两种人基质干细胞与同源退变髓核细胞共培养条件下相互作用的细胞生物学比较研究

Stem cells offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. However, the cellular biological mechanisms of interactions between stem cells and degenerative IVD still remain uncertain. The current lack of specific marker(s) for IVD cells limits the ability to meaningfully explore the differentiation of stem cells to an IVD phenotype. Moreover, there have been no studies investigating the proper candidate stem cells specifically for disc regeneration. The purpose of this study is thus to begin to investigate the cellular biological interactions between mesenchymal stem cells and degenerative nucleus pulposus cells, and to compare the ability of differentiation toward disc phenotype of marrow and adipose tissue derived stem cells introduced by homologous degenerative nucleus pulposus cells in direct and indirect contacting co-culture systems. Homologous human mesenchymal stem cells (MSCs), adipose derived stem cells (ADSCs) and degenerative nucleus pulposus cells (NPCs) will be harvested with informed consent from bone marrow, adipose and nucleus pulposus of forty patients undergoing lumbar spine surgery(grouped with age and IVD Pfirrmann classification) . MSCs and ADSCs will be co-cultured respectively with indirect (Transwell system) and direct cell-cell contact with homologous degenerative NPCs for 14 days. Functinoal cell factors(TGF-β1, IGF-1, BMP-7) and changes of matrix-associated genes and protein (collagen type I,II,III, aggrecan, SOX9 and Versican)of MSCs, ADSCs and degenerative NPCs are to be assessed by quantitative real-time PCR and ELISA in 3rd ,7th and 14th day. We are trying to answer two questions of biological treatment for intervertebral disc degenerative diseases by this comparative study: Does co-culture effectively induce stem cell differentiation toward nucleus pulposus cell phenotype? Which stem cell is the most promising candidate of cell-based therapy for disc degenerative degeneration

尽管多种干细胞在实验研究中表现出修复椎间盘退变的良好前景，但目前普遍倾向于应用研究，而忽视了深入探讨干细胞修复椎间盘退变的生物学机制。这也是拓展干细胞在椎间盘退行性疾病临床应用的重要瓶颈。既缺乏促进干细胞向类髓核细胞分化的明确手段，也不清楚哪种干细胞适宜作为椎间盘修复的最佳种子细胞来源。本研究拟在实施脊柱外科手术同时，采集同源人骨髓、脂肪与髓核组织，体外分离骨髓间充质干细胞、脂肪干细胞与髓核细胞，将两种干细胞与同源的退变髓核细胞分别以直接及非直接接触的方式进行共培养，观察共培养前后不同干细胞向类髓核细胞分化表型及髓核组织功能性细胞因子表达的变化，及其对退变髓核细胞的生物学效应，并进行对比性研究。探讨基质干细胞与退变髓核细胞相互作用的细胞生物学机理，并以此回答两个问题：基质干细胞与髓核细胞共培养能否达到"近朱者赤"的定向诱导分化作用？两种来源的干细胞哪种更适宜于椎间盘退行性疾病的生物学治疗？

干细胞修复椎间盘退变已经成为脊柱外科基础研究的重点。但目前对外源性干细胞如何与退变髓核发生相互作用从而产生生物学修复效果尚缺乏了解，也是其深入研究及临床应用的瓶颈。本研究拟在实施脊柱外科手术同时，采集同源人骨髓、脂肪与髓核组织，体外分离骨髓间充质干细胞、脂肪干细胞与髓核细胞，将两种干细胞与同源的退变髓核细胞分别以直接及非直接接触的方式进行共培养，观察共培养前后不同干细胞向类髓核细胞分化表型及髓核组织功能性细胞因子表达的变化，及其对退变髓核细胞的生物学效应，并进行对比性研究。.研究结果表明，式共培养较真实的模拟干细胞治疗退变椎间盘微环境，方法简单可行，具有良好的实用价值和前景。通过接触式共培养，BMSCs和ADSCs均能刺激自身退变NPCs，产生明显的激活和营养作用，使退变NPCs中Ⅱ型胶原、蛋白多糖、SOX-9等细胞外基质表达显著升高。同时，相对于ADSCs，BMSCs对退变NPCs的激活效应更强。因而，BMSCs可能更加适合于椎间盘退行性疾病的生物学治疗。