人脂肪干细胞的提取和鉴定

Extraction and identification of human adipose-derived stem cells

背景:脂肪干细胞是存在于脂肪中的全能干细胞,具备自我更新能力与多向分化潜能,遗传背景相当稳定,体内植入后免疫排斥少,是一种比较理想的种子细胞。目的:提取人大网膜脂肪干细胞,并进行成脂和成骨分化能力鉴定。方法:收集手术患者大网膜的脂肪组织,经Ⅰ型胶原酶消化、过滤、离心后进行原代培养,观察细胞生长状态;用细胞计数法绘制人脂肪干细胞增殖曲线,计算处于对数生长期的倍增时间;用相应的定向诱导液诱导人脂肪干细胞向脂肪细胞及骨细胞方向分化,并用油红O染色、茜素红染色进行鉴定。结果与结论:从手术患者大网膜脂肪组织中成功分离人脂肪干细胞,贴壁生长的人脂肪干细胞为长梭形,形态类似成纤维细胞。第3代人脂肪干细胞生长曲线呈S形,对数生长期人脂肪干细胞的倍增时间为45.90 h。人脂肪干细胞经成脂、成骨定向诱导分化2,3周后,油红O染色显示细胞内有大小不等的橘红色脂肪滴,茜素红染色可见着橘红色的典型钙化结节,提示所培养的脂肪干细胞具有向脂肪细胞、骨细胞系分化的能力。

BACKGROUND：Adipose-derived stem cel s are totipotent stem cel s in the adipose tissue, and have the function of self-renewal and multi-directional differentiation. Human adipose-derived stem cel s are ideal seed cel s with stable genetic milieu and few rejections. OBJECTIVE：To extract human adipose-derived stem cel s from human omental adipose tissue and to identify the cel s by adipogenic and osteogenic induction. METHODS：Omental adipose tissues were col ected from surgical patients to isolate and culture adipose-derived stem cel s using type I col agenase digestion, filtration and centrifugation. Cel growth was observed and proliferative curve of human adipose-derived stem cel s were drawn by cel counting method to calculate the doubling time at logarithmic growth phase. After adipogenic and osteogenic induction, induced cel s were identified using oil red O and alizarin red staining, respectively. RESULTS AND CONCLUSION：Human adipose-derived stem cel s were successful y isolated from the omentum tissues of surgical patients. Adherent cel s were fusiform-shaped and like fibroblasts. The growth curve of passage 3 cel s was in S shape, and the doubling time was 45.90 hours. After adipogenic and osteogenic induction for 2 and 3 hours, respectively, oil red O staining showed unequal-sized orange fat droplets, and alizarin red staining showed typical calcified nodules that were in orange. These findings indicate that adipose-derivedstem cel s have the adipogenic and osteogenic capacity.