Effects And Mechanisms Of Toll Like Receptor -3 And -4 To Chondrogenesis Of Stem Cells

Objective To explore the effects of Toll Like Receptor(TLR)-3 and-4 to the formation of proteoglycan and type II collagen in chondrogenesis of Bone Marrow Mesenchymal Stem(BMSCs) and possible mechanisms. Methods After abstracted from Sprague-Dawley(SD) rats by Ficoll density gradient centrifugation, BMSCs were cultured in vitro. CD34、CD44、CD54 and CD90 were detected by flow cytometry for identification. Before the formal group intervention, we firstly use Western Blot to detect the expression of proteoglycan and type II collagen in un-induced BMSCs as a follow-up test comparison of baseline. Cells were then divided randomly into controlled group, TLR-3 activated group(Poly[I:C],SIGMA P9582-5MG 1!g/ml) and TLR-4 activated group(LPS,SIGMA L2630-10 MG 1!g/ml), treating with PBS, TLR-3 agonist and TLR-4 agonist respectively. After 14 days’ chondrogenesis induction, alcian blue staining was porformed to detect proteoglycan, immunofluorescence and Western blot were porformed to detect type II collagen, real time fluorescent quantitative PCR was porformed to detect Sox-9. Results Fibroblast-like adherencing cells were abstracted with Ficoll density gradient centrifugation method.Flow cytometry showed the expression of CD34 negative and CD44, CD54, CD90 positive,that verified the cells we abstract were SD rats BMSCs.Proteoglycan and type II collagen were not expressed in BMSCs before handled.After 2 weeks of chondrogenesis induction, alcian blue staining were positive in all groups, however, there was no significant difference of staining intensity among groups.Expression of type II collagen were positive in all groups(F=0.628,P=0.547>0.05). Expression of type II collagen was less in the controlled group than that in the TLR-3 and-4 activated group(t3=3.993, P3=0.003<0.05; t4=7.650, P4=0.000<0.05). However, there was no difference of type II collagen expression between TLR-3 and-4 activation group(t=0.934,P=0.372>0.05). The results of PCR showed, compared with the expression of Sox-9 in the controlled group, that in the TLR-3 and-4 groups was respectly 2.2 and 2.14 mutpled.there have significant difference(t3=4.47,P3=0.00<0.05;t4=3.61,P4=0.02<0.05),the expression of Sox-9 in TLR-3 and-4 activated group has no significant difference(t=1.12,P=0.27>0.05). Conclusion After chondrogenesis induction, BMSCs was able to form the matrix proteoglycan and type II collagen, which was regarded as the characteristics of chondrocytes. TLR-3 and-4 have no significant effect on the formation of matrix protein, but can promote the formation of type II collagen. Up-regulation of TLR-3 and-4 to Sox-9 is a probable mechanism.