连续传代培养对hUC-MSCs上NLR家族mRNA表达的影响  

Effect of continuous subculturing of hUC-MSCs on mRNA expression of NLR family

作　　者：陈智聪[1] 刘俊[1] 廖继东[1] 谷景义[1] 杨晓蕾[1] 李扬秋[2] 

机构地区：[1]暨南大学医学院田家炳医学实验中心,广东广州510632 [2]暨南大学医学院血液病研究所,广东广州510632

出　　处：《中国病理生理杂志》2014年第6期1082-1087,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81100353)

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。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 .

关 键 词：人脐带间充质干细胞 天然免疫受体 受体  NOD样 

分 类 号：R363[医药卫生—病理学]