摘要
[目的]探索大鼠骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)成骨过程各阶段,不同Toll样受体(Toll Like Receptor,TLR)亚型的作用。[方法]利用贴壁培养法分离纯化SD大鼠BMSCs,传代扩增,免疫荧光分析检测细胞表面分子CD44、CD105、Vimentin的表达并进行细胞鉴定。选取活力好的第3代细胞,随机分为3组:空白组,TLR-3激活组和TLR-4激活组,均以成骨诱导培养液在相同的环境下培养。Westernblot法检测Collagen-I和Osteocalcin的表达,观察干细胞向成骨细胞(Osteoblast,OB)分化。于培养10、14 d进行碱性磷酸酶染色及茜素红染色,观察矿化结果。[结果]细胞表面抗原CD44、CD105、Vimentin呈阳性。Collagen-I和Osteocalcin的蛋白表达依次为,TLR-4激活组>TLR-3激活组>空白组。3组碱性磷酸酶染色及茜素红染色均呈阳性,TLR-3激活组强度最高,TLR-4激活组和空白组差异不明显。[结论]在BMSC成骨过程中,不同TLR亚型在不同的阶段起作用,TLR-3和TLR-4均可在BMSCs向OB分化过程中起促进作用,TLR-4的效果更明显。在OB成熟矿化为骨细胞的过程中,TLR-3起促进作用,而TLR-4则起抑制作用。
[Objective]To explore the role of toll- like receptor （TLR） subtypes in the ossification of rat bone marrow mesenchymal stem cells （ rat - BMSCs）. [ Method ] After isolation and purification using the adherence method, rat - BMSCs were identified by the presence of surface makers such as CD44, CD105, and vimentin by immunofluorescence. BMSCs in the 3rd passage were randomly divided into 3 groups ： control group, TLR3 - primed group, and TLR4 - primed group. Alkaline phospha- tase （ALP） and alizarin red staining were performed on the 10th day and 14th day. Expression of collagen - I and osteocalcin was detected by western blotting, and statistical analysis was performed. [ Result] CD44, CD105, and vimentin were detected. Colla- gen- I and osteocalcin were expressed in all three groups;the expression was strongest in the TLR- 4 -primed group and wea- kest in the control group. ALP and alizarin red staining were positive for all groups ;the staining was stronger in the TLR -3 - primed group than in the other two groups. The staining results for the TLR - 4 - primed group and control group showed no sta- tistical difference. [ Conclusion] The differentiation of BMSCs to osteoblasts （OB） was promoted by TLR - 3 and TLR -4 ,with TLR-4 showing a stronger effect. However,the ossification of OB to osteocytes was promoted by TLR -3 and inhibited by TLR -4.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2014年第16期1498-1503,共6页
Orthopedic Journal of China
基金
镇江市科技发展基金项目(编号:SH2002019)
关键词
TOLL样受体
骨髓间充质干细胞
成骨分化
矿化
Toll -like receptor, bone marrow mesenchymal stem cells, osteogenic differentiation, mineralization
