乳腺干细胞培养基的建立及有效性验证

Creation and effectiveness of mammary stem cell medium

背景:干细胞培养基,尤其是乳腺干细胞培养基现阶段尚无有效的制备方法。目的:利用Sca-1+乳腺细胞验证自制乳腺干细胞培养基的有效性。方法:用BM培养基培养乳腺器官样小体,6 d后以免疫荧光方法监测成纤维细胞Sca-1和vimentin的表达。筛选出MaECM培养基,培养乳腺细胞6 d后,免疫磁珠筛选Sca-1+和Sca-1-细胞群,流式细胞术分析Sca-1阳性细胞的纯度,1×104 Sca-1+或Sca-1-细胞种植在4只鼠双侧乳腺脂肪垫上,6-8周后取出乳腺脂肪垫,以卡红整体染色和苏木精-伊红染色分析长出乳腺的结构数量。结果与结论:乳腺器官样小体用BM培养基培养6 d后,检测到大量Sca-1和vimentin阳性的成纤维细胞,说明BM培养基不适合分离Sca-1+乳腺干细胞。筛选出的MaECM培养基能够抑制成纤维的生长。磁珠筛选后,流式细胞术检测Sca-1+细胞在Sca-1+和Sca-1-细胞群的纯度分别是92%和5%。移植实验显示在8个种植Sca-1+细胞的脂肪垫生长出6个乳腺结构;而在8个种植Sca-1-细胞的脂肪垫中除1只鼠死亡,其余脂肪垫中均未长出乳腺结构。提示MaECM培养基适用于培养鼠乳腺干细胞。

BACKGROUND：Now in mammary stem cellresearch, no proper mammary stem cellmedium is provided to culture mammary stem cells. OBJECTIVE：To create a mammary stem cellmedium and validate its application by isolating Sca-1＋mammary stem cells. METHODS：We first used BM medium to culture mammary organoids, and after 6 days, the expression of Sca-1 and vimentin was detected in fibroblasts by immunofluoresence method. Then, we established MaECM medium which arrested fibroblasts growth. After 6 days culture of mammary organoids by MaECM medium, Sca-1＋and＆amp;nbsp;Sca-1-cellpopulations were sorted out by magnetic sorting and the purity was analyzed by flow cytometry. Sorted 1×10^4 Sca-1＋or Sca-1-cells were transplanted into the bilateral mammary fat pads of four mice, and after 6-8 weeks, the fat pads were harvested for whole-mount immunohistochemical analysis and hematoxylin-eosin staining. RESULTS AND CONCLUSION：After 6 days culture of mammary organoids under BM medium, smal-sized colonies were generated around lots of fibroblasts. Immunofluoresence staining detected strong expression of vimentin and Sca-1 in fibroblasts, indicating that the BM medium is not suitable to isolate Sca-1＋mammary stem cell. The MaECM medium promoted the proliferation of mammary epithelial cells whereas arrested fibroblasts growth. After 6 days culture of mammary organoids under MaECM medium and magnetic sorting, the flow cytometry showed that the purity of Sca-1＋cellreached 92%and 5%in the Sca-1＋and Sca-1-population, respectively. The results from transplantation test showed that six mammary outgrowths were regenerated out of eight injected fat pads in the Sca-1＋cells transplantation, but in the Sca-1-transplantation population, one mouse died and the other transplants failed to produce outgrowths. We developed the MaECM medium which promoted the proliferation of mammary epithelial cells whereas arrested Sca-1＋fibroblasts growth. Using the medium, we confirmed that Sca-1＋mammary cells have capacity of isolating mammary stem cells.