人脂肪干细胞的分离培养与生物学特性

背景:高效、稳定地获得大量较高纯度的人脂肪干细胞,是其在组织工程学及再生医学中广泛应用的基础和前提。目的:探索体外培养脂肪干细胞的适宜条件,从而提高其增殖能力。方法:采用胶原酶消化的方法,从人腹部皮下块状脂肪中分离出脂肪干细胞,经贴壁筛选法纯化细胞、低糖培养基体外培养扩增。Giesam染色后观察细胞形态;绘制细胞生长曲线并进行细胞周期分析,观察分析传代后染色体核型改变;选取第3代脂肪干细胞做流式细胞鉴定、EdU细胞增殖能力检测以及克隆形成实验。结果与结论:分离培养的原代脂肪干细胞形态不一,经传代后的细胞形态趋于长梭形,排列紧密呈漩涡状生长。细胞生长曲线呈S形,细胞周期分析及EdU掺入法结果显示体外培养的脂肪干细胞具有较强的增殖能力。经染色体核型分析结果提示,体外培养不会引起脂肪干细胞的核型改变。第3代脂肪干细胞经流式细胞仪检测CD29,CD44,CD90,CD105均呈阳性表达,而CD34和CD45呈阴性;克隆形成率为8.8%;在一定的诱导条件下,脂肪干细胞能够向脂肪细胞及成骨细胞分化。结果说明采用胶原酶消化法可成功分离培养人脂肪干细胞,且具有较强的增殖能力。

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

BACKGROUND：Human adipose-derived stem cells are widely used as seed cells in tissue engineering and regenerative medicine. Therefore, harvesting a large number of high-purity human adipose-derived stem cells is important for above-mentioned studies. OBJECTIVE：To explore the suitable culture condition of human adipose-derived stem cells in vitro, and to enhance the proliferative ability of human adipose-derived stem cells. METHODS：Collagenase I was used to digest and isolate human adipose-derived stem cells from intact fat of human abdomen. Human adipose-derived stem cells were purified using attachment method, cultured with low-glucose medium and passaged in vitro. Morphology was observed after Giemsa staining; the growth curve was drawn and cell cycle was analyzed. The karyotype of the passaged human adipose-derived stem cells was analyzed. Cells at the third passage were subjected to flow cytometry analysis, EdU incorporation and colony forming experiments. RESULTS AND CONCLUSION：The morphology of primary cultured human adipose-derived stem cells was not the same, passaged human adipose-derived stem cells were spindle-shaped and arranged tightly in a vortex-like appearance. The growth curve was ＂S＂ shaped. Cell cycle analysis and EdU incorporation results showed that human adipose-derived stem cells cultured in vitro could maintain strong proliferative ability. Karyotype mapping showed that in vitro culture could not cause chromosome abnormalities of adipose-derived stem cells. Flow cytometry analysis showed that passage 3 adipose-derived stem cells were positive for CD29, CD44, CD90 and CD105, but they were negative for CD34 and CD45, with a clone formation rate of 8.8%. Under certain induction condition, adipose-derived stem cells could differentiate into adipocytes and osteoblasts. Human adipose-derived stem cells were successfully isolated by collagenase digestion method, cultured in vitro and showed strong proliferative ability.