Study On Primary Culture Of Human Mammary Epithelial Cells Of Gland-associated Hypertrophic Breast And Its Proliferation And Apoptosis In Vitro

Part I primary culture in vitro of human mammary epithelial cells of gland-associated hypertrophic breast (hMEC-GAHB)Objective To investigate the proper methods of primary cell culture of human mammary epithelial cells of gland-associated hypertrophic breast (hMEC-GAHB), and find out its morphological and biological characteristics in vitro.Methods①Harvesting the primary mammary epithelial cells from 10 cases of gland-associated hypertrophic breasts and 10 cases of breasts for normal volume, using 0.25% trypsin and type I collagenase, which interact with the mammary tissue under 4℃for a long time and then under 37℃for a while, companying the employment of low-speed centrifugation, and then culture hMEC-GAHB in vitro.②culturing passage cells of human normal mammary epithelial cell line HBL-100 at the same time.③evaluating the result of harvest of primary cells, observing their morphological characteristics.④identifying the primary cell species by multiantibody immunocytochemistry.⑤analyzing their biological characteristics in cell cycle and proliferation and apoptosis in vitro by PI staining method and MTT method and flow cytometry.Results About 5×106 cells could be obtained from 1g mammary tissue, the livability of the cells was about 70%, and the adherence rate after 48h was about 60%. The adherent cells showed good activity, and cytoplast stretched after 72h and started to connect each other after 120h. Obvious proliferation could be observed, without apparent apoptosis. hMEC- GAHB seemed to have no distinct difference between others in cell morphology, and it expressed normally typical epithelial skeleton protein (CK8/ CK14/CK18/a-SMA). Under the environment containing proper 17b-estradiol in vitro, the population doubling rate and the cell number ratio of G2/M and G0/G1 phase of hMEC-GAHB were both higher than those of normal volume breast group with statistical difference in logarithmic growth phase (P<0.05). hMEC-GAHB could be passaged for 3 to 4 times, then it stepped into senescence phase gradually.Conclusion Satisfactory cell number and activity could be obtained by the improved methods of enzyme digestion. hMEC-GAHB possessed typical biological and morphological characteristics of epithelial cells. Compared with hMEC-NVB,it showed relative great proliferative activity in the environment containing estrogen in vitro, and had normal senescence phase.PartⅡThe location and expression of estrogen receptor alpha (ERa) in mammary tissue and epithelial cells of gland-associated hypertrophic breastObjective To investigate the location and expression of estrogen receptor alpha (ERα) in mammary tissue and epithelial cells of gland-associated hypertrophic breast, and analyze their signification in etiology of gland-associated hypertrophic breast.Methods①Primary cells culturing in vitro of hMEC-GAHB and human mammary epithelial cells from normal volume breast.②culturing passage cells of human normal mammary epithelial cell line HBL-100 and epithelial cell line human mammary cancer MCF-7 (ERα+) at the same time.③analyzing qualitatively and quantitatively the histopathological characteristics of mammary tissue and the expression of ERa in tissue and cells by immunohistochemistry (IHC) and immunofluorescence histochemistry (IFHC) and western blot.Results ERa expressed in the luminal cells of ducts and lobules, and ERa+ cells,which expressed in the mammary tissue of gland-asoociated hypertrophic breast, were more in ducts than those in lobules with the constant ratio of 1.7:1. However, it didn't express in myoepithelial cells or mesenchymal cells at all. The positive expression rate (PER) of ERα+ cells in mammary tissue of gland-associated hypertrophic breast and normal volume breast were respectively 17.25% and 6.13%. The protein expression value (PEV) of ERa in hMEC-GAHB/hMEC-NVB /HBL-100/MCF-7 were respectively 1.965/ 1.002/0.060/2.338. PER and ERa PEV in gland-associated hypertrophic breast were both higher than those in normal volume breast and HBL-100 with distinct statistical difference(P<0.05), and they had a positive correlation with the severity of hypertrophic breast.The relative data of ERa in MCF-7 were distinctly different with others((P<0.05).Conclusion The expression of ERa had a characteristic location. Compared with normal volume breast, the PER and PEV of ERa in hMEC-GAHB increased obviously, and have a positive correlation with severity of hypertrophy in gland-associated hypertrophic breast. Therefore, we could infer that the happen of gland-associated macromastia has a very close relationship with the expression and location of ERa in mammary tissue and epithelium.. PartⅢThe influence of tamoxifen in proliferation and apoptosis of human mammary epithelial cells of gland-associated hyper-trophic breast in vitroObjective To investigate the influence of tamoxifen, which is one of selective estrogen receptor modulators, in proliferation and apoptosis of human mammary epithelial cells of gland-associated hypertrophic breast(hMEC-GAHB) in vitro, and analyse the mechanism.Methods①Intervening hMEC-GAHB(Ml)/hMEC-NVB/HBL-100/MCF-7 res-pectively with tamoxifen in the environment containing proper estrogen in vitro, and setting control group hMEC-GAHB M2.②detecting the cell cycle of each group, by PI staining method, and detecting the proliferation and apoptosis of cells by immunofluorescence cytochemistry (IFCC) and MTT method and Annexin V-FITC apoptosis kit.③analyzing qualitatively and quantitatively the change in expression of ERa protein and the amplification of ERa mRNA of hMEC-GAHB M group after intervention, respectively by western blot and real-time RT-PCR skills.Results After intervention, pyknosis and cataclasm of karyon were observed in part of hMEC-GAHB under fluorescence microscope, companying the occurrence of apoptotic body and microkernel. With the process of time, the population doubling rate (PDR) of hMEC-GAHB M1 decreased obviously with increased apoptotic cells, and the apoptosis rate of cells stayed between that of HBL-100 and MCF-7. hMEC-GAHB was arrested in G0/G1 phase of cell cycle, and the cell proportion of this phase enlarged,as well as S phase.But the cell proportion of G2/M phase decreased. The expression of ERa protein decreased obviously, and the amplification multiple of ERa mRNA minished, the gene expression was down as well. The relative data of control group M2, however, showed reversely.Conclusion Tamoxifen could interdict the agonist effect on estrogen in ERα+ cells, induced apoptosis of ERα+ cells and restrained obviously the proliferation of hMEC-GAHB. It could also lead to the decrease on intracellular expression of ERa protein, and regulated down the ERαmRNA gene expression, while it seemed to have no apparent effect on ERα- cells. The influence of induced apoptosis was time-depedent. Therefore, we could infer that tamoxifen could antagonise estrogen competively, prevent estrogen from binding with ERa. The target cells are ERa+ mammary epithelial cells, with the possible mechanism by regulating the ERa expression at gene level.