大鼠腹腔脂肪干细胞的分离培养及诱导成骨

Isolation,culture and osteogenic differentiation of adipose-derived stem cells from the abdominal cavity of rats

背景:脂肪干细胞取材方便、增殖旺盛、具有多向分化潜能,有望取代骨髓间充质干细胞而成为新一代组织工程的种子细胞。然而,脂肪干细胞的分离培养仍存在诸多困难与不足。目的:优化脂肪干细胞的分离培养方法,并鉴定其成骨分化潜能。方法:选取200g成年雌性大鼠1只,无菌环境下切取大鼠肾脏、子宫周围及腹后外侧白色脂肪组织,多次胶原酶消化法分离消化,低糖培养基培养脂肪干细胞,倒置显微镜下观察脂肪干细胞的形态变化及增殖能力。采用成骨诱导培养液对第3代细胞进行成骨诱导,并采用碱性磷酸酶、茜素红染色方法进行鉴定。结果与结论:脂肪干细胞形态以长梭形为主,增殖活跃呈旋涡状生长。经成骨诱导10d后,碱性磷酸酶染色为阳性;成骨诱导28d后,茜素红染色阳性。提示采用多次酶消化法得到的大鼠腹腔源性脂肪干细胞在体外易于分离培养,可以稳定传代。在一定条件诱导下,可以向成骨细胞分化。采用以上方法进行脂肪干细胞的分离培养可以为组织工程提供大量、优质的种子细胞。

BACKGROUND： Adipose-derived stem cells could be obtained easily with rapid proliferation and multi-directiona differentiation potential, so they are promising to be seed cells of tissue engineering instead of bone marrow stromal stem cells. However, the isolation and culture of adipose-derived stem cells still remain lots of difficulties. OBJECTIVE： To optimize the methods of isolation and culture of adipose-derived stem cells, and identify the ability of osteogenic differentiation. METHODS： The adipose tissues were obtained from the kidney, peri-uterus and abdominal cavity of one adult female rat weighing 200 g under sterile conditions. Adipose-derived stem cells were isolated by the method of collagenase digestion for several times and cultured with low-glucose medium. The morphologic changes and proliferation of adipose-derived stem cells were observed under inverted microscope. The passage 3 adipose-derived stem cells were cultured in osteogenic medium and then identified with alkaline phosphatase staining and alizarin red staining. RESULTS AND CONCLUSION： Adipose-derived stem cells were mainly spindle-shaped and proliferated quickly with whirlpool-shaped arrangement. After osteogenic induction for 10 days and 28 days, the results of alkaline phosphatase staining and alizarin red staining were both positive. Experimental findings indicate that, adipose-derived stem cells isolated from abdominal cavity of rat by the method of （~.oUagenase digestion for several times can easily be cultured and passaged in vitro, and allow stable passaging and differentiation into osteoblasts under certain induction conditions. The isolation and culture methods for the adipose-derived stern cells may offer more and better seed cells for tissue engineering.