CAJ | 학술논문

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。
목적：탐토련속전대배양대인제대간충질간세포（hUC-MSCs）표체NOD양수체（NLR）가족소유23개성원mRNA적영향，위개진hUC-MSCs전대배양질량여증가hUC-MSCs재실험화림상응용중적수량급안전성심조절입도경。방법：취부궁산무균신생인제대，경효원매II소화결합첩벽선택분리순화hUC-MSCs，병련속전대배양；채용류식세포술、유도분화화RT-qPCR대련속배양전대전후적hUC-MSCs（제3화28대）진행감정병대23개NLR가족성원mRNA적표체진행정량분석。결과：련속전대배양전후hUC-MSCs적세포표형균위CD29＋／CD44＋／CD105＋／CD31-／CD34-／CD40-／CD45-／CD106-／HLA-DR-，능피유도위성골화지방세포，부합국제세포치료협회건의적MSCs기본특정。전부NLR가족성원적mRNA재배양제3대hUC-MSCs상균유표체，차NOD1、NLRC4、NL-RC5、NLRP1、NLRP3、NLRP10、NAIP、NLRX1화APAF1고표체，기여성원저표체。배양전대지제28대제NLRP10적mRNA표체상승、NLRC5화NLRX1 mRNA표체기본불변외，기여성원적mRNA표체균유소하강，기중NLRP1 mR-NA표체차이유통계학의의（P＜0．05）。결론：체외련속전대배양대hUC-MSCs표체NLR가족성원적영향시다향성적。저사영향여MSCs적증식、분화급기면역조절공능적련계유대진일보실험탐토。
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 colla-genase II digestion and adherence screening methods .These cells were continuously subcultured .The hUC-MSCs at pas-sage 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 expres-sion between passage 3 and passage 28 hUC-MSCs was observed with statistical significance (P<0.05).CONCLU-SION:The effects of subculturing on the expression of NLR family in hUC-MSCs are pleiotropic .It requires further investi-gation to confirm whether these effects are related to the proliferation , differentiation and immunomodulation of MSCs .