【摘要】 目的:探讨连续传代培养对人脐带间充质干细胞(hUC-MSCs)表达NOD样受体(NLR)家族所有23个成员mRNA的影响,为改进hUC-MSCs传代培养质量与增加hUC-MSCs在实验和临床应用中的数量及安全性寻找切入途径。方法:取剖宫产无菌新生儿脐带,经胶原酶II消化结合贴壁选择分离纯化hUC-MSCs,并连续传代培养;采用流式细胞术、诱导分化和RT-qPCR对连续培养传代前后的hUC-MSCs(第3和28代)进行鉴定并对23个NLR家族成员mRNA的表达进行定量分析。结果:连续传代培养前后hUC-MSCs的细胞表型均为CD29+/CD44+/CD105+/CD31-/CD34-/CD40-/CD45-/CD106-/HLA-DR-,能被诱导为成骨和脂肪细胞,符合国际细胞治疗协会建议的MSCs基本特征。全部NLR家族成员的mRNA在培养第3代hUC-MSCs上均有表达,且NOD1、NLRC4、NLRC5、NLRP1、NLRP3、NLRP10、NAIP、NLRX1和APAF1高表达,其余成员低表达。培养传代至第28代除NLRP10的mRNA表达上升、NLRC5和NLRX1 mRNA表达基本不变外,其余成员的mRNA表达均有所下降,其中NLRP1 mRNA表达差异有统计学意义(P<0.05)。结论:体外连续传代培养对hUC-MSCs表达NLR家族成员的影响是多向性的。这些影响与MSCs的增殖、分化及其免疫调节功能的联系有待进一步实验探讨。更多还原

【Abstract】 AIM: To investigate the influence of continuous subculturing of human umbilical cord mesenchymal stem cells( hUC-MSCs) on the mRNA expression of all 23 family members of NOD-like receptors( NLRs),and to search for the way of improving the subculture quality of hUC-MSCs and increasing the quantity and safety in the experimental and clinical application. METHODS: Neonatal umbilical cord was collected to isolate and purify the hUC-MSCs with the collagenase II digestion and adherence screening methods. These cells were continuously subcultured. The hUC-MSCs at passage 3 and passage 28 were identified by flow cytometry and induced differentiation. The mRNA expression of NLRs in the passage 3 and passage 28 hUC-MSCs was detected by RT-qPCR. RESULTS: The cell phenotypes of both passage 3 and passage 28 hUC-MSCs were CD29+/CD44+/CD105+/ CD31-/CD34-/CD40-/CD45-/CD106-/HLA-DR-,and both of the cells were induced into osteoblasts and adipocytes,which were conformed to the criteria of International Society for Cellular Therapy to define MSCs. All the NLR family members were expressed in passage 3 hUC-MSCs. NOD1,NLRC4,NLRC5,NLRP1,NLRP3,NLRP10,NAIP,NLRX1 and APAF1 at mRNA levels were highly expressed,and the rest were lowly expressed. When hUC-MSCs were subcultured to passage 28,NLRP10 mRNA was increased,NLRC5 mRNA and NLRX1 mRNA were hardly changed,and all of the rest members were decreased. The difference of NLRP1 mRNA expression between passage 3 and passage 28 hUC-MSCs was observed with statistical significance( P < 0. 05). CONCLUSION: The effects of subculturing on the expression of NLR family in hUC-MSCs are pleiotropic. It requires further investigation to confirm whether these effects are related to the proliferation,differentiation and immunomodulation of MSCs.更多还原