| Mammary epithelial cells play an important role in dairy cows.They are also the only functional cells with the ability to produce milk.At the same time,they can also hinder the invasion of external pathogens to the mammary gland,thereby forming an important mammary immune barrier.Moreover,there is also an important relationship between mammary epithelial cells and the occurrence of breast cancer.When breast tissue develops a tumor,the cancer cells in the breast lose the characteristics of mammary epithelial cells.The connections between cancer cells will become loose,causing the cancer cells to fall off,and the shed cancer cells will enter the circulatory system for metastasis,forming metastases in distant organs such as the lungs.The patient lost his life because the lungs could not be removed.At present,There is a great deal of debate about how breast cancer cells metastasize.Therefore,this paper uses mice as the research object to study the physiological and pathological processes of mammalian mammary glands,so as to trace the proliferation,transformation and migration process of mammary epithelial cells.In this study,mice were used as experimental models to construct mouse Kit CreER,a tool that can specifically target and label mammary epithelial cells by genetic lineage tracing technology,and to study the proliferation,regeneration and tumor metastasis of mouse mammary epithelial cells.Using Kit CreER;R26-Ai47 mice to specifically label mammary luminal epithelial cells,combined with MMTV-PyMT in spontaneous mammary tumor model mice,to observe the metastasis of mammary tumor cells to the lungs in young and old mice.The main results of this study are as follows:1.Kit CreER;R26-Ai47 specifically marked mammary luminal epithelial cellsBased on the inducible CreER-loxP recombination system in genetic lineage tracing technology,we combined the inducible Cre tool mouse Kit CreER driven by a specific promoter Kit with the reporter gene mouse R26-Ai47 of Rosa26-LSL-GFP.The inducible Cre tool mouse Kit CreER driven by a specific promoter is used in combination with the reporter gene mouse R26-Ai47 to and further control the active expression of Cre recombinase in a specific time by regulating the in vivo time of Tamoxifen.We performed Tam induction in Kit CreER;R26-Ai47 double genotype adult female mice and analyzed mammary gland tissue.The results show that Kit CreER can efficiently and specifically label mammary luminal epithelial cells,which provides an experimental basis for further research.2.Establishment of Kit CreER-labeled Breast Tumor Epithelial Cell Tracking in the Early Stage of TumorKit CreER;R26-Ai47 double-gene mice were mated with mammary tumor model mice MMTV-PyMT to obtain triple-genotype female mice.Early labeling and tracing of mammary luminal epithelial cells during breast tumor development and metastasis in cell motility and fate transition.Three-gene female mice were 8 weeks old and induced by Tam for three consecutive times,and the mouse mammary gland and lung tissue samples were collected on the 4th day.The results showed that there were clonal in situ mammary gland tumors labeled with green fluorescence signal in situ,but no tumor-like morphology appeared in the lungs.Immunofluorescence staining showed that most of the mammary glands had abnormal non-tubular morphological mammary in situ tumors.But no GFP-positive tumor metastases were found in lung tissue.This indicated that early breast epithelial cell proliferation and transformation contributed to the formation of breast tumor cells,and no obvious lung metastasis was formed.3.Establishment of Kit CreER-labeled Breast Tumor Epithelial Cell Tracking in Advanced TumorKit CreER;R26-Ai47;MMTV-PyMT tri-genotype female mice were induced by Tam for three times by gavage at the age of 8 weeks,and the mammary gland and lung tissues were collected at the age of 19 weeks.To verify whether advanced breast tumors can migrate to form lung metastases.The results showed that advanced breast tumors filled the entire breast and formed clonal GFP-positive lung metastases.It shows that advanced breast tumor epithelial cells can migrate and form lung metastasis.In summary,this study provides an in vivo tracking tool for the proliferation,transformation and migration of dairy cow mammary epithelial cells.This method of tracking the biological characteristics of cells in vivo can be used for the proliferation,migration,transformation,pathological changes,etc.of mammalian mammary glands,which has a good identification effect,and has important application value for solving the occurrence and development of related diseases. |
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