Research On Isolation, Culture, Identification Of The Adult Mammary Stem Cells And Drugs Intervention Studies

The breast cancer is a common kind of malignant tumor which endangers thelife and health of the female. In our country, it occupies about 7%-10% in themalignant tumor all over the body. The morbility ranks the first of the femaletumor in our country. And now it is increasing by 2% every year. The feature ofthe breast cancer invasion makes more youthful increasingly. In our country, thereis two problems in the studies of the breast cancer: firstly, In the fundamentalstudies of the breast cancer, there is no ready-made tumour generation model, sowe can not observe development and growth of the breast cancer in it's mostprimitive stage. Secondly, in the therapy aspect of the breast cancer stage, it ischanging from the surgery to combined therapy along with we know more aboutthe biology feature of the breast cancer. Nevertheless the surgery is the principaltherapy of the breast cancer. Although lumpectomy and axillary dissection hasbeen the first therapy in the Europe and America, it is developing in minorityhospital in our country. Breast defect after the radical mastectomy brings about thetrauma and pain in the patient's spirit. The breast reconstructive operation afteroperation has been considered as the part of the treatment for the breast cancer.Now various methods can reconstruct the breast, including installing the artificialimplant, transplanting the rectus abdominis muscular flap and the latissimus dorsimuscular flap, but they are imperfect. The ideal breast reconstruction should beenno concomitant impair, safety, imitating the breast tissue drooping, and format thesoft, temp, feeling and plastic breast. If we can use autospecific breast tissue toreconstruct breast, it is perfect. Recently, the studies about the mice mammarygland stem cells has confirmed that they can grow the integrity and functionalbreast, and it includes normal mammary gland cells and can lactate. The successof animal studies makes the research for mammary gland stem cells of mankindeasier. The mammary gland grown from stem cells can be used in reconstructiveoperation of mammary gland postmastectomy, and its prospect of application isspacious.The research on the mammary stem cells began in 1980s, the scientificresearch team leaded by Denmark scientists Peterson and Gudjonsson declaredthat they had separated and cultured the mammary stem cells from tissues ofreduction mammoplasties by the immunomagnetic beads sorting and proved thatthe stem cell are able to divide by themselves and convert to the other types in themammary glands cells and give rise to terminal ductal lobulous unit.At present paper we separated the human mammary gland cells from thenormal tissue surrounding the breast cancer, and do primary culture in vitro, andwe do the immunohistochemistry on the normal mammary gland. The mammarygland epithelial cell in the stationary phase comprises two distinct lineages: theluminal epithelial cell and the myoepithelial cell. The results ofimmunohistochemistry staining show that the result of ASMAimmunohistochemistry staining of myoepithelial is positive, and the ESAimmunohistochemistry staining of luminal epithelial is also positive. According tothe MUC,ESA and ASMA polyclonal antibody, by magnetic cell separationsystem (MACS), it can combine the surface-specific antigen of the mammarygland cell directly. They combine the beads also, and passing through the magnet,we can separate the mammary gland cells. By immunohistochemistry staining, themammary gland luminal epithelial cell comprises two lineages: MUC expressesnegative and ESA expresses positive;MUC expresses positive and ESA expressesnegative. And ASMA immunohistochemistry staining of myoepithelial cell ispositive. The MUC-/ESA+ luminal epithelial cell could convert to myoepithelialcell, which is tested by immunohistochemistry staining. These studies confirmedthat the MUC -/AMSA -/ESA+ luminal epithelial cell had capability ofdifferentiated, and had the characteristic of the adult stem cells. In order to obtainthe immortalization, we transfected with a plasmid carrying an origin-defectiveSimian virus 40 genome gene fragment to the mammary epithelial cell, theseparated MUC-/ESA+ luminal epithelial cell by magnetic cell separation systemhad the characteristic of the adult stem cells. And these cells cultured with LIF for3-5 days, the observation showed that they could not convert to the myoepithelialcell. Matrigel is a dissolubility basement membrane which extracted from thesarcoma of the EHS mice. Its ingredients comprised laminin, collagen IV, heparinsulfate 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 whichcould not verificate by tissue staining.Adding different concentration of panaxosideRg3, milkvetch root, paclitaxeland fluorouracil to the separated MUC-/ESA+ luminal epithelial cell, by MTTassay we drawed the cell growth curve. We observed that the fluorouracil (20.0and 100.0ug/ml) and paclitaxel (10.0ug/ml) had depressant effect on themammary gland stem cells;panaxoside Rg3 (5 and 10ug/ml) cultured for 48 to 72hours had promotion for them and the highest concentration (400mg/ml) ofmilkvetch root cultured for 72 hours had promotion, too.Our studies confirmed that the MUC-/AMSA-/ESA+ luminal epithelial cellseparated from the normal tissue surrounding the breast cancer could convert tomyoepithelial cells, and formed the terminal ductal lobulous unit culturing on thesystem of three dimension made up of laminin and we transfected with a plasmidcarrying an origin-defective Simian virus 40 genome gene fragment to themammary epithelial cell, they obtain the immortalization, the separated MUC-/ESA+ luminal epithelial cell by magnetic cell separation system had thecharacteristic of the adult stem cells. And these cells cultured with LIF for 3-5days, the observation showed that they could not convert to the myoepithelial cell.The drugs intervention studies on the mammary gland stem cells showed that thefluorouracil and paclitaxel had depressant effect on the mammary gland stem cells;panaxoside Rg3 and milkvetch root had promotion on them. Our studiesestablished the cytology foundation for the mammary reconstruction ofmastectomy by using mammary gland stem cells.