兔脂肪干细胞的分离培养鉴定及成软骨分化的实验研究

发布时间：2018-11-03 16:04

【摘要】：【目的】对兔脂肪来源的干细胞（rabbitadipose-derivedstemcells，RADSCs）的分离、培养和鉴定进行探讨，并初步探讨不同浓度转化生长因子β3（TransformingGrowthFactor-β3，TGF-β3）促进RADSCs向软骨细胞分化的可行性。为软骨组织工程中种子细胞的选择提供新思路。 【方法】取成年大耳白兔腹股沟部或颈背部脂肪组织，利用酶消化法和密度梯度离心法获得细胞；进行原代和传代培养，，观察细胞变化并绘制细胞生长曲线；流式细胞术和免疫荧光技术鉴定细胞表面CD分子；将获取的第三代细胞进行体外成软骨细胞诱导，分别于诱导的第1W、2W、3W进行形态学观察、甲苯胺蓝染色、阿利新蓝染色和Ⅱ型胶原（ColⅡ）的免疫组化染色。 【结果】所获取细胞呈类成纤维细胞样贴壁快速生长，生长曲线呈比较典型的“S”型；传代后生长、增殖稳定可达20代以上；细胞表面抗原CD34、CD45呈阴性反应，CD44、CD29呈阳性反应，阳性率达97%以上；在TGF-β3的诱导下，细胞形态逐渐变圆，甲苯胺蓝染色、阿利新蓝染色和Ⅱ型胶原的免疫组化染色均呈强阳性，而对照组和空白组则不明显。 【结论】初步鉴定成功分离得到了ADSCs，细胞体外增殖稳定快速，并且具有较好的成软骨分化的潜能，含10ng/mlTGF-β3浓度的诱导液更有利于ADSCs向软骨细胞分化。ADSCs有望成为理想的种子细胞应用于软骨组织工程以修复软骨损伤。
[Abstract]:[objective] to investigate the isolation, culture and identification of rabbit adipose derived stem cells (rabbitadipose-derivedstemcells,RADSCs), and to explore the feasibility of promoting the differentiation of RADSCs into chondrocytes with different concentrations of transforming growth factor 尾 3 (TransformingGrowthFactor- 尾 3 TGF- 尾 3). To provide a new idea for the selection of seed cells in cartilage tissue engineering. [methods] Adipose tissue of inguinal region or neck and back of adult white rabbits were obtained by enzyme digestion and density gradient centrifugation, primary and passage culture were carried out, cell changes were observed and cell growth curve was drawn. Flow cytometry and immunofluorescence techniques were used to identify the CD molecules on the cell surface. The third generation cells were induced by chondroblasts in vitro. Morphological observation, toluidine blue staining, alimine blue staining and type 鈪

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