Experimental Study Of EGF's Effects On Mammary Stem Cells In Vitro And Transplantation Of MSC In Vivo

1,Objective and significance:mammary stem cells are the quiescent and undifferentiated cells,which at presence in the breast tissue , and have the ability to maintain long-term self-renewal. It can differentiate to various types of cells in breast tissue with specific morphological features and unique functions. Breast stem cells are characterized by asymmetric splitted : offspring maintain stem cells or cells begin to differentiate. Stem cells can also symmetrical split to two progeny cells which were stem cells, it suggest the existence of a mechanism to regulate the balance between self-renewal and differentiation. Breast ductal epithelial stem cells can differentiate into muscle cells and epithelial cells. And the specificity of the cell-specific patterns in performance and functional characteristics are the result of cell differentiation. The study propose to find features of the signs in mammary stem cell's differentiation, metabolism , and separation purification methods of mammary stem cell , and the technical means of cultured system to control the proliferation and differentiation.Nevertheless the surgery is the principal therapy of the breast cancer. Although lumpectomy and axillary dissection has been the first therapy in the Europe and America; it is developing in minority hospital in our country. Breast defect after the radical mastectomy brings about the trauma and pain in the patient's spirit. The breast reconstructive operation after operation has been considered as the part of the treatment for the breast cancer. Now various methods can reconstruct the breast; including installing the artificial implant; transplanting the rectus abdominis muscular flap and the latissimus dorsi muscular flap; but they are imperfect. The ideal breast reconstruction should been no concomitant impair; safety; imitating the breast tissue drooping; and format the soft; temp; feeling and plastic breast. If we can use autospecific breast tissue to reconstruct breast; it is perfect. Recently; the studies about the mice mammary gland stem cells has confirmed that they can grow the integrity and functional breast; and it includes normal mammary gland cells and can lactate. The success of animal studies makes the research for mammary gland stem cells of mankind easier. The mammary gland grown from stem cells can be used in reconstructive operation of mammary gland postmastectomy; and its prospect of application is spacious.The research on the mammary stem cells began in 1980s; the scientific research team leaded by Denmark scientists Peterson and Gudjonsson declared that they had separated and cultured the mammary stem cells from tissues of reduction mammoplasties by the immunomagnetic beads sorting and proved that the stem cell are able to divide by themselves and convert to the other types in the mammary glands cells and give rise to terminal ductal lobulous unit.2,Material and Methods :In the Second part of the experiment. We select nude mice to transplant mammary stem cells in vivo as a breast stem cells transplantation's model. Nude mice are lack of thymus,as T cell-deficient mice, the tumors's transplantation in vivo has been widely used in research. MCF-7 human breast cancer cell lines and as a control group. From the experiments in vivo we can observe that the significant difference in the formation of stem cell transplantation and breast tumors cells. From the histological sections we can find that adult stem cells are similar to breast mammary duct-like structures, ductal epithelial cells in the basal and myoepithelial cells may be seen as the spindle cell. Separated by the surrounding connective tissue the fat cells can be seen , but not see the typical acinar structures. We need to consider the role of estrogen and progesterone, breast ductal structure resulting from the possible structure of the acini. tumor mitotic phase and disappearing were arranged in nests. Central common cancer nests of hemorrhage and necrosis. These results suggest that adult stem cells have mammary hyperplasia and differentiation in vivo activity continues,it provide test materials to further study the development and the formation of breast formation and breast cancer .At present paper we separated the human mammary gland cells from the normal tissue surrounding the breast cancer; and do primary culture in vitro; and we do the immunohistochemistry on the normal mammary gland. The mammary gland epithelial cell in the stationary phase comprises two distinct lineages: the luminal epithelial cell and the myoepithelial cell. The results of immunohistochemistry staining show that the result of ASMA immunohistochemistry staining of myoepithelial is positive; and the ESA immunohistochemistry staining of luminal epithelial is also positive. According to the MUC,ESA and ASMA polyclonal antibody; by magnetic cell separation system(MACS); it can combine the surface-specific antigen of the mammary gland cell directly. They combine the beads also; and passing through the magnet; we can separate the mammary gland cells. By immunohistochemistry staining; the mammary gland luminal epithelial cell comprises two lineages: MUC expresses negative and ESA expresses positive; MUC expresses positive and ESA expresses negative. And ASMA immunohistochemistry staining of myoepithelial cell is positive. The MUC-/ESA+ luminal epithelial cell could convert to myoepithelial cell; which is tested by immunohistochemistry staining. These studies confirmed that the MUC-/AMSA-/ESA+ luminal epithelial cell had capability of differentiated; and had the characteristic of the adult stem cells. In order to obtain the immortalization ;we transfected with a plasmid carrying an origin-defective Simian virus 40 genome gene fragment to the mammary epithelial cell; the separated MUC-/ESA+ luminal epithelial cell by magnetic cell separation system had the characteristic of the adult stem cells. And these cells cultured with LIF for 3-5 days; the observation showed that they could not convert to the myoepithelial cell. Matrigel is a dissolubility basement membrane which extracted from the sarcoma of the EHS mice. Its ingredients comprised laminin ; collagen IV;heparin sulfate glycoprotein and some growth factors. By feeding the separated MUC-/ESA+ luminal epithelial cell on the system of three dimension made up of laminin ;we could discover that the cell lineages form the terminal ductal lobulo unit which could not verificate by tissue staining.Our studies confirmed that the MUC-/AMSA-/ESA+ luminal epithelial cell separated from the normal tissue surrounding the breast cancer could convert to myoepithelial cells; and formed the terminal ductal lobulous unit culturing on the system of three dimension made up of laminin and we transfected with a plasmid carrying an origin-defective Simian virus 40 genome gene fragment to the mammary epithelial cell; they obtain the immortalization; the separated MUC-/ESA+ luminal epithelial cell by magnetic cell separation system had the characteristic of the adult stem cells. And these cells cultured with LIF for 3-5 days ;the observation showed that they could not convert to the myoepithelial cell. The drugs intervention studies on the mammary gland stem cells showed that the fluorouracil and paclitaxel had depressant effect on the mammary gland stem cells; panaxoside Rg3 and milkvetch root had promotion on them. Our studies established the cytology foundation for the mammary reconstruction of mastectomy by using mammary gland stem cells.3,Result1.MACS technology can seperated mammary stem cells in vitro and cultured to proliferation.2.EGF can be used as a supportive factor for breast stem cell proliferation. It's role has realationship with its concentration.3.BRCA-1 gene can control the proliferation of adult mammary stem cells. BRCA-1 gene positive expression has correlation with Adult mammary stem cell proliferation. 4.EGF gene expression can be controlled by BRCA-1 for positive control. BRCA-1 gene and protein expression and the concentration of EGF.