论文标题:小鼠类乳腺干细胞的分离、培养和鉴定
Isolation, Culture and Evaluation of Mammary Epithelial Stem Cells in Mice
论文作者 方昌阁
论文导师 李宁,论文学位 博士,论文专业 生理学
论文单位 中国农业大学,点击次数 79,论文页数 89页File Size5409k
2004-06-01论文网 http://www.lw23.com/lunwen_373019447/ 小鼠乳腺;类乳腺干细胞;Scal-1;Cytokeratin;分化;培养;流式细胞仪
Mouse mammary gland;Stem cells;Scal-1;Cytokeratin;Differentiation;Culture;Flow cytometry analysis sorting
哺乳动物的乳腺是唯一在动物出生后可以多次再生、退化的器官。不同发育时期的动物乳腺中都存在一定比例的干细胞，正是这些干细胞维持和保证了乳腺发育的特性。利用类乳腺干细胞再生乳腺是近两年来兴起的一项高新技术，具有以下四个方面的重要意义：为那些因乳腺肿瘤或癌症而切除乳腺的妇女修复或再生功能性乳腺；为那些需要隆胸的女性提供安全可行的填充新材料；为将健康成年雌性家畜乳腺改造成为分泌药用或保健用蛋白的乳腺提供一个实验动物的技术平台：为其他器官的发育、再生或修复提供方法、思路利理论借鉴。 我们以小鼠为模型，运用组织化学、免疫荧光组织(细胞)化学、流式细胞仪分选方法(FACS)以及分子生物学手段，研究了小鼠乳腺的发育规律：小鼠乳腺组织中类乳腺干细胞：小鼠乳腺细胞的分离、培养以及类乳腺干细胞的鉴定；小鼠类乳腺干细胞分化的潜能；小鼠乳腺类腺体体外短期培养富集类乳腺干细胞体系的优化等。研究结果表明： 1．3周龄的balb／c小鼠是去除乳腺腺体的最佳时期； 2．不同发育时期乳腺组织中都存在类乳腺干细胞； 3．6～8周龄和怀孕期小鼠是获得大量类乳腺干细胞的理想时期； 4．首次证明体外短期培养的乳腺类腺体体系中，有一些细胞可以表达一定量的胚胎干细胞的特异性表达抗原基因Oct-4、Rex-1；上皮干细胞的特异性表达抗原基因integrin β-1；大部分已知干细胞共表达的抗原基因bcrpl／acbg21； 5．小鼠类乳腺干细胞移植到去除乳腺腺体的小鼠乳脂垫中，可以再生功能性乳腺，因此，类乳腺干细胞在体内具有分化潜能； 6．小鼠类乳腺干细胞在体外诱导泌乳体系中培养7天，可以表达β-酪蛋白及其基因，表明类乳腺干细胞在体外具有分化潜能； 7．首次建立了优化的乳腺类腺体体外短期培养富集类乳腺干细胞体系，由此体系培养的类腺体获得类乳腺干细胞(Scal-1~+)的比例远远高于文献报道使用体系获得类乳腺干细胞比例； 8．小鼠乳腺类腺体结构体外短期培养获得的细胞中有一定量的类乳腺干细胞存在：优化体系培养22小时获得的细胞的类乳腺干细胞(Scal-1~+)比例远远高于培养72小时获得细胞的比例；相反，表达造血干细胞特异性抗原CD34的细胞(CD34~+)比例在培养72小时比培养22小时高；同时，培养的体系中还有一定比例的Scal-1~+／CD34~+细胞存在。 总之，随着对类乳腺干细胞研究的深入，我们急需找到类乳腺干细胞的特异性表达抗原。在此之前，仍然只能以造血干细胞的特异性表达抗原Scal-1作为一个标记，进行类乳腺干细胞的分析和分选研究。进一步研究类乳腺干细胞的特异性表达谱，以期能够找到类乳腺干细胞的特异性表达抗原，分离得到类乳腺干细胞，作为发育生物学和再生生物学研究的模型。另外，如果能对乳腺类腺体体外短期培养富集类乳腺干细胞体系进行优化，或许可以摸索适合类乳腺干细胞体外扩增的培养体系。
Mammary gland is a unique organ, which can postnatally regenerate, terminally differentiate, and involute upon successive cycle of pregnancy, lactation, and involution, respectively, and provides an optimal model for the study of organogenesis, differentiation, apoptosis, and pattern formation involved in development. This impressive renewal capacity is due to the function of a multipotent mammary gland stem cell population. The research of mammary epithelial stem cells has many significant meaning such as rebuilding woman breasts after surgical sectioning because of the breast tumors or cancers, enlarging lady breast for more beautiful, regenerating transgenic mammary glands secreting medical or tonic proteins for theraping genetic deficient diseases or for healthier in human, and establishing models for organogenesising, cell differentiation and regeneration, and so on.Thus, in order to investigate the developmental pathways not only involved in the regulation of growth and patterning, but also in the determination of cell lineages and differentiation, we utilized the fluorescent immunohistochemical methods, flow cytometry analysis sorting (FACS) and molecular methods to investigate the developmental law of mammary gland at the different developmental stages , distribution of the stem cells in mammary gland, the methods of isolation, culture and evaluation for the stem cells, the multipotent abilities in vivo and in vitro, and the efficient cultural system for stem cells enriched in vitro.The results showed below:1. The optimal stage of clearing the mammary gland from fat pad is the 3-week-old in balb/c mice.2. There are stem cells in the mammary glands at any stage of postnatal development.3. The best stages of gaining the stem cells from the mammary glands are when the mice are 6 to 8 weeks old or in pregnant stage.4. The mammary epithelial stem cells, sorted with anti-Scal-1 antibody by the FACS, have multipotent abilities because they could be induced to mimic the function of mammary gland to produce the milk component B-casein in vitro and could regenerate the functional mammary glands as injecting into the cleared fat pads.5. Firstly in mammary epithelial stem cells, we investigated that there were some cells to express the specific genes, defined in embryonic stem cells or other known stem cell lineages, such as Oct-4, Rex-1, IntegrinB1, bcrp1/acbg 2 and CD 34.6. Estabished an optimal cultural system for enriched mammary epithelial stem cells, firstly. After being cultured in optimal cultural system in vitro for 72 hours, we could sort 30 % of mammary stem cells (Scal-1+) in the cells derived from the mammary orgnoids and the percentages of stem cells was higher than that of the reference, but less that of the cells cultured in vitro for 22 hours.7. We sorted several percentages of CD34+ cells derived from the cultured mammary gland orgnoids and the percentages of CD34+ cells in 22-hour cultural system was lower than that of the 72-hour cultural system. These results need to be investigated further.In summary, we should investigate the more efficient cultural system for mammary orgnoids in vitro for enriched more mammary epithelial stem cells, and to screen the expression profiles in mammary epithelial stem cells to discover the specific marker for mammary epithelial stem cells in order to utilize the model of mammary epithelial stem cells to regenerate transgenic mammary glands in cow or sheep for human health, to uncover the mechanism of organogenesis, and differentiation.
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