有限稀释法分离鼠髓核间充质干细胞及对细胞活性的影响
发布时间：2019-04-25 20:46
【摘要】：背景椎间盘退行性疾病(degenerative disc disease,DDD)是腰痛的主要原因,给患者带来巨大痛苦,也给家庭和社会造成巨大的经济负担,而目前仍缺乏有效的治疗手段。随着细胞治疗的发展,干细胞移植成为治疗椎间盘退变的研究热门。然而,椎间盘恶劣的微环境使外源性干细胞难以存活,成了研究的瓶颈。近年来,随着髓核来源干细胞——髓核间充质干细胞(nucleus pulposus mesenchymal stem cells,NPMSCs)的发现及其更好的椎间盘微环境适应力的证实,椎间盘退变的细胞治疗开启了新的一页。然而,目前存在多种干细胞筛选方式,使得NPMSCs的分离纯化尚未得到共识。其中,控制细胞种植密度实现干细胞分离的有限稀释法,有助维持细胞活性及干性潜能,因其操作便捷,费用较低得到推广应用。既往研究报道已经利用该方法成功分离获得牙周膜干细胞、单克隆脐血间充质干细胞、齿龈间充质干细胞和髌腱干细胞。但有限稀释法是否适用于NPMSCs的体外分离,尚未见文献报道。目的本研究通过高、中、低细胞接种密度(高密度接种处理为对照组,低、中密度处理即有限稀释法为实验组)分离培养NPMSCs,从细胞形态、增殖、集落形成、迁移、细胞周期、多系诱导分化、免疫表型和干性基因表达,评估其对NPMSCs的生物学活性影响,进一步优化分离和纯化NPMSCs,为干细胞治疗DDD及组织工程种子细胞的探索打下坚实基础。方法取三月龄SD雄性大鼠髓核组织,体外培养,P1代以细胞种植密度分为三组:低密度组(5个细胞/cm2)、中密度组(100个细胞/cm2)、高密度组(10000个细胞/cm2)。各组继续培养皆以10000个细胞/cm2进行传代,观察细胞形态并检测细胞周期分布、集落形成、迁移、增殖和多系诱导分化能力,干性基因(Oct4、Sox2 和 Nanog)及干细胞免疫表型(CD29、CD34、CD44、CD45、CD90)表达情况。结果细胞外观上,各组NPMSCs均贴壁生长,局部旋涡样排列。低密度组细胞为集落样增殖,均一长梭形外观;高、中密度组细胞中出现多角形、类圆形细胞,细胞形态异质性增大,出现宽大扁平且折光性差的细胞。相比另外两组,低密度组细胞具有更强的集落形成能力、迁移能力,并且有更多的细胞进入S期,更少的细胞滞留在G0/G1期(P0.05)。三组细胞均能够进行成骨、成脂、成软骨诱导分化,但低密度组细胞三系分化能力较其他两组增强(P0.05)。低密度组细胞的干性相关基因Nanog、Oct4和Sox2表达量均高于其余两组(P0.05)。低、中密度组免疫表型CD29、CD44和CD90的阳性率高于高密度组(P0.05),而CD34、CD45在三组间无明显统计学差异(P0.05)。结论本研究首次应用有限稀释法(5个细胞/cm2)从髓核中分离得到活性良好且干性潜能得以维持的NPMSCs,更好地应用于干细胞移植及组织工程相关研究,为椎间盘退变的研究奠定基础。
[Abstract]:Background Intervertebral disc degenerative disease (degenerative disc disease,DDD) is the main cause of low back pain, which brings great pain to patients, and also causes great economic burden to family and society. However, there is still no effective treatment. With the development of cell therapy, stem cell transplantation has become a hot topic in the treatment of intervertebral disc degeneration. However, the poor microenvironment of intervertebral disc makes it difficult for exogenous stem cells to survive, which has become the bottleneck of research. In recent years, with the discovery of nucleus pulposus-derived mesenchymal stem cells (nucleus pulposus mesenchymal stem cells,NPMSCs) and its better microenvironmental adaptation to intervertebral disc, the cell therapy of intervertebral disc degeneration has opened a new page. However, there are a variety of stem cell screening methods, so the isolation and purification of NPMSCs has not been agreed. The limited dilution method, which can control the density of cells to separate stem cells, is helpful to maintain the cell activity and dry potential. Because of its convenient operation and low cost, it has been popularized and applied. It has been reported that periodontal ligament stem cells, umbilical cord blood mesenchymal stem cells, gingival mesenchymal stem cells and patellar tendon stem cells have been successfully isolated by this method. However, whether the finite dilution method is suitable for in vitro separation of NPMSCs has not been reported in the literature. Objective to isolate and culture NPMSCs, from cell morphology, proliferation, colony formation, migration and cell cycle by high, middle and low cell density (high density inoculation as control group, low and medium density treatment i.e. limited dilution method as experimental group), and through cell morphology, proliferation, colony formation, migration, cell cycle, cell morphology, proliferation, colony formation, migration, cell cycle. Multi-line differentiation, immunophenotype and dry gene expression were used to evaluate the biological activity of NPMSCs, and further optimization of isolation and purification of NPMSCs, laid a solid foundation for the treatment of DDD and tissue engineering seed cells by stem cells. Methods Nucleus pulposus tissue of 3-month-old male SD rats was cultured in vitro. P1 generation was divided into three groups: low density group (5 cells / cm2), medium density group (100 cells / cm2) and high density group (10000 cells / cm2). Cell morphology and cell cycle distribution, colony formation, migration, proliferation and multilineage inducing differentiation ability, stem gene (Oct4,Sox2 and Nanog) and stem cell immunophenotype (CD29,) were observed in each group by 10000 cells / cm2. CD34,CD44,CD45,CD90). Results on the surface of the cells, NPMSCs was adherent to the wall and arranged in a local vortex-like manner. The cells in the low density group showed colony-like proliferation with a long spindle-shaped appearance, and in the high and middle density groups, polygonal and circular cells appeared, and the heterogeneity of the cell morphology increased, and the cells with wide flattened and poor refractive property appeared in the high-density and middle-density groups. Compared with the other two groups, the cells in the low density group had stronger colony forming ability and migration ability, and more cells entered the S phase and fewer cells remained in the G0 / G1 phase (P0.05). All the cells in the three groups were able to induce differentiation by osteogenesis, lipogenesis and chondrogenesis, but the differentiation ability of the three cell lines in the low density group was higher than that in the other two groups (P0.05). The expression levels of Nanog,Oct4 and Sox2 in the low density group were higher than those in the other two groups (P0.05). The positive rates of immunophenotype CD29,CD44 and CD90 in the low and middle density groups were higher than those in the high density group (P0.05), but there was no significant difference in CD34,CD45 among the three groups (P0.05). Conclusion in this study, the limited dilution method (5 cells / cm2) was first used to isolate NPMSCs, from nucleus pulposus and maintain its dry potential, which could be used in stem cell transplantation and tissue engineering. It lays a foundation for the study of intervertebral disc degeneration.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.5
[Abstract]:Background Intervertebral disc degenerative disease (degenerative disc disease,DDD) is the main cause of low back pain, which brings great pain to patients, and also causes great economic burden to family and society. However, there is still no effective treatment. With the development of cell therapy, stem cell transplantation has become a hot topic in the treatment of intervertebral disc degeneration. However, the poor microenvironment of intervertebral disc makes it difficult for exogenous stem cells to survive, which has become the bottleneck of research. In recent years, with the discovery of nucleus pulposus-derived mesenchymal stem cells (nucleus pulposus mesenchymal stem cells,NPMSCs) and its better microenvironmental adaptation to intervertebral disc, the cell therapy of intervertebral disc degeneration has opened a new page. However, there are a variety of stem cell screening methods, so the isolation and purification of NPMSCs has not been agreed. The limited dilution method, which can control the density of cells to separate stem cells, is helpful to maintain the cell activity and dry potential. Because of its convenient operation and low cost, it has been popularized and applied. It has been reported that periodontal ligament stem cells, umbilical cord blood mesenchymal stem cells, gingival mesenchymal stem cells and patellar tendon stem cells have been successfully isolated by this method. However, whether the finite dilution method is suitable for in vitro separation of NPMSCs has not been reported in the literature. Objective to isolate and culture NPMSCs, from cell morphology, proliferation, colony formation, migration and cell cycle by high, middle and low cell density (high density inoculation as control group, low and medium density treatment i.e. limited dilution method as experimental group), and through cell morphology, proliferation, colony formation, migration, cell cycle, cell morphology, proliferation, colony formation, migration, cell cycle. Multi-line differentiation, immunophenotype and dry gene expression were used to evaluate the biological activity of NPMSCs, and further optimization of isolation and purification of NPMSCs, laid a solid foundation for the treatment of DDD and tissue engineering seed cells by stem cells. Methods Nucleus pulposus tissue of 3-month-old male SD rats was cultured in vitro. P1 generation was divided into three groups: low density group (5 cells / cm2), medium density group (100 cells / cm2) and high density group (10000 cells / cm2). Cell morphology and cell cycle distribution, colony formation, migration, proliferation and multilineage inducing differentiation ability, stem gene (Oct4,Sox2 and Nanog) and stem cell immunophenotype (CD29,) were observed in each group by 10000 cells / cm2. CD34,CD44,CD45,CD90). Results on the surface of the cells, NPMSCs was adherent to the wall and arranged in a local vortex-like manner. The cells in the low density group showed colony-like proliferation with a long spindle-shaped appearance, and in the high and middle density groups, polygonal and circular cells appeared, and the heterogeneity of the cell morphology increased, and the cells with wide flattened and poor refractive property appeared in the high-density and middle-density groups. Compared with the other two groups, the cells in the low density group had stronger colony forming ability and migration ability, and more cells entered the S phase and fewer cells remained in the G0 / G1 phase (P0.05). All the cells in the three groups were able to induce differentiation by osteogenesis, lipogenesis and chondrogenesis, but the differentiation ability of the three cell lines in the low density group was higher than that in the other two groups (P0.05). The expression levels of Nanog,Oct4 and Sox2 in the low density group were higher than those in the other two groups (P0.05). The positive rates of immunophenotype CD29,CD44 and CD90 in the low and middle density groups were higher than those in the high density group (P0.05), but there was no significant difference in CD34,CD45 among the three groups (P0.05). Conclusion in this study, the limited dilution method (5 cells / cm2) was first used to isolate NPMSCs, from nucleus pulposus and maintain its dry potential, which could be used in stem cell transplantation and tissue engineering. It lays a foundation for the study of intervertebral disc degeneration.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.5
【参考文献】
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1 张燕;陶晖;顾韬;李欢迎;李海峰;吴剑宏;何R，
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