA and estrogen-progestogen combinationsObjective To induce the carcinogenesis in rat mammary gland by DMBA and estrogen-progestogen combinations, and to observe the histomorphological changes under a light microscope.Methods Fifty Sprague-Dawley (SD), twenty days old, female rats were randomly divided into five groups, G0(Group blank control), G I (Group I, DMBA 15mg/kg), GⅡ(GroupⅡ, DMBA 20mgl/kg), GⅢ(Group Ⅲ, DMBA 25mg/kg), GⅣ(GroupIV, DMBA 30mg/kg), and the rats in G Ⅰ - G Ⅳ were treated with β-estradiol-3-benzoate 0.5mg/kg and progesterone 4mg/kg with a cycle of 5 days respectively. All the rat samples in each group were fixed in 10% neutral formalin liquid, paraffin-embedded, cut into slices with 4 μ m and stained with hematoxylin-eosin(HE), to observe histomorphological alternations under a light microscope, and record the mammary gland lesions of each rat in experimental groups at 120th,148th,180th, including dutal hyperplasia, lobular hyperplasia, stroma hyperplasia, cystic hyperplasia, atypical hyperplasia, lobular hyperplasia, carcinoma in situ and invasive carcinoma, the determination standard refered the World Health Organization classification of human breast lesions.Results1 The histomorphological alternations would be observed under a light microscope, including the terminal duct hyperplasia(TDH), lobules hyperplasia(LH), cystic hyperplasia(CH), stromal hyperplasia(SH), atypical duct hyperplasia(ADH), atypical lobule hyperplasia(ALH), carcinoma in situ(CIS) and invasive carcinoma(IC). Among the above lesions, TDH, LH, CH, SH belonged to benign lesion, ADH and ALH were premalignant lesions.2 At 120th,148th,180th, the incidence of breast lesions in experimental groups was 4.38%,14.06%,34.06% respectively, showing an increasing trend, the highest incidence was at 180th; the incidence of TDH, ADH, CIS and IC was 57.5%,45%, 32.5%,22.5% respectively, showing a decreasing trend.ConclusionsDMBA combined with estrogen-progestogen could induce the carcinogenesis in rat mammary gland, and the series changes from benign lesions to premalignant lesions, to carinoma lastly would be observed in experimental groups at 120th,148th, 180th.Part TwoThe research on expression of P63, CK5/6, CK34 β E12 during carcinogenesis of rat mammary gland and human breastObjective To observe the expression of P63, CK5/6, CK34 β E12 in ductal lesion of rat mammary gland, to investigate the differences between them, and to analyze the similarities and differences betwwen the carinogenesis of bresat in SD rat and human.Methods Ten mammary gland samples in rat and ten in human breast, paraffin-embedded, were collected respectively from the part one experiment and from the Department of Pathology, Affiliated Hospital of Shandong Academy of Medical Sciences during 2013-2014 years which the patients accepted radical mastectomy, the pathological results of rat mammary gland tissue and human breast tissues were diagonise as invasive ductal carcinoma, and including the usual ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in stiu, invasive ductal carcinoma. The expression and distribution of P63, CK5/6, CK34 β E12 in ductal lesions of rat and human breast were detected by immunohistochemical staining PV-9000 two step method, and to observe staining results under a light microscope. And to analyse by the SPSS17.0 software:(1) the differential expression of P63, CK5/6, CK34 β E12 between each breast lesions in rat and human respectively; (2) the differential expression of P63, CK5/6, CK34 β E12 in each breast lesions between in rat and human.Results1. The expression of P63 in ductal lesion of rat breast was similar to that in human breast, there was no difference between them(P>0.05); the expression of P63 in usual ductal hyperplasia, atypical ductal hyperplasia, ductal carcinSma in situ, invasive ductal carcinoma was decreased gradually with the alteration of the lesion in rat breast, there was significantly differences(P<0.0.1). The same trend was observed in human breast.2. In rat and human breast tissues, the expression of CK5/6, CK34 β E12 was gradually decreased from usual ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ, invasive ductal carcinoma, there was significantly difference respectively(P<0.0.1); the expression of them in the ductal lesions, there was no difference between in rat and human breast(P>0.05).Conclusions1 The expression of P63, CK5/6, CK34 β E12 in rat mammary gland lesions in each groups was similar to that in human breast, we guessed the carcinogenesis of rat mammary gland carcinoma was resemble to that of human breast, including the process from usual ductal hyperplasia, atypical hyperplaisa, ductal carcinoma in situ to invasive carcinoma.2 As the most common myoepithelial cell markers, P63, CK5/6, CK34 β E12 played an important role in pathologic diagnosis clinically, and they have the same application in identifying the mammary gland lesions in rat as they did in human.3 It could be concluded that the morphological changes in rat mammary gland lesions was alike basically to that in human. It could provide the histopathological basis for study on the breast carcinoma model and be as an ideal model for breast carcinoma prevention and therapeutic methods.Part ThreeThe research on expression of E-Cadherin, PCNA, Bcl-2 during carcinogenesis in rat mammary gland and human breast and its correlationObjective To observe the expression of E-Cadherin, PCNA, Bcl-2 in the usual ductal hyperplasia, atypical ductal hyperplasia, dutal carcinoma in situ, invasive ductal carcinoma and normal breast tissues of rat mammary gland and human breast, and to investigate the differential expression and correlation of them.Method Ten paraffin-embeded samples of rat mammary gland lesions from part one experiment and ten in human breast from the Department of Pathology, Affiliated Hospital of Shandong Academy of Medical Sciences were collected, the standard for selecting the breast samples was same to the part two experiment, the immunohistochemial staining PV-9000 two step method was used to detect the expression of E-Cadherin, PCNA, Bcl-2. and to detect the proliferation index(PI) by staining PCNA and the anti-apoptosis index(AI) by staining Bcl-2, to observe the staining results and record the positive cell. And to analyse by the SPSS17.0 software: (1) the differential expression of E-Cadherin, PCNA, Bcl-2 between each breast lesions in rat and human respectively; (2) the correlation between the expression of PCNA and E-Cadherin; (3) the correlation between PI and AI.Results1 In rat mammary gland, the expression of E-Cadherin in normal duct, usual dutal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive carcinoma was decreased gradually, there was significantly difference between them(P<0.0.1); the same trend was observed in human breast lesions(P<0.0.1).2 In rat mammary gland, the expression of PCNA in normal ducta, usual ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive carcinom was increased gradually (P<0.01); and its expression between in ND, UDH, ADH and DCIS had significantly difference by multiple comparision (P<0.01); its expression trend of human breast lesion in each group was resemble to that of rat mammary gland(P<0.01).3 The expression trend of Bcl-2 was increasing with the changes of rat mammary gland lesions which from ND, UDH, ADH, DCIS to IDC(P<0.01), but there was no statistically difference in human breast lesions (P>0.0.5).4 In rat mammary gland and human breast lesions, the intensity score of PCNA had negative correlation with the expression of E-Cadherin (P<0.05), but PI had positive correlation with the expression of Bcl-2 (P<0.05).Conclusions1 With the develoment of breast lesions which from usual hyperplasia, atypical hyperplasia, carcinoma in situ to invasive carcinoma, the expression of E-Cadherin decreased gradually, and the intercellular adhesion also decreased, it could be concluded that the loss expression of E-Cadherin have relationship with breast carcinogenesis.2 The expression of PCNA and Bcl-2 increased by the alterations of breast lesions, the cell proliferation index and anti-apoptosis index also increased. Moreover, PI had positive correlation with expression of Bcl-2, we guessed that the interaction between cell proliferation and cell apoptosis could spur the breast carcinogenesis.3 The intensity score of PCNA had negative correlation with the expression of E-Cadherin, we conjectured the cell proliferation could have relationship with cell adhesion, it might play a role in breast carcinogenesis.4 The expressions of E-Cadherin, PCNA and Bcl-2 in rat mammary gland lesons were highly consistent with that of them in human, we speculated the carcinogenesis in rat mammary gland was similar to that in human breast.