髓核样细胞与温敏型支架复合培养的探索研究

发布时间：2018-06-22 20:59

  本文选题：脂肪间充质干细胞 + 髓核细胞　； 参考：《第四军医大学》2010年硕士论文

【摘要】： 椎间盘(Intervertebral Disc, IVD)由软骨终板、髓核以及纤维环组成,髓核(Nucleus pulposus)是椎间盘结构与功能的核心,包含髓核细胞和细胞外基质。人类椎间盘在生命中“第二个十年”就开始退变,出现细胞凋亡、基质成分改变、纤维环松弛等病理变化。而椎间盘退变是一系列脊柱退行性疾病发生的前提和始动环节,特别是下腰痛发生的首要因素。如若可以控制椎间盘退变进程,就可以从一方面防止这些疾病的发生。组织工程技术是利用种子细胞结合载体、支架材料重建组织器官、修复病变的方法,这一技术的成功应用,可以从根本上解决椎间盘退变的问题。目前多选用间充质干细胞作为种子细胞,在体外以适宜的方法诱导其分化为髓核样细胞。复合生长因子诱导以及细胞共培养诱导在业界均有一定研究,但二者对于脂肪干细胞向髓核样细胞分化的效果有何不同,尚需探索。另外,本实验把处于软骨组织工程领域内研究热点的壳聚糖基可注射支架与诱导后的髓核样细胞复合,评估其是否具备良好的相容性并维持髓核样细胞的分化表型、保证正常的细胞外基质分泌,为“可注射支架复合髓核样细胞微创修复退变椎间盘”这一临床应用构想进行体外实验。 实验一选取2月龄新西兰兔,从肩胛间脂肪组织提取脂肪干细胞并提取髓核细胞,分别进行体外单层培养扩增,倒置显微镜观察细胞形态;脂肪干细胞传3代后,流式细胞术鉴定细胞表面抗原;将3代脂肪干细胞分为两组,一组以成髓核诱导培养基诱导分化2周,另一组与髓核细胞在Transwell培养板中共培养2周,比较两组细胞在蛋白聚糖、Ⅱ型胶原mRNA表达水平上的差异。 实验二以兔脂肪干细胞作为种子细胞,采用实验一中成髓核诱导培养基诱导为髓核样细胞;以氯化壳聚糖(C)与β-甘油磷酸钠(GP)构建温敏型可注射支架。将细胞与支架混合,37℃成胶后,在成髓核诱导培养基中培养3周,通过AO-PI染色法和扫描电镜观测细胞存活情况,PR-PCR检测蛋白聚糖、Ⅱ型胶原mRNA表达水平。 结果证明,生长因子诱导与共培养诱导对细胞在蛋白聚糖、Ⅱ型胶原mRNA表达水平上均无统计学差异,仅蛋白聚糖表达水平前者略高;髓核样细胞在温敏型壳聚糖支架上生存状况良好,培养3周后细胞功能状态(蛋白聚糖、Ⅱ型胶原mRNA表达水平)较复合培养前的髓核样细胞有一定提高,其蛋白聚糖表达水平差异有统计学意义。
[Abstract]:Intervertebral disc (IVD) consists of endplate of cartilage, nucleus pulposus and fibrous ring. Nucleus pulposus is the core of the structure and function of intervertebral disc, including nucleus pulposus cells and extracellular matrix. In the second decade of human intervertebral disc degeneration, apoptosis, matrix composition changes, fiber ring relaxation and other pathological changes. Intervertebral disc degeneration is the premise and initiation of a series of spinal degenerative diseases, especially the primary factor of low back pain. If the process of disc degeneration can be controlled, these diseases can be prevented on the one hand. Tissue engineering is a method to reconstruct tissues and organs and repair lesions by using seed cell and carrier. The successful application of this technique can fundamentally solve the problem of disc degeneration. Mesenchymal stem cells are often used as seed cells to induce them to differentiate into nucleus pulposus like cells in vitro. The effects of co-culture and co-culture on the differentiation of adipose stem cells into nucleus pulposus cells have been studied in the industry, but it is still necessary to explore the difference between them in the differentiation of adipose stem cells into nucleus pulposus like cells. In addition, chitosan based injectable scaffolds in cartilage tissue engineering were combined with induced nucleus pulposus like cells to evaluate their compatibility and maintain the differentiation phenotype of nucleus pulposus cells. To ensure normal extracellular matrix secretion, in vitro experiments were carried out for the clinical application of "injectable scaffold combined with minimally invasive repair of degenerative intervertebral disc with nucleus pulposus cells". In experiment one, adipose stem cells were extracted from interscapular adipose tissue and nucleus pulposus cells were extracted from 2-month-old New Zealand rabbits. The third generation of adipose stem cells were divided into two groups: one group was induced to differentiate into two groups on myeloblastic medium for 2 weeks, the other group was co-cultured with nucleus pulposus cells on Transwell culture plate for 2 weeks, and the two groups were compared in proteoglycan. The difference of mRNA expression level of type 鈪，

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