人软骨终板干细胞与退变髓核细胞体外非接触共培养的实验研究

An in vitro study on the interaction of human cartilage endplate stem cells with nucleus pulposus cells in co-culture system

中文关键词:  软骨终板干细胞  退变髓核细胞  共培养

英文关键词:Cartilage endplate stem cells  Degenerative nucleus pulposus cells  Co-colture

中文摘要:

  【摘要】 目的：探究人软骨终板干细胞（cartilage endplate stem cells，CESCs）与退变髓核细胞（nucleus pulposus cells，NPCs）在Transwell非接触共培养条件下的相互作用。方法：对取自因腰椎退行性疾病行腰椎间盘切除椎弓根螺钉内固定患者的软骨终板、退变髓核进行CESCs及NPCs的分离培养及鉴定。通过琼脂糖悬浮培养法获得CESCs的克隆，扩增后行流式技术及免疫荧光对CESCs进行干细胞表面标记的检测，取第三代CESCs与第一代NPCs进行实验，建立三个细胞培养组：CESCs单独培养组、NPCs单独培养组、CESCs与NPCs共培养组（分别接种于Transwell底部和上层插槽中进行非接触共培养）。在培养之后的不同时间点（3、5、7d），采用实时荧光定量PCR（Real-time PCR，RT-PCR）检测各组细胞中蛋白聚糖（Agg）、SOX-9及Ⅱ型胶原蛋白（Coll Ⅱ）mRNA的表达变化情况，共培养7d后，采用Western-blot检测各组细胞中蛋白聚糖、Ⅱ型胶原蛋白、Sox-9蛋白的表达变化。结果：经过分离培养及鉴定，筛选出的细胞为CESCs（流式细胞计数分析，CESCs细胞表面的CD73、CD90、CD105阳性率分别为97.5%、98.7%、98.7%）；共培养后，RT-PCR显示单独培养的CESCs在各检测时间点几乎无Agg、Coll Ⅱ、SOX-9基因表达；而共培养组的CESCs在第5天开始出现Agg、Coll Ⅱ、SOX-9基因表达（其表达量分别为0.10、0.11、0.15），与单独培养CESCs相比有统计学意义（P<0.05）；共培养组NPCs的Agg、Coll Ⅱ及SOX-9基因表达量（其表达量分别为1.32、1.25、0.92）高于单独培养NPCs的表达量（P<0.05），且随着时间延长逐渐升高，共培养与单独培养相比，差异有统计学意义（P<0.05）。共培养7d后，Western-blot结果与RT-PCR结果一致，共培养组CESCs的Agg、Coll Ⅱ、SOX-9蛋白表达量高于单独培养组（P<0.05）。结论：CESCs与NPCs共培养时的相互作用能促使CESCs表达髓核细胞特异性标记物Agg、Coll Ⅱ和SOX-9；通过相互作用，CESCs可增强NPCs表达自身特异性相关分子。

英文摘要:

  【Abstract】 Objectives: To investigate the biological effects of Cartilage endplate stem cells(CESCs) and degenerative nucleus pulposus cells(NPCs) when co-cultured under a transwell system. Methods: The Cartilage endplate organization and degenerative nucleus pulposus tissue were obtained from the patients undergoing discectomy and fusion for lumbar degenerative disease. The cells were subcultured in the agarose culture and the cell clones were selected and expanded in vitro for the assays of stem cell markers by using immunofluorescence and flow cytometry assay. The third generation of CESCs and the primary NPCs were co-cultured in a transwell plate. The three groups were assigned as follows: CESCs group, NPCs group and co-colture group with CESCs and NPCs. The expressions of type Ⅱ collagen, aggrecan and Sox-9 of CESCs and NPCs were detected by RT-PCR at 3 days, 5 days and 7 days respectively. On the 7th day after co-culture, the Western-blot assay was performed to detect the expression of the protein. Results: Cultured cells in vitro exhibited a positive expression of the stem cell markers(the percentage of stem cell markers tested by flow cytometry showed CD73, CD90 and CD105 were detected and their positive rate was 97.5%, 98.7% and 98.7% respectively). The expressions of Agg, Coll Ⅱ and SOX-9 were not detected in CESCs group by RT-PCR. Higher levels of aggrecan, type Ⅱ collagen and Sox-9 expressions from RT-PCR were detected in NPCs from the co-cultured group ( their value was 1.32, 1.25, 0.92 respectively) than those in control group from the 5th day（P<0.05）, similarly, compared with the CESCs that not cultured with NPCs, the CESCs from the co-culture group showed statistically significant expressions of aggrecan, type Ⅱ collagen and Sox-9 from the 5th day(P<0.05)(the value in co-culture group was 0.10, 0.11, 0.15 respectively). On the 7th day after co-culture, the Western-blot showed that the CESCs from the co-culture group had a significantly higher expression of collagen protein Ⅱ, proteoglycan and SOX-9. Conclusions: During the co-colture process, the existence of NPCs can assist the CESCs diffierentiation into NPCs-like cells; furthermore, CESCs have the ability to enhance the expression of NPCs′s specific molecules.