鸡肌腱干细胞的分离培养与初步鉴定

Isolation and primary identification of chicken tendon-derived stem cells

为了建立鸡肌腱干细胞的分离培养体系和鉴定方法,试验使用Ⅰ型胶原酶消化20日龄鸡胚肌腱组织,低密度接种得到鸡肌腱干细胞,用结晶紫染色细胞克隆团块,选择50,500,5 000个/cm^2密度接种7~10 d,镜下观察细胞形态,用细胞计数仪测定细胞增殖能力,用分化诱导试剂对细胞进行成脂与成骨分化诱导,采用油红O和碱性磷酸酶进行染色鉴定。结果表明:原代细胞及传代细胞为长梭形,接种7~10 d后开始形成克隆团块,细胞生长曲线呈S型。500个/cm^2的细胞密度接种细胞克隆能力与增殖能力最强;将该密度接种细胞消化后,按1×10~4个/cm^2接种,分别对其进行成脂与成骨诱导,可发现成脂分化过程中有脂滴形成,油红O染色呈阳性;成骨分化过程中有钙结节形成,碱性磷酸酯酶染色呈阳性。说明消化20日龄鸡胚肌腱组织可成功分离出鸡肌腱干细胞,克隆增殖能力强,且具有分化多种细胞的潜能。

The aim of the present study was to establish an isolation and primary identification system of chicken tendon - derived stem cells （TSCs）. The TSCs were obtained by low density culture of 20 -day -old chicken embryo tendon tissue digested with type I collagenase. The clonogenicity of the cells could be observed by staining with crystal violet. Then the TSCs were digested and seeded with 50 cells/cm2, 500 cells/cm2 , 5 000 cells/cm2, respectively. After incubating for 7 - 10 days, the morphology of TSCs was observed under the microscope. Cell proliferation of TSCs was evaluated by cells counter. Then their adipogeuic and ostcogcuic differentiations were chemically induced. The differentiated cells were identified by oil red O and alkaline phosphatase （ALP） staining. The results showed that the primary cultured and sub- cultured cells were fibroblast - like morphology under the microscope. While the clonogenicity could be observed after culturing for 7 to 10 days. The growth curve of the cells showed an S - type. The ability of the cell replication and proliferation was the strongest when inoculated at 500 cells/cm2. The cells were induced to adipogeuic and osteogenic differentiations after the cells were digested and inoculated at I x 104 eells/cm2. Some lipid droplets could be found in the adipogenic differentiation process. The lipid droplets showed positive by oil red O stai- ning. Similarly, some calcifications could be found in the process of osteogcnic differentiation. The calcifications showed positive by ALP stai- ning. The results indicated that the tendon - derived stem cells could be successfully isolated from 20 - days - old chick embryos. The prolifera- tion ability of the cells was relatively strong. The cells also had a potential for differentiation.