膝骨关节炎滑膜间充质干细胞多向分化及免疫抑制能力

Multipotent differentiation and immunosuppression of synovium-derived mesenchymal stem cells from patients with knee osteoarthritis

目的观察骨关节炎(OA)患者膝关节滑膜来源的间充质干细胞(SMSCs)多向分化能力以及免疫抑制能力。方法无菌环境下通过关节镜取出中山大学孙逸仙纪念医院10例OA患者膝关节滑膜组织,分离培养出SMSCs,采用CCK-8法检测细胞增殖能力,通过成骨、成软骨和成脂肪诱导分化培养基对SMSCs诱导分化,于诱导第21天分别行茜素红、甲苯胺蓝和油红O染色。将SMSCs和CD3、CD28抗体刺激后的人外周血单核细胞(PBMC)进行共培养,通过流式细胞术对PBMC的增殖率进行检测,组间比较通过方差分析总体差异后再通过Bonferroni法进行组间两两比较。结果OA患者SMSCs第3天开始进入对数增长期,第7天细胞增殖进入平台期。SMSCs经过成骨、成软骨和成脂肪诱导分化后,茜素红、甲苯胺蓝和油红O染色均为阳性。SMSCs和CD3、CD28抗体刺激后的PBMC进行共培养5 d后,阴性对照组PBMC增殖率为(3.8±0.4)%,几乎不增殖,阳性对照组PBMC增殖率为(80.9±8.1)%,实验组PBMC的增殖率为(52.3±5.1)%,实验组与阳性对照组的差异具有统计学意义(t=8.97,P<0.01)。结论 OA患者SMSCs同样具有多向分化能力和免疫抑制能力,可作为组织工程理想的种子细胞来源。

Objective To examine the multipotent differentiation and immunosuppression of knee synovium-derived mesenchymal stem cells（ SMSCs） from patients with osteoarthritis（ OA）. Methods The synovial membranes were obtained with arthroscope and the SMSCs were separated and purified. CCK-8 assay was used to detect cell proliferation ability. Osteogenesis,chondrogenesis and adipogenesis were induced,and the cells were stained with alizarin red,toluidine blue and oil red O respectively. Peripheral blood mononuclear cells（ PBMC） stimulated with anti-human CD3 and anti-human CD28 were co-cultured with SMSCs,and the cell proliferation rate of PBMC was examined with flow cytometry. Results among groups were analyzed by one-factor analysis of variance（ ANOVA） followed by Bonferroni correction.Results After SMSCs inoculation,the latent phase was 1-2 days,the logarithmic phase was 3-6 days and the stagnate phase began from the 7thday. After osteogenesis,chondrogenesis and adipogenesis inductions,the cells were positive for alizarin red,toluidine blue and oil red O staining. The cell proliferation rate of PBMC at the 5thday without anti-human CD3 and anti-human CD28 stimulation was（ 3. 8 ± 0. 4） %,while it was（ 80. 9 ± 8. 1） % with anti-human CD3 and anti-human CD28 stimulation. However,when cocultured with SMSCs at the 5thday,the cell proliferation rate of PBMC stimulated with anti-human CD3 and anti-human CD28 was（ 52. 3 ± 5. 1） %,which was significantly lower than that one with anti-human CD3 and anti-human CD28 stimulation only（ t = 8. 97,P〈0. 01）. Conclusion Because of multipotent differentiation and immunosuppression,SMSCs from the patients with osteoarthritis could become potentially ideal seed cells for the tissue engineering.