酶消化法分离和培养人源原代跟腱干细胞

目的建立人胎儿来源跟腱干细胞的分离方法,并对人源跟腱干细胞的生物学特性进行鉴定。方法采用酶消化法从胎儿跟腱组织中分离培养跟腱干细胞;通过光学显微镜观察细胞形态;CCK-8法测定细胞增殖能力;流式细胞术检测表面抗原的表达情况;分别用成骨、成脂和成软骨诱导培养液诱导其分化,RT-qPCR鉴定分化标记基因表达;同时对跟腱干细胞蛋白表达进行检测,探究其生物学特性。结果从胎儿来源的跟腱组织中提取的跟腱干细胞具有特征性的细胞形态;在P3代以内,细胞增殖差异无统计学意义;细胞高表达CD44、CD90,不表达CD34、CD45;细胞通过诱导分化后成功向骨、脂肪及软骨细胞分化,相关性基因表达升高;人源跟腱干细胞表达CollagenⅠ、CollagenⅢ、Tenascin-C、Scleraxis。结论酶消化法是分离、培养跟腱干细胞的有效办法,跟腱干细胞表达与跟腱损伤修复有关的蛋白,为跟腱干细胞的鉴定及临床应用提供理论依据。

Isolation and culture of human primary achilles tendon-drived stem cells by enzymatic digestion

Objective To establish a method for isolating primary human fetal achilles tendon-drived stem cells and to identify the biological characteristics of human fetal achilles tendon-drived stem cells. Methods We isolated achilles tendon-drived stem cells from fetal achilles tendon tissue by enzymatic digestion. Cell morphology was observed by inverted microscope; cell proliferation ability was determined by CCK-8; surface antigen expression was detected by flow cytometry; multiple differentiation was induced by adipogenic, osteogenic and chondrogenic medium respectively; RT-qPCR was used to identify the expression of differentiation marker genes; the protein expression of achilles tendon-drived stem cells was detected to explore its biological characteristics. Results Anthropogenic achilles tendon-drived stem cells possessed characteristic cell morphology. Within the P3 generation, the cell proliferation and differentiation abilities had no significant difference. Cell surface markers were expressed as CD34-, CD44+, CD45- and CD90+. The cells had the potential of osteogenesis, chondrogenesis and adipogenic differentiation. Anthropogenic achilles tendon-drived stem cells expressed Collagen I, Collagen III, Tenascin-C and Scleraxis. Conclusion Enzymatic digestion is an effective method to isolate and culture achilles tendon-drived stem cells. Achilles tendon-drived stem cells express proteins related to the repair of achilles tendon injury, which provides a theoretical basis for the identification and clinical application of achilles tendon-drived stem cell lines.