微球培养脂肪间充质干细胞分化为髓核样软骨细胞的实验研究

发布时间：2018-03-14 05:26

  本文选题：脂肪间充质干细胞　切入点：髓核细胞　出处：《第四军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 椎间盘退变是一类严重影响人们生活质量的常见病,为腰背痛主要原因之一,其退变主要是由于椎间盘内髓核细胞减少和细胞外基质成份的改变。近年椎间盘组织工程研究中,利用种子细胞移植修复组织损害和促进功能恢复作用的治疗方法成为目前医学领域中最引人关注的热点之一。在诸多种子细胞的研究中,脂肪组织中的脂肪干细胞具有含量多、位置表浅易取材及增殖能力强的优点,是一种具有多分化潜能的成体干细胞,很多实验已经证明脂肪间充质干细胞能分化为髓核样软骨细胞。细胞的微球培养技术在组织工程中应用表明,在细胞微球的中心由于缺氧和低营养状态,这与体内的髓核细胞的生存环境相类似,更利于种子细胞向髓核样软骨细胞分化。基于诱导脂肪间充质干细胞向髓核样软骨细胞分化的各项研究,本实验旨在通过与髓核细胞共培养和细胞因子两种诱导方式做比较,探索体外细胞诱导分化的最佳方法,为今后种子细胞移植治疗椎间盘退变性疾病提供实验依据。 方法 1.取新西兰大白兔腹股沟、腋下脂肪,0.3%的Ⅱ型胶元酶胶原酶溶液中下剪碎并消化40 min (37℃),所得的消化液过滤、离心获取脂肪间充质干细胞。取新西兰大白兔整段脊柱,切开纤维环取髓核组织,0.3%的Ⅱ型胶元酶胶原酶溶液中下剪碎并消化30 min (37℃),所得的消化液过滤、离心获取髓核细胞。所收集的脂肪间充质干细胞和髓核细胞用DMEM/F12(1:1)培养液分别单层培养。 2.脂肪干间充质干细胞培养至3代经鉴定后,按5×106个髓核细胞和脂肪间充质干细胞分别置于Eppendorf管中离心,培养3-4天制成细胞微球。 3.将脂肪间充质干细胞微球置于Transwell下层培养,分别通过5×106个髓核细胞微球及TGF-β1/IGF-1诱导;于诱导前、诱导7、14天,观察细胞形态变化,并通过RT-PCR对Ⅱ型胶原、蛋白多糖含量进行测定。 结果 在体外诱导7、14天,两组脂肪间充质干细胞微团体积、形态无明区别,RT-PCR检测结果显示7天TGF-β1/IGF-1组与髓核诱导组均有Ⅱ型胶原及蛋白多糖mRNA表达,但TGF-β1/IGF-1组表达更强;诱导14天髓核诱导组Ⅱ型胶原及蛋白多糖mRNA表达明显增高,优于但TGF-β1/IGF-1组。 结论 在体外NPCs和TGF-β1/IGF-1对ADSCs进行诱导均有促进其向NPCs分化作用,而TGF-β1、IGF-1为正常髓核细胞分泌重要因子,本实验结果说明NPCs对ADSCs的诱导之间除细胞因子作用外,尚存在相互促进增殖分化作用,为更好向髓核样细胞分化仍其它诱导条件存在。 髓核细胞与脂肪间充质干细胞的三维共同培养较细胞因子诱导经济有效,不但能更好促进种子细胞向目的细胞分化,而且能模拟椎间盘内环境以利于今后的体内研究。
[Abstract]:Intervertebral disc degeneration is a common disease that seriously affects people's quality of life and is one of the main causes of low back pain. The degeneration is mainly due to the decrease of nucleus pulposus cells and the changes of extracellular matrix in intervertebral disc. The treatment of repairing tissue damage and promoting functional recovery by seed cell transplantation has become one of the most attractive topics in the field of medicine. In many seed cell studies, adipose stem cells are abundant in adipose tissue. The position surface is a kind of adult stem cells with multiple differentiation potential because of its advantages of easy access to materials and strong proliferative ability. Many experiments have shown that adipose mesenchymal stem cells can differentiate into nucleus pulposus like chondrocytes. This is similar to the living environment of nucleus pulposus cells in vivo and is more conducive to the differentiation of seed cells into nucleus pulposus chondrocytes, based on studies on inducing adipose mesenchymal stem cells to differentiate into nucleus pulposus chondrocytes. The purpose of this study was to explore the best method of cell differentiation in vitro by comparing with the co-culture of nucleus pulposus cells and cytokines in order to provide experimental evidence for the treatment of degenerative disc diseases by seed cell transplantation in the future. Method. 1. Adipose mesenchymal stem cells were obtained from the inguinal region of New Zealand white rabbits and 0.3% of type 鈪，

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