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目的:探讨建立喉部黏膜间充质干细胞的分离培养方法,并对其生物学特性进行鉴定,为进一步研究其在喉部瘢痕中形成的作用及喉部组织工程提供参考。方法:以比格犬喉部会厌背侧(舌面)黏膜为研究对象,采用消化培养的方法分离具有间充质干细胞样细胞。选取第3代细胞对其进行生物学特性鉴定,首先利用MTT法检测其增殖活性及克隆形成情况,然后通过流式细胞术检测细胞表面分子标记物CD29及CD34的表达情况,最后应用第3代细胞对其进行成脂肪细胞和成骨细胞分化培养,观察其分化能力。结果:分离培养细胞形态较为一致,绝大多数呈梭形,排列不规则。MTT增殖活性实验及克隆形成试验结果显示,所分离的细胞具有良好的增殖活性和克隆形成率;流式细胞术结果显示,该细胞表达CD29间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34;同时,该细胞诱导分化成脂肪细胞和成骨细胞实验表明,其具有多向分化潜能。结论:从比格犬会厌背侧黏膜分离的细胞具有间充质干细胞样特性,为进一步研究喉部瘢痕形成机制及喉部组织工程提供了技术基础。
OBJECTIVE: To explore the method of isolation and culture of throat mucosa-derived mesenchymal stem cells and to identify the biological characteristics of the laryngeal mucosa-derived mesenchymal stem cells for further study of its role in the formation of laryngeal scar and laryngeal tissue engineering provide a reference. Methods: The mucous membrane of dorsal (lingual) mucosa of beagle dogs was collected from Beagle dogs. The mesenchymal stem cells were isolated by digestion culture. The third generation of cells were selected for biological characterization. Firstly, MTT assay was used to detect the proliferative activity and clonogenicity. Then the expression of CD29 and CD34 on cell surface was detected by flow cytometry. Finally, Cells were differentiated into adipocytes and osteoblasts to observe their differentiation ability. Results: The morphology of the isolated and cultured cells was more consistent, most of them were fusiform and arranged irregularly. The results of MTT assay and colony formation assay showed that the isolated cells had good proliferative activity and colony formation rate. The results of flow cytometry showed that the cells expressed CD29 mesenchymal stem cell surface marker and low expression of hematopoietic stem cells Surface markers CD34; the same time, the cells induced differentiation into adipocytes and osteoblasts experiments show that it has a multi-directional differentiation potential. CONCLUSION: The cells isolated from the mucosa of the beagle dorsal patella possess mesenchymal stem cell-like features, which provide the technical basis for further research on the mechanism of laryngeal scar formation and laryngeal tissue engineering.