大鼠尾椎间盘髓核细胞的分离、培养与鉴定-东南大学学报医学版

大鼠尾椎间盘髓核细胞的分离、培养与鉴定

单位：1. 东南大学医学院, 江苏南京 210009;
2. 东南大学附属中大医院脊柱外科, 江苏南京 210009

分类号：R681

出版年·卷·期（页码）：2016·35·第一期（1-6）

摘要：

目的:利用单纯Ⅱ型胶原酶消化法从大鼠尾椎间盘获取髓核细胞,拓展组织工程髓核细胞的获取途径。方法:单纯Ⅱ型胶原酶消化法分离培养大鼠尾椎间盘髓核细胞,镜下观察细胞形态,行HE、甲苯胺蓝、免疫组化及免疫荧光染色鉴定观察;取原代和传代细胞行细胞活性检测;取P1代细胞检测,绘制生长曲线并行细胞周期分析。结果:成功分离出大鼠尾椎间盘髓核细胞。P0代细胞中包含多角形类软骨样细胞、体积大且胞浆中含有大量空泡的脊索样细胞;经传代纯化后最终得到较高纯度的软骨样髓核细胞。台盼蓝细胞活力测定P0代活力高达99%,到P3代细胞活力稍下降,为95%~97%。细胞生长曲线及周期测定示P1代细胞有较强增殖潜能,活力旺盛。胞浆内蛋白聚糖被甲苯胺蓝染成天蓝色,免疫组化呈阳性,表现为黄褐色沉淀;Ⅱ型胶原免疫荧光染色示强阳性表达。结论:大鼠尾椎髓核组织可以提供代谢旺盛、维持类软骨表型的髓核细胞。

Objective:To isolate nucleus pulposus cells from rat caudal disc by Ⅱ collagenase and extent the way of access to nucleus pulposus cells for tissue engineering. Methods:Nucleus pulposus from rat caudal disc were collected to extract nucleus pulposus cells by solo Ⅱcollagenase. The morphology and growth of nucleus pulposus cells was observed under the inverted phase contrast microscope everyday. And the growth curve was draw. Cell morphology and expressions of collagenⅡ and aggrecan were observed by HE staining,toluidine blue staining, immunohistochemical staining and immunofluorescence staining. Trypan blue staining was performed to determine the cell viability of primary and subcultured cells. Cell cycle was detected by flow cytometry. Results:The nucleus pulposus cells were successfully isolated from rat caudal disc. The cultured primary nucleus pulposus cells were consisted of round or polygonal chondrocyte-like cells and irregular notochord cells which large in size and full of vacuoles. High purity nucleus pulposus cells with the chondrocyte-like phenotype could be obtained while the cell passaged more times. Trypan blue staining showed that the primary cell viability was 99%, and P3 cell was 95%-97%. The growth curve and cell cycle analysis indicated that P1 cells proliferated rapidly and vigorously. Aggrecan in the nucleus pulposus cells could be stained as azure and brown respectively by toluidine blue and immunocytochemistry. Immunofluorescence staining showed strongly positive expressions of collagenⅡ. Conclusion:In vitro isolating and culturing the rat caudal disc nucleus pulposus cells with strong activity and maintaining the chondrocyte-like phenotype by solo Ⅱcollagenase is a feasible scheme.

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