接触式细胞共培养诱导人脐带华通胶间充质干细胞向类髓核细胞分化

Differentiation of human umbilical cord Wharton′s jelly-derived mesenchymal stem cells into nucleus pulposus-like cells by coculture with cell-cell contact in vitro

中文关键词:  华通胶间充质干细胞  共培养  诱导分化  髓核细胞

英文关键词:Wharton′s jelly-derived mesenchymal stem cells  Coculture  Differentiation  Nucleus pulposus cells

中文摘要:

  【摘要】 目的：观察接触式细胞共培养条件下，人髓核细胞对脐带华通胶间充质干细胞（Wharton′s jelly-derived mesenchymal stem cells，WJMSCs）的诱导分化效应，为椎间盘退变性疾病的治疗寻找种子细胞来源。方法：取足月产健康新生儿脐带约30cm，分离、纯化、培养WJMSCs；取人椎间盘髓核组织，酶消化法分离培养髓核细胞。取第三代稳定增殖的WJMSCs，利用流式细胞方法检测细胞免疫表型CD73/CD90/CD105/CD34/CD45/HLA-ABC/HLA-DR。用羧基荧光素乙酰乙酸琥珀酰亚胺酯（CFSE）标记WJMSCs，与髓核细胞以1∶1比例进行混合，在6孔板中进行接触式细胞共培养；以单独培养的WJMSCs为对照组。培养7d后利用高速流式细胞仪分选荧光标记阳性的WJMSCs，提取细胞总RNA，进行反转录获得cDNA，利用Real-Time PCR方法检测其Ⅰ型胶原、Ⅱ型胶原、Ⅵ型胶原、蛋白多糖、SOX-9和多能聚糖基因的相对表达，管家基因GAPDH作为内参，单独培养的WJMSCs作为对照，利用2-ΔΔCt方法计算基因相对表达变化。结果：WJMSCs原代细胞接种24h可见部分细胞贴壁生长，形态为梭形和多角形，1周后形成集落，2周时细胞融合达到90%。流式细胞检测结果显示CD73/CD90/CD105/和HLA-ABC（+），CD34/CD45和HLA-DR（-）。与髓核细胞共培养7d后WJMSCs的Ⅱ型胶原、蛋白多糖和SOX-9基因相对表达较对照组显著性增加（P<0.01），Ⅰ型胶原、Ⅵ型胶原和多能聚糖基因相对表达未见显著性变化（P>0.05）。结论：通过接触式细胞共培养人脐带WJMSCs能够被髓核细胞诱导分化为类髓核细胞，可为组织工程技术和细胞治疗修复退变椎间盘提供种子细胞来源。

英文摘要:

  【Abstract】 Objectives: To investigate the differentiation of human Wharton′s jelly-derived mesenhymal stem cells(WJMSCs) into nucleus pulposus-like cells by coculture with cell-cell contact in vitro. Methods: Umbilical cord was obtained from normal birth, and the mesenchyamal stem cells were isolated from the umbilical cord Wharton′s jelly. Human nucleus pulposus cells(NPCs) were isolated from human intervertebral nucleus pulposus. WJMSCs were taken for flow cytometric analysis(CD73/CD90/CD105/CD34/CD45/HLA-ABC/HLA-DR). Fluorescent labeled WJMSCs and no-labled NPCs were cocultured for 7 days by cell-cell contact method. Cells were seeded at ratio of 1∶1. WJMSCs cultured alone served as controls. MoFlo high-speed cell sorting was conducted to separate fluorescent labeled WJMSCs and NPCs after coculture. Total RNA was extracted from cells using Trizol reagent. After the cDNA was obtained by reverse transcription, relative gene expressions of type Ⅰ collagen, type Ⅱ collagen, type Ⅵ collagen, aggrecan, SOX-9 and versican were determined by Real-Time PCR and normalized to the GAPDH housekeeping gene. WJMSCs cultured alone served as controls. The 2-ΔΔCt value was then used to calculate relative expression of each target gene. Results: WJMSCs were isolated from human umbilical cord and expanded as primary cultures. After attachment, the cells gradually spread out and were shown as fibroblast-like morphology. According to the immunophenotypic analysis of flow cytometry, the WJMSCs expressed CD73, CD90, CD105, and HLA-ABC, but did not express CD34, CD45 and HLA-DR. While WJMSCs and NPCs were cocultured with cell-cell contact, SOX-9, type Ⅱ collagen and aggrecan mRNA increased significantly in WJMSCs(P<0.01). There were no significant changes in type Ⅰ collagen, type Ⅵ collagen and versican mRNA expression(P>0.05). Conclusions: WJMSCs display the characteristics of mesenchymal stem cells. Coculture of NPCs and WJMSCs with cell-cell contact can induce WJMSCs differentiating into a NPCs phenotype within 7 days, which indicates that WJMSCs may be a promising seed cell for tissue engineering therapies or cell-based therapies for degenerated intervertebral discs.