克隆筛选纯化在大鼠脂肪源性干细胞原代培养中的作用

背景：研究中发现从成体的脂肪组织中可以分离出一种间质干细胞，即脂肪源性干细胞，但传统的分离方法仍存在一些问题需要改进。 目的：在脂肪源性干细胞传统的分离方法的基础上进行改进，克隆筛选纯化脂肪源性干细胞。 方法：消化离心法分离Lewis大鼠脂肪源性干细胞，有限密度稀释法克隆化及条件培养基筛选，进行原代培养，以克隆筛选的脂肪源性干细胞为实验组，未克隆筛选的脂肪源性干细胞为对照组，观察细胞形态，对比生长曲线、倍增时间，流式细胞仪鉴定脂肪源性干细胞表面标记CD49d、CD29、CD44。 结果与结论：经克隆筛选的脂肪源性干细胞与未经克隆筛选的脂肪源性干细胞相比，前者形态均一，生长力旺盛，倍增时间短，多代培养后生长曲线无明显改变，通过流式细胞仪鉴定，实验组与对照组在3~6代之间均表达表面标记CD49d、CD29、CD44。结果证实克隆筛选的纯化方法是脂肪源性干细胞原代培养的有效、经济的纯化方法。

Influence of monoclonal screening on the purification of primary cultured rat adipose tissue-derived stem cells

BACKGROUND: It was found that a kind of mesenchymal stem cell can be isolated from animal adult adipose tissues, that is, adipose tissue-derived stem cells (ADSCs). However, there were still some problems need to be improved of the traditional isolation methods. OBJECTIVE: To investigate a new method that using monoclonal screening in the purification of primary cultured ADSCs. METHODS: After isolated the Lewis rat ADSCs with the digestion centrifuge process, the ADSCs were screened with the limited density dilution cloning and condition culture medium screening in the original generation culture. The group with clone screening ADSCs served as the experimental group, without clone screening as the control group. Cellular appearance, growth curve and cell doubling generation time were compared between groups. The surface markers CD49d, CD29, CD44 of ADSCs were determined by flow cytometry. RESULTS AND CONCLUSION: The appearance of ADSCs which underwent clone screening had been homogeneously, with vigorous growing power and short doubling generation time. The growth curve was not changed obviously after many generations. The surface marks CD49d, CD29, and CD44 were expressed in both groups at 3-6 passages. The clone screening purification method is effective, economical purification method of ADSCs.