Toll样受体在强直性脊柱炎患者MSC中的作用研究

An experimental study of Toll-like receptors regulation of mesenchymal stem cell in ankylosing spondylitis

目的研究强直性脊柱炎(AS)患者间充质干细胞(MSC)中的Toll样受体表达及其对MSC免疫调节的调控作用。方法首先利用流式细胞分析的方法鉴定AS患者骨髓腔中分离培养MSC是否满足特定细胞表型;然后利用RT-PCR的方法检测AS患者MSC中的Toll样受体表达情况;接着分别应用q RT-PCR检测AS患者MSC中TLR3和TLR4的表达水平;最后分别用特定激动剂激活TLR3和TLR4后,利用ELISA检测AS患者MSC中IL-6的分泌情况。结果 AS患者MSC不表达CD34、CD45和HLA-DR,表达CD29、CD90和CD105;RT-PCR结果显示ASMSC表达TLR1、2、3、4、5、6、8,几乎不表达TLR7、9、10;q RT-PCR显示AS患者MSC中TLR3较正常人降低47.5%,TLR4较正常人降低了34.8%;TLR3和TLR4激活后,AS患者MSC中IL-6的分泌较前增加,分别增加了8.3倍13.8倍。结论 AS患者MSC中TLR3和TLR4表达和功能均异常,在AS患者MSC免疫调节中起重要的调控作用。

Objective To detect the expression of Toll-like receptors（TLRs） on mesenchymal stem cells（MSCs） from ankylosing spondylitis（AS） patients and investigate their effect on immunoregulatory function of AS-MSCs. Methods First, flow cytometry was used to determine whether the specific immunophenotype were expressed on the cells cultured from the bone marrow of AS patients. Then, the expression of all TLRs on AS-MSCs was detected by the RT-PCR and the TLR3 and TLR4 were further measured by the q RT-PCR. Finally, after the TLR3 and TLR4 were activated by their specific ligands, ELISA was used to detect the secretion of IL-6. Results The ASMSC expressed CD29, CD44 and CD105, but not CD34, CD45 or HLA-DR. The results from RT-PCR showed that AS-MSC expressed TLR1,TLR2, TLR3, TLR4, TLR5, TLR6 and TLR8 and seldom expressed TLR7, TLR9 or TLR10. In addition, the expression of TLR3 and TLR4 in AS-MSC were respectively reduced to 47.5% and 34.8%, compared to the healthy donors. Ligation of TLR3 and TLR4 can improve the IL-6 secretion of MSCs to 8.3 folds and 13.8 folds respectively in AS patients.Conclusion The expression and function of TLR3 and TLR4 were abnormal in MSCs from AS patients,and they may play a key role in the immunoregulatory dysfunction of AS-MSCs.