| AIM: Mammary stem cells can be isolated successfully from the normal mammary gland, tissue beside the benign or malignant breast tumor and mammary epithelium separated from latex. This experiment chose the normal mammary para-cancer tissue, which was cultured in vitro. And human mammary epithelial cells that had characteristic of stem cells were isolated by magnetic-activated cell sorting (MACS). To detect c-erbB-2 and maspin gene expression in breast stem cells, breast cancer cell line MCF-7 cells, breast cancer and normal mammary para-cancer tissue by immunoblotting.Then we were going to validate that the adult mammary tissue contains seed cells that can be used in the tissue engineering.METHODS:①The origin of tissues and cells: The tissue samples were from breast cancer and normal mammary para-cancer tissue (>3 cm) identified as the normal mammary gland by pathological methods in the resected specimens of thirty female patients with breast cancer in the Department of Mammary Surgery of the First Hospital of Jilin University from March in 2007 to March in 2008. Informed consent was obtained from each patient; Breast cancer cell line MCF-7 cells .②The fat and capillary vessels around the mammary gland were removed and cut into pieces about 0.3 cm3 by scissors. After centrifugation, supernatant removal and digestion by I collagenase, the DMEM/F12 basic culture medium that contained 10% fetal bovine serum was added for perimary culture. Two weeks later, the cells coated 80% of the culture flask bottom at least. Then trypsinase and D-HANKS were used to digest and separate the cells that adhered to the culture plate. We passaged the cells according to the ratio of 1:2 or 1:3. The adult mammary stem cells were isolated by MACS.③The growth characteristics of the mammary cells after primary culture and passage were observed. The shape and growth characteristics of the mammary stem cells after isolation by MACS were also observed. Immunohistochemistry was used to identify the mammary stem cells after isolation and transformation.④Detection of c-erbB-2 and maspin gene expression in breast stem cells, breast cancer cell line MCF-7 cells ,breast cancer and normal mammary para-cancer tissue by immunoblotting.⑤Detection of c-erbB-2 and maspin gene expression in breast cancer and normal mammary para-cancer tissue by immunohistochemistry.RESULTS:①Growth characteristic of the mammary cells cultured in vitro: The cells adhered to the culture plate 72 hours after isolation, and showed fusiform and polygon on the day 5. Ten days later, the cells productively grew with intensive arrangement, and as time passed, a large percentage of the cells grew into the cells like shuttle gradually. The cells proliferated fast and grew better with well-ordered and well-proportioned arrangement after passage.②Shape and growth characteristics of the mammary stem cells: Human adult mammary stem cells, which were elliptic, grew very well after isolation. Cultured for three days, the cells showed polygon.③Identification by immunohistochemistry: The mammary stem cells isolated by MACS all expressed the epithelial specific antigen, not the Mucoprotein-1; The myoepithelial cells after transformation from mammary stem cells expressed the ASMA.④Identification by immunoblotting show that mammary stem cells and normal mammary para-cancer tissue do not express c-erbB-2 protein and express maspin protein highly. The rate of expression is 100%. The breast cancer cell line and the corresponding isogenous breast cancer tissue express both c-erbB-2 protein and maspin protein . Nine of thirty breast cancer tissues express c-erbB-2 protein. The rate of expression is 30%. Fourteen of thirty breast cancer tissues express maspin protein. The rate of expression is 46.7%.⑤Identification by immunohistochemistry contrastively show that there were significant differences of c-erbB-2 positive expression rates between normal mammary gland tissue and the corresponding isogenous breast cancer tissue(P < 0.005); there were significant differences of maspin positive expression rates between normal mammary gland tissue and the corresponding isogenous breast cancer tissue(P<0.005).CONCLUSION:①The experiment isolated the human mammary cells, which are epithelial specific antigen+, Mucoprotein-1- and have the characteristics of stem cells, from the human mammary para-cancer tissue by MACS successfully. One economical and handy method for isolating mammary stem cells was found in laboratory.②Breast stem cells and normal mammary para-cancer tissue do not express c-erbB-2 protein. The corresponding isogenous breast cancer tissue and breast cancer cell line MCF-7 cells express both c-erbB-2 protein and maspin protein.③It was verified that the adult human tissue has the'seed'cells can be used in tissue engineering. Such this cell can be used to research the molecule mechanism of breast cancer and breast reconstruction.
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