中文摘要：

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、NL-RC5、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 mR-NA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。

英文摘要：

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 hUCMSCs 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 .