无血清培养富集乳腺癌干细胞的初步研究

目的：探讨富集乳腺癌干细胞的方法，以获得稳定数量的肿瘤干细胞。方法：（1）采用无血清悬浮培养方法，从乳腺癌MDA-MB-435细胞中筛选和培养肿瘤干细胞。（2）采用相差显微镜观察含胰岛素和不含胰岛素的无血清培养基中干细胞球形成大小、数量及随时间生长变化情况。（3）采用荧光显微镜观察Hoechst33342染料对MDA-MB-435肿瘤干细胞染色情况。（4）将MDA-MB-435肿瘤干细胞转人有血清培养基培养诱导分化，观察是否能有分化现象。结果：（1）采用无血清悬浮培养方法筛选和培养MDA-MB-435肿瘤干细胞成功。（2）发现不含胰岛素的无血清培养基中，形成的肿瘤干细胞团数量多且体积大。（3）随着培养时间延长，肿瘤干细胞球数量逐渐增多，体积逐渐变大。（4）Hoechst33342染料对肿瘤干细胞染色鉴定，约有90％肿瘤干细胞蓝色荧光染料较弱，呈暗淡区。（5）MDA-MB-435肿瘤干细胞经含血清培养基再次培养，具有分化功能。结论：含生长因子的无血清培养基培养MDA-MB-435细胞，可生成肿瘤干细胞球；不含胰岛素的无血清培养基更适合作为筛选肿瘤干细胞的培养基。

Enrichment of Breast Cancer Stem Cells in Serum-free Medium

Objective： To explore the methods for gathering breast cancer stem cells to obtain a stable quantity of tumor stem cells. Methods： （ 1 ） The breast cancer MDA-MB-435 tumor stem cells were screened and cultured by using serum-free suspension culture method. （2） The size, quantity of mi- crospheres （tumor stem cell sphere） cultured in serum free medium with and without insulin and the changes with the time passing was observed with phase contrast microscopy. （3） The MDA-MB-435 tumor stem cells stained with Hoechst 33342 were observed under fluorescent microscope. （4） The MDA-MB-435 tumor stem ceils were transferred into serum medium to induce differentiation, and whether there was differentiation were observed. Results： （1）The MDA-MB-435 tumor stem cells were successfully screened and cultured by serum-free suspension culture medium. （2） The size and quantity of microspheres cultured in serum free medium without insulin were larger and more than those cultured in serum free medium with insulin. （3） The quantity and size of microspheres increased grad-ually with the culture time passing. （4） 90% of tumor stem cells stained by Hoechst 33342 showed weak blue fluorescence. （$）The MDA-MB-435 tumor stem cells cultured in serum medium again showed differentiation ability. Conclusions： The tumor stem cell spheres can be obtained when the MDA-MB-435 cells are cultured in serum free medium containing growth factors. The serum free medi- um without insulin is more suitable for collecting tumor stem cells than the medium with insulin.