改良分次酶消化法分离培养兔肌腱干细胞及诱导分化鉴定

Isolation and culture of rabbit tendon stem cells by modified fractional enzyme digestion and identification of their induced differentiation

目的采用改良分次酶消化法体外分离培养家兔肌腱干细胞,观察其生物学特性,并进行诱导分化及鉴定。方法无菌条件下取出家兔髌腱组织,分别运用改良的分次酶消化法和酶消化后低密度稀释接种法进行分离、培养、传代,用倒置相差显微镜观察细胞形态特征并绘制两种方法的生长曲线,通过流式细胞鉴定仪检测肌腱干细胞表面抗原标志物的表达,取P3-P4代肌腱干细胞向成骨细胞、成软骨细胞诱导分化并鉴定。结果改良的分次酶消化法较酶消化后低密度稀释接种法细胞增殖速度加快,形态均一,杂质细胞少。分离出的肌腱干细胞表面抗原标志CD90、CD44呈阳性,而CD34、CD14呈阴性,证实之前分离的细胞为肌腱干细胞。鉴定出肌腱干细胞具有向成骨细胞和成软骨细胞分化能力。结论采用改良的分次酶消化法分离培养兔肌腱干细胞简单易行,肌腱干细胞生长及传代速度可观,活性良好,纯度较高。另外,肌腱干细胞的成功分离培养,也为肌腱相关疾病的研究开辟了一条新途径。

Objective To isolate and culture rabbit tendon stem cells in vitro by a modified fractional enzyme digestion method, observe their biological characteristics, and induce and identify their differentiation. Methods Rabbit patellar tendon tissue was removed under sterile condition. After the tissue was digested by a modified fractional enzyme digestion method and enzyme digestion method respectively, a low-density dilution inoculation method was adopted to isolate, culture and passage the rabbit tendon stem cells. Morphological characteristics of the cells were observed under an inverted phase contrast microscope and the growth curves of the cells isolated from the tendon tissue with the two methods were drawn. The expressions of tendon stem cell surface antigen markers were identified by a flow cytometer. The P3-P4 generations of the tendon stem cells were induced for the differentiation into osteoblasts and chondroblasts, and the differentiation was identified. Results The proliferation of tendon stem cells isolated by the modified fractional enzymatic digestion and inoculated with low-density dilution inoculation method was accelerated, and the cell shapes were uniform with few impure cells. Surface antigen markers CD90 and CD44 of the isolated tendon stem cells were positive, but CD34 and CD14 were negative, indicating that the isolated cells were the tendon stem cells and the identified tendon stem cells had the ability of differentiation into osteoblasts and chondroblasts. Conclusions The modified fractional enzyme digestion for isolation and culture of rabbit tendon stem cells is simple and practicable. The growth and passage speed of the cells are satisfactory, their viability is good, and the purity is high. In addition, the successful isolation and culture of tendon stem cells may create a new way for the research on tendon-related diseases.