| The stem cells have the ability of self-renewal and differentiate potentiality. At present the research of human stem cells already became a new commanding point in life science research. In 1999 the development of stem cell investigations was evaluated as the head of top ten world scientific advance by "Science" magazine. The stem cell bio-engineering technology can treat disease. The most remarkable characteristic is it can reconstruct many kinds of normal and younger organs. Using the stem cell treats disease has many merits compared with the conventional routes, such as safe, does not need to completely understand the accurate pathogenesy,can also transplant with own stem cell to avoid immunological rejection. In March, 2002 Peterson in Denmark announced that they had isolating cultured human breast stem cell by immunomagnetic beads from patients undergoing reduction mammoplasty. Proved that has the ability of differentiate each kind of mammary epithelial cell and forms the mammary gland structure. In January, 2006 Mark Shackleton proclaimed that they cultivated a functional mammary gland throμgh a single breast stem cell. These two researches provided the theory and the practice foundation to realize the mammary gland regeneration by stem cell. This experiment base on the two researches; take mammary gland regeneration for the patients with breast cancer as the final goal. Approach the isolating culture technique for breast cancer patients'mammary gland stem cell in vitro. Analyze the influence for stem cell grows in vitro with different density of Radix Astragali, Salvia Miltiorrhiza, Pirarubicin, Tegafur and Paclitaxel. Examine the disparity of 22000 gene levels between mammary gland stem cell and breast cancer cell in the homologous cancer tissue. Further determine whether the mammary gland stem cell genome doesn't have the multi-gene variation.The first part, 34 example, 29-45 year-old breast cancer patients, undergo the modified radical mastectomy, primary culture the breast cancer tissue and the normal tissue which far away from the tumor and confirmed by pathology. Passage when the cells overspread entire surface of culture bottle in 10th to 14th days. Collect breast cancer cell in 7th to 10th days after one generation, meanwhile separate the normal mammary gland cell with the immunomagnetic beads. After the cells which stained by Muc-1, ESA and goat-anti-mouse IgG pass the magneto-insulator, collect Muc-,ESA+cells, namely mammary gland stem cell. The second part, analyze the influence of the different density of Radix Astragali, Salvia Miltiorrhiza ,Radix Astragali and Salvia Miltiorrhiza compound parenteral solution to the mammary gland stem cell in vitro by the means of MTT and flow cytometry. Get the result that the Salvia Miltiorrhiza and the small dosage Radix Astragali may promote the mammary gland stem cell multiplication(P<0.01). The compound parenteral solution has more obvious promotion to the mammary gland stem cell multiplication in vitro(P<0.01). The third part, analyze the influence of the different density of Pirarubicin, Tegafur and Paclitaxel to the mammary gland stem cell in vitro by the means of MTT and flow cytometry. Get the result that Tegafur and small dosage Pirarubicin have no influence to the mammary gland stem cell. Dosage Pirarubicin has the inhibitory action to the mammary gland stem cell growth (P<0.05). Large dose Pirarubicin and every experimental dosage Paclitaxel have more obvious depressant effect to the mammary gland stem cell growth in vitro(P<0.01). The fourth part, in recent years the gene chip technology is a rapidly developed high-tech in the life sciences area. The important target gene expression level has close correlation with the disease progress, may supply a big help for the disease clinical diagnosis and the prognosis judgment. In the numerous genes, some genes are called oncogene, in fact in the normal cells the oncogene participate in cell growth, desintegration and differentiated. These genes exist in normal cell by non-activated modality, therefore are called proto-oncogene. Proto-oncogene activates into oncogene when receive the effectiveness of many kinds of factors and change the structure. The genes inhibit cancer occurrence namely anti-oncogene, which is as important as oncogene in cancer occurrence, even more important. The gene DNA sequence mutation or the gene absence can cause the absence of restrain cancer occurrence function, may also appear in process of named methylation, during this process one kind of named methyl group combine with the DNA region of neighbor gene and prevent its activation, finally causes the gene"silence". If the oncogene is the difficult controlled cell growth accelerator, then anti- oncogene is the brake which does not hope the cell grows. In the normal condition, anti-oncogene control cell growth by their protein product, or restrain cancer occurrence throμgh combined the enzyme activation and anti-oncogene inactivation. This experiment takes the mammary gland stem cell as the experimental group, the homologous breast cancer cell as the control group, finds out difference gene 798, declines gene 174, upward gene 624. In the difference gene altogether has 16 with breast cancer inverse correlation gene,declines gene 6, upward gene 10.Conclusions:1)In the adult mammary gland includes the seed cells which may use for the organization project, can obtain throμgh the cell primary culture and the immunomagnetic beads separation technique.2)The mammary gland stem cell culture in vitro can unite the traditional Chinese medicine to enhance the ability of proliferation and differentiation.3)Some chemotherapy medicine have no or little influence to the mammary gland stem cell growth, when carrying on the stem cell mammary gland regeneration need prudent choice chemotherapy medicine, reduces the effect.4)Analysis the difference gene screened by the gene chip examination, may prove that the mammary gland stem cell does not have the malignancy changes molecular foundation.Innovation point:1)Unified the traditional Chinese medicine with the modern stem cell technology, provides the experiment evidence for further discusses the influence of Chinese medicine to the stem cell growth, differentiation and development and so on.2)In view of breast stem cell research finally item, after surgery the patients must carry on chemotherapy in the earlier period, this experiment examined the commonly used chemotherapy medicine, with screens have no influence to the growth of stem cell or have small impact. Establish experiment foundation for the stem cell mammary gland regeneration applies in the clinical.3)Use the gene chip technology analyzes genome team expression between the homologous mammary gland stem cell and cancer cell. Confirm the security of the mammary gland stem cell. The excessive activation of oncogenes and relative deactivation of anti-oncogenes is the root of tumorigenesis. It caused the tumor cell dissymmetry fission. Analyze the expression level and correlation between some oncogenes and anti-oncogenes in the stem cell and filial generations, possibly enlighten the studies of stem cell and the tumor stem cell dissymmetry fission and the self-renewal mechanism. |
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