犬脂肪间充质干细胞体外分离培养及其多向分化潜能

Isolation,culture and multi-directional differentiation potency of canine adipose-derived mesenchymal stem cells

目的:为进一步分析脂肪间充质干细胞的生物学特性,建立犬脂肪间充质干细胞的体外分离培养方法,并对其表面标志和多向分化潜能进行鉴定。方法:实验于2006-09/2007-04在解放军第四军医大学组织工程中心完成。①实验方法:1岁龄家犬麻醉后,无菌条件下取犬腹股沟皮下脂肪组织,用Ⅰ型胶原酶消化,离心弃上层脂肪及上清,沉淀用含体积分数为0.1胎牛血清的D/F12培养基制成单细胞悬液,按2×108L-1接种于培养瓶中,细胞长满80%时用胰酶消化传代。②实验评估:分别取第3,5,10代脂肪间充质干细胞,倒置显微镜下连续观察细胞形态变化;MTT法检测细胞增殖活性;采用免疫细胞化学和体外诱导分化方法对脂肪间充质干细胞表面分子标志和多向分化潜能进行鉴定。结果:①细胞形态观察:原代培养1d多数贴壁细胞呈圆形;3d时贴壁细胞数量明显增加,大部分呈梭形、三角形;6d后细胞呈团簇状生长,形成集落;9～10d后细胞融合超过80%。传代后2～4h开始贴壁,经过较短的潜伏期后开始增殖,3d即可长满培养瓶底壁。②细胞增殖活性:培养的细胞分裂增殖能力强,无明显的生长滞缓期,第3天进入对数生长期,第7天达到生长峰值,第8天后进入平台期。第3,5,10代细胞传代接种后的潜伏期、对数生长期、平台期无明显差异,传10代后细胞无明显的衰老征象。③细胞表面分子标志的鉴定:第3代脂肪间充质干细胞CD29,CD44均呈阳性表达。④脂肪间充质干细胞的多向分化潜能:向脂肪细胞诱导第6天胞质中出现透亮的小脂滴,油红染色呈阳性;向神经细胞预诱导24h后,细胞由梭形变为栅栏样,正式诱导0.5h后细胞呈双极或多极,3h后细胞形态不再发生变化,神经元特异性烯醇化酶、胶质原纤维酸性蛋白均呈阳性;向成骨细胞诱导第21天可见钙化结节。结论:体外分离培养的犬脂肪间充质干细胞生长稳定、增殖较快,存在脂肪组织来源干细胞的相关标志分子CD29及CD44,诱导后能向脂肪细胞、神经细胞和成骨细胞分化。

AIM：To establish a method for the isolation and culture of canine adipose-derived mesenchymal stem cells （ADMSCs） in vitro and identity the surface marker and multi-directional differentiation potency, in order to further analyze the biological characteristics of ADMSCs. METHODS： The experiment was conducted in Tissue Engineering Center, the Fourth Military Medical University of Chinese PLA from September 2006 to April 2007. （1）Empirical method： One-year old canine was anesthetized to excise the inguinal fat tissues with sterile technique and perform a digestion with collagenase type Ⅰ. The floating adipocytes and supernatant were separated by centrifugation. Cells were seeded in culture flasks at a density of 2×10^8 L^-1 in D/F12 supplemented with 10% fetal calf serum. After reaching 80% confluence, the cell was digested with pancreatic enzyme and passage-cultured. （2）Evaluation： The morphology of ADMSCs at passages 3, 5, 10 was observed by inserted microscope constantly. Cellular proliferation was detected with MTT. Surface markers and multi-directional differentiation potency of ADMSCs were identified by immunocytochemistry and inductive differentiation experiment in vitro. RESULTS： （1）Cell morphology： In primary culture, the attached cells were round in one day. At three days, attached cells increased in number and cell morphology appeared as a spindle-shaped and triangle. At six days, cells showed clump growth and formed settlement. Cellular confluence exceeded 80% after 9-10 days. After passaged 2-4 hours, cells adhered and began to proliferate through a short latency period, then overgrew on culture flask in three days. （2）Cell proliferation activity： The division and growth capability of cultured cells were powerful and no obviously growth stoppage period was found. Cultured cells entered exponential phase of growth in the third day, reached grow peak in the seventh day, and cellular proliferation got in platform period in the eighth day. The latency period, exponential phase of growth and platform period were not notably different in the third, fifth and tenth passage cells. After the tenth passage, cells had no obviously apolexis. （3）ldentification of cell surface antigen： CD29 and CD44 expressed positively in the three passage ADMSCs.（4）Multi-directional differentiation potency： After induced 6 days to adipose cells, kytoplasm appeared small lucent lipid droplet, showing positive by oil red staining. After preinduced 24 hours to neurocytes, cell morphology changed from spindle-shaped to bars. When formal induction 0.5 hour, cells showed dipolar or multipolar potency. And three hours later, cell morphology did not change. Both neurone specific enolase and glial fibrillary acidic protein expressed positively. After osteoblast was induced for 21 days, calcific nodus emerged. CONCLUSION： Canine ADMSCs stably grow and rapidly proliferate in vitro. They can differentiate into adipocytes, neurecytes and osteoblasts after induction, expressing CD29 and CD44.