Research On Isolation, Culture, Identification Of The Adult Mammary Stem Cells And Instability Of Genomic

The breast cancer is a common kind of malignant tumor which endangers the life and health of the female. In our country, it occupies about 7%-10% in the malignant tumor all over the body. The morbidity ranks the first of the female tumor in our country. And now it is increasing by 2% every year. The feature of the breast cancer invasion makes more youthful increasingly. it is changing from the surgery to combined therapy along with we know more about the biology feature of the breast cancer. Nevertheless the surgery is the principal therapy of the breast cancer. Although lumpectomy and auxiliary 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 abdominal 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 auto specific 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.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. 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 negative and ESA expresses negative. And ASMA immunohistochemistry staining of myoepithelial cell is positive. The MUC-/ESA+ luminal epithelial cell can 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. 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 lobule unit which could not verificate by tissue staining.The analysis of the chromosome karyotype through the GIMESA staining indicate that this cell is the same as the number of chromosome as the normal lymphocyte of the peripheral blood. The number of the chromosome is constancy and has no defect or loss of short arm. We found it forms a big and circle appearance. The nucleus looks like a ellipse at one side of the cell, and there is some lipid droplet in the cytoplasm. The cell has redundant mitochondria and rough endoplasmic reticulum while the Golgi body around the nucleus. The mammary epithelial cell separated by MACS possessed the character of SHMEC and couldn't have malignancy transformation.The microsatellite instability can induce the changing length of DNA according to the replication errors. It seems that the function defect of the errors repair alleles cause one defect protein. So the alleles can not repair the replication mistakes normally, and changing the mirosatellite DNA, this makes the abnormally proliferation and differentiation and leads to the carcinoma. MSI is a novel molecular marker of carcinogenesis, reflecting replication errors induced by the defective function of the mismatch repair genes .it indicates the appearance of a novel, noninherited microsatellite allele in tumor cells.Microsatellites ,also called short tandem repeats (STRS), They contain only 1-6 base pairs and they has more polymorphism, wide-spread throughout the whole genome of all vertebrates. Human genome is estimated to contain almost 100000 microsatellite loci. This tandem repeats local as promoter,coding region and intervening sequence of chromosome. So it has important effect on the rearrangement and variation, the expression of gene. Even in maintaining the genomic stability. MS can bind with the protein of specificity and encode for protein directly. Some of them have possible effect on the folding of chromatid and forming of telomere, MS processes this effect through the alteration of the DNA structures or combining with the specific protein, and so it has become a novel, more polymorphism marker. It makes great contribution in DNA detection technology.How to choose the MS as the DNA marker is a primary problem, The marker should be polymorphism. The MS is elected on the region which has DNA regiment lost by karyotype detection in reference. So it is possible to check out the oncogene or anti-oncogene if we choose the MS in these regions. We elected some oncogene and anti-oncogene which have been confirmed. And we get the LOH in these loci of gene though the MS marker detection which prove the conclusion of reference. nevertheless, it is necessary to compare with the tumor cells and normal tissue whatever in MSI or LOH detection. So the 7 MS markers for 15 breast cancer patients detection for SHEMC and tumors has been made by a cross-check analysis, and the peripheral blood cells are also as the control group. The analysis shows that the SHEMC has the same DNA sequences as the peripheral blood cells while it has distinction from the tumor cells. The cells separated by MACS is safety as normal tissue cells and has no tendency to carcinoma in any environment. It seems that the tendency to carcinoma when the LOH exists on the oncogene in one allege, if the defection of chromosome or gene mutation in another allege. So it is significance for new oncogene by detecting the MS LOH beside the oncogene instead. We elect 3 markers for 15 patients on 7q21-22 as the reference to detect the LOH. And the result compare with the LOH of confirmed oncogene and anti-oncogene from the same tumor cells. By this experiment the three MS markers local as 7q21-22 has the same LOH as the confirmed oncogene and anti-oncogene through statistics. It seems that one or more oncogene expression and anti-oncogene inactivation exist in this chromosome region.