人脂肪间充质干细胞的分离培养与生物学特性

背景：脂肪来源间充质干细胞取材于吸脂手术所获得的脂肪抽吸物，可反复取材，原料来源充足。目的：建立一种体外分离培养人脂肪来源间充质干细胞的方法，并分析其生物学特性。方法：采用胶原酶消化法分离获取人脂肪来源间充质干细胞并进行体外培养，倒置相差显微镜下观察细胞形态；观察分析传代第3，7，10 代细胞生长曲线；采用流式细胞仪检测传代第5 代细胞表面标志表达。取传代第4代细胞行体外成骨细胞诱导和成脂诱导，采用碱性磷酸酶染色及油红O染色鉴定。冻存传代细胞，分别于 2，6 个月后复苏，锥虫蓝染色计数复苏后细胞存活率。结果与结论：原代细胞3 d 贴壁，6 d 后开始快速增长并形成集落，11 d左右达80%~90%融合，多呈纤维样；传代后细胞维持纤维样形态。传代细胞潜伏期24~48 h，对数增殖期三四天，对数增殖期后第五六天进入平台期。间充质干细胞表面CD34、CD14、HLA-DR 呈阴性表达，CD44、CD105、CD13呈阳性表达，HLA-ABC呈弱阳性表达。具有向脂肪细胞、成骨细胞分化的能力，冻存复苏后细胞存活率达 90% 以上，且与未冻存传代细胞具有相同的生长特性。

Isolation,culture and biological characteristics of human adipose mesenchymal stem cells in vitro

BACKGROUND:Adipose-derived mesenchymal stem cells (MSCs) are taken from fat tissue obtained by liposuction aspirates, which can be repeatedly drawn, and there are adequate sources of raw materials.OBJECTIVE:To establish the isolation and culture method of MSCs derived from human adipose in vitro, and to explore their biological properties.METHODS:Separation by collagen enzyme digestion was used to obtain MSCs from human adipose that were cultured in vitro until the cells reached 80% confluence and passaged. The P3, P7, P10 generation cells were collected to observe and analyze the cell growth curve. The surface markers of the P5 generation cells were detected by using flow cytometry. The P4 cells were induced into osteoblasts in vitro. The cryopreserved passage cells were recovered respectively in 2 and 6 months to detect cell survival rate after resuscitation.RESULTS AND CONCLUSION:Primary cells adhered to the wall at 3 days, then started the swift growth and formed the colony after 6 days, and reached 80%-90% fusions at 11 days, showing the desmoid shape. After passage, the cells maintained desmoid shape. The passage cells growth curves had the common characteristics: latent period was 24-48 hours, logarithm multiplication period was 3-4 days, and the cells entered the platform period at 5-6 days after the logarithm multiplication period. The flow cytometry examination showed that the expressions of CD34, CD14, HLA-DR were negative, CD44, CD105, CD13 were positive, and HLA-ABC were weakly positive on the surface of MSCs. After the recovery, the cell survival percentage reached above 90%; compared with the uncryopreserved passage cells, the cryopreserved passage cells had the same growth characteristics.