Toll样受体-3和-4对干细胞成软骨分化的影响及机制  

Effects of Toll Like Receptor-3 and-4 on Chondrogenic Differentiation of Stem Cells

作　　者：徐小峰[1] 曹学书 陈奇[1] 吴巍[2] 张志坚[3] 陈平波[1] 吴康健[1] 江潮[1] 

机构地区：[1]江苏大学附属医院骨科,镇江212000 [2]华中科技大学同济医学院附属同济医院骨科,武汉430030 [3]江苏大学基础医学与医学技术学院,镇江212001

出　　处：《华中科技大学学报（医学版）》2016年第3期263-267,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81401762);江苏大学博士创新基金资助项目(No.JDFYRC-2013017)

摘　　要：目的探索Toll样受体(Toll like receptor,TLR)-3和-4在骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成软骨分化过程中对蛋白多糖和Ⅱ型胶原蛋白形成的影响及机制。方法用Ficoll密度梯度离心法分离SD大鼠BMSCs,并进行传代扩增。流式细胞术检测细胞表面标志物CD34、CD44、CD54、CD90的表达进行细胞鉴定。以Western blot法检测第3代细胞的蛋白多糖和Ⅱ型胶原的表达。再随机分为空白组、TLR-3激活组、TLR-4激活组,分别以PBS、TLR-3激动剂、TLR-4激动剂处理后,进行成软骨诱导培养。培养第14天,以阿利新蓝染色检测蛋白多糖,以免疫荧光及Western blot法检测Ⅱ型胶原的表达,以实时荧光定量PCR检测Sox-9的表达。结果 Ficoll密度梯度离心法分离所得细胞,其表面标志物表达为CD34(-)、CD44(+)、CD54(+)、CD90(+),符合BMSCs特点。分组处理前,BMSCs不表达蛋白多糖及Ⅱ型胶原。经过2周成软骨诱导培养,空白组、TLR-3激活组和TLR-4激活组阿利新蓝染色均为阳性,且3组间无明显差异。免疫荧光及Western blot结果显示,3组均表达Ⅱ型胶原,但空白组表达明显低于TLR-3激活组和TLR-4激活组,且TLR组间无明显差异。PCR结果显示,TLR-3和TLR-4激活组Sox-9相对表达量较空白组增高1倍,而TLR组之间无明显差异。结论经过成软骨诱导培养,BMSCs能够形成基质蛋白多糖及Ⅱ型胶原,初步具有软骨细胞特性。TLR-3和-4对基质蛋白多糖的形成无明显作用,却能够促进Ⅱ型胶原的形成,其机制可能与TLR-3和-4对Sox-9的上调有关。Objective To explore the effects of Toll like receptor （TLR）-3 and-4 on the expression of proteoglycan and type Ⅱ collagen in bone marrow mesenchymal stem cells （BMSCs ） during chondrogenesis and the possible mecha-nisms.Methods BMSCs in Sprague-Dawley （SD）rats were isolated by Ficoll density gradient centrifugation,cultured and pas-saged in vitro,and identified by detection of their surface markers （CD34,CD44,CD54 and CD90）by flow cytometry.Western blotting was used to detect the expression of proteoglycan and type Ⅱ collagen in BMSCs at 3rd passage.Cells were then divid-ed randomly into blank group,TLR-3 activated group and TLR-4 activated group,in which PBS,TLR-3 agonist and TLR-4 ago-nist were added to treat cells,respectively.Fourteen days later,Alcian blue staining was used to detect the expression of proteo-glycan,immunofluorescence and Western blotting to detect the expression of type Ⅱ collagen,and real time fluorescent quanti-tative PCR to detect the expression of Sox-9.Results Cells were negative for CD34 and positive for CD44,CD54 and CD90, suggesting that the cells were BMSCs.Alcian blue staining revealed positive results in all the groups after 2 weeks of chondro-genesis induction,and no statistical difference was found between groups （P〉0.05）.Immunofluorescence staining and Western blotting showed that type Ⅱ collagen was expressed in all the three groups,its expression was significantly lower in blank group than that in TLR-3 and TLR-4 activated groups （P〈0.05）and no statistical difference was found between the latter two groups （P〉0.05）.PCR showed that the relative expression level of Sox-9 was a 1-fold increase in TLR-3 and TLR-4 activated groups as compared with blank group.There was no significant difference in Sox-9 expression between TLR-3 and TLR-4 acti-vated groups.Conclusion Matrix proteoglycan and type Ⅱ collagen were found to expresse in BMSCs after chondrogenesis in-duction,suggesting the differentiation of BMSCs into chondrocytes.TLR-3 and-4

关 键 词：TOLL样受体 干细胞 成软骨分化 蛋白多糖 Ⅱ型胶原 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]