大鼠尾椎与腰椎髓核组织及细胞的分离培养以及髓核表型的对比研究-东南大学学报医学版

大鼠尾椎与腰椎髓核组织及细胞的分离培养以及髓核表型的对比研究

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中心, 江苏南京 210009

分类号：Q813.1;R-33;R681.5

出版年·卷·期（页码）：2016·35·第三期（293-301）

摘要：

目的:对大鼠尾椎与腰椎髓核组织及细胞进行体外分离、培养与观察,并对髓核软骨样细胞表型进行对比研究。方法:分别取12周龄Sprague-Dawley(SD)大鼠腰椎与尾椎椎间盘,用分析天平称取髓核组织的质量。组织贴壁法分离培养髓核细胞;倒置相差显微镜观察HE染色的髓核组织及不同代次细胞形态变化;流式细胞术检测细胞凋亡率;实时荧光定量PCR检测第2代尾椎与腰椎髓核细胞髓核表型的差异。结果:腰椎髓核组织总质量及单个节段的质量均显著低于尾椎(P<0.05)。成功分离培养腰椎与尾椎髓核细胞。腰椎原代髓核细胞贴壁时间及70%融合时间显著高于尾椎原代髓核细胞(P<0.05);第2代腰椎髓核细胞中脊索样细胞数量显著低于尾椎髓核细胞(P<0.05)。HE染色示尾椎髓核组织中脊索样细胞较多,细胞外基质丰富,髓核样软骨细胞数目较少。第2代腰椎与尾椎髓核细胞凋亡率差异无统计学意义。实时PCR示第2代尾椎髓核细胞与腰椎髓核细胞相比,前者显著下调低氧诱导因子(Hif)2α、aggrecan、Brachyury(T)、细胞角蛋白(Krt)8、Krt18及Krt19的表达(P<0.05),且Brachyury(T)、Krt8、Krt18及Krt19的下调量更明显;显著上调碳酸酐酶(Car)3、Car12的表达(P<0.05)。结论:与腰椎髓核组织相比,尾椎髓核组织中的脊索样细胞数目更多,细胞活性更佳,组织量大,易取材,更适合作为椎间盘退变研究的种子细胞。体外单层培养的腰椎与尾椎髓核细胞表型存在差异。

Objective:To isolate, culture and observe nucleus pulposus tissues and cells from the lumbar and caudal intervertebral disc of rat, and to compare the nucleus pulposus phenotypic markers. Methods:The whole spine was separated from 12-week-old Sprague Dawley rats under the sterile condition. The weights of lumbar and caudal nucleus pulposus tissues were measured by analytical balance. Lumbar and caudal nucleus pulposus cells were cultured by adherent cultivation approach. Lumbar and caudal nucleus pulposus tissues morphology and cellular morphologic changes were noted by HE staining and continuous observation under inverted phase contrast approach, respectively. Lumbar and caudal nucleus pulposus cells apoptosis rates were analyzed with flow cytometry. Messenger RNA expression levels of nucleus pulposus phenotypic markers of lumbar and caudal nucleus pulposus cells at P2 were determined by real-time polymerase chain reaction array analysis. Results:The total and single weights of lumbar nucleus pulposus tissues were significantly lower than those of caudal nucleus pulposus tissues(P<0.05). Lumbar and caudal nucleus pulposus cells were isolated and cultured successfully. The adherence time of the primary cells(the cell fusion reached 70%) in lumbar group was significantly longer than that in caudal group in prithany generation(P<0.05). The numbers of notochordal cells in lumbar group were significantly lower than those in caudal group at P2(P<0.05). HE staining showed that the numbers of notochordal cells, the extracellular matrix in caudal group were abandant than those in lumbar group, however, the numbers of nucleus pulposus cartilage cells were lower than those in lumbar group. The cell apoptosis rates had no significant differences between lumbar and caudal nucleus pulposus cells at P2(P>0.05). Messenger RNA expression levels of nucleus pulposus phenotypic markers in caudal group were significant lower expression of Hif2α, aggrecan, Brachyury(T), Cytokeratin8, 18, 19(P<0.05), and lower expression of Brachyury(T), Cytokeratin8, 18, and 19 was more steeper, and significant higher expression of Carbonic anhydrase3, 12. Conclusion:Compared with the lumbar nucleus pulposus, caudal nucleus pulposus has more notochordal cells, a better cellular status, more tissue, easier assessible, which is more suitable for experiment as a seed cell. Messenger RNA expression levels of nucleus pulposus phenotypic markers have a difference between lumbar and caudal nucleus pulposus cells cultured in monolayer in vitro.

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