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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。
OBJECTIVE: To observe the differentiation effect of human nucleus pulposus cells on cord blood WJMSCs under the condition of contact cell co-culture, in order to find out the treatment of degenerative disc disease Seed cell source. Methods: Umbilical cord of about 30cm healthy newborn babies were isolated, purified, and cultured. The nucleus pulposus of human intervertebral disc was harvested and nucleus pulposus cells were isolated and cultured by enzyme digestion. Take the third generation of stable proliferation of WJMSCs, the use of flow cytometry cell phenotype CD73 / CD90 / CD105 / CD34 / CD45 / HLA-ABC / HLA-DR. WJMSCs were labeled with carboxyfluorescein acetoacetate succinimidyl ester (CFSE) and mixed with nucleus pulposus cells in a 1: 1 ratio. Contact-type cells were co-cultured in 6-well plates. WJMSCs cultured alone were used as control. After cultured for 7 days, the fluorescent labeled positive WJMSCs were sorted by high-speed flow cytometry, total cellular RNA was extracted and reverse transcribed to obtain cDNA. Real-time PCR was used to detect the expression of type Ⅰ collagen, type Ⅱ collagen, type Ⅵ collagen, The relative expression of polysaccharides, SOX-9 and pluripotency genes, the housekeeping gene GAPDH as an internal control, WJMSCs cultured alone were used as controls to calculate the relative gene expression changes by 2-ΔΔCt method. Results: Some of the primary cells of WJMSCs were observed adherent growth after 24 hours of inoculation. The morphology of the primary cells was fusiform and polygonal. After 1 week, colonies formed and reached 90% after 2 weeks. Flow cytometry results showed that CD73 / CD90 / CD105 / and HLA-ABC (+), CD34 / CD45 and HLA-DR (-). Compared with the control group, the expression of type Ⅱcollagen, proteoglycan and SOX-9 in WJMSCs increased significantly (P <0.01), and the expression of collagen type Ⅰ, type Ⅵ collagen and pluripotency gene No significant change (P> 0.05). CONCLUSION: Co-culture of human umbilical cord WJMSCs with contact cells can be induced to differentiate into nucleus pulposus cells by nucleus pulposus cells, which may provide seed cell sources for tissue engineering and cell therapy for degenerative disc repair.