| Objective:To isolate and identify the tendon-derived stem cells(TDSCs).To investigate the clonogenicity and multi-lineage differentiation potential of Tendon-derived stem cells(TDSCs)effected by IL-6 in vitro.To investigate whether IL-6 promotes proliferation but inhibits tenogenic differentiation via the JAK/Stat3 pathway of TDSCs.Methods:Tendon-derived stem cells(TDSCs)were isolated from the Achilles tendons in SD rats as we previously reported.Briefly,the Achilles tendons were dissected and incubated with 600 U/ml(3 mg/ml)type I collagenase in PBS for 2h at 37? with gentle shaking.The dissociated cells were plated at a density of 140 cells/cm2 in 100-mm dishes and cultured in DMEM containing 20%FBS.The TDSCs at passage 3 or 4 were used in the following experiments.We usually isolated TDSCs from 4 Achilles tendons and plated on two 100-mm dishes.All aspects of the research were conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee of Nanfang Hospital,Southern Medical University.Cell ProliferationTo perform cell proliferation assay,TDSCs were plated at 103 cells/well in a 96-well plate and allowed to adhere overnight.DMEM containing 10%FBS medium was supplemented with 0,0.1,1,10 and 100 ng/ml rat IL-6 for 1,3,5 days.Proliferation activity was then determined using manual counting and a CCK8 cell counting kit following the manufacturer's protocol.All assays were carried out in triplicate for each sample.Cell cycle analysisTDSCs that were either untreated or treated with IL-6 for 3 days were washed once in phosphate-buffered saline(PBS)and fixed with 500 ?l of cold 70%ethanol in PBS for 2h or overnight at 40?.The cells were then centrifuged at 2000 rpm for 5 min,washed again in phosphate-buffered saline(PBS)and resuspended in 100 ?l RNase A,and incubated at 37? for 30 min.Afterwards,after mixed with 400 ?l propidium iodide(PI),the cells were incubated at 4? for 30 min,and then analyzed by flow cytometry.All assays were carried out in triplicate for each sample.RNA isolation and gene expression assayAfter appropriate treatments,total RNA was isolated using TRIzol following the manufacturer's protocol and reverse-transcribed into cDNA.The resulting cDNA was subjected to a quantitative polymerase chain reaction(qPCR)assay.The qPCR was performed with a LightCycle480 Real-Time PCR System using SYBR green reagents.The average threshold cycle value(Ct value)was calculated from triplicate reactions.Standard curves were generated using 10-fold serial dilutions of cDNA of each gene with a correlation coefficient of>0.98.Relative expression levels were calculated based on a standard curve and normalized to glyceraldehyde 3-phosphate dehydrogenase(Gapdh).Immunoblot analysisThe TDSCs were plated on a density of 4×105/well in a 60-mm dish and cultured in DMEM containing 10%FBS in the presence or absence IL-6 at the concentration of 10 ng/ml for 3 days and then lysed in SDS sample buffer.Scleraxis(Scx),tenomodulin(Tnmd),collagen 1(Coll)collagen 3(Col3)and GAPDH contents were examined by Immunoblot using the corresponding antibodies.To demonstrate that IL-6 exerts functions through the J AK/Stat3 pathway,the TDSCs were plated at a density of 4×105/well on a 60-mm dish and allowed to adhere overnight.DMEM containing 10%FBS medium was supplemented with the 10 ng/ml IL-6,with or without Stat3 inhibitor WP1066 at the concentration of 5 ?M for 3 days.After the above treatment,the cells were collected and used in further experiments.Results:After one week culture of the cells,we observed that all the cells in the culture dish could form clone colonies by the optical microscope,and all of them showed a fusiform cell form,it's in accordance with the previously reported morphology of tendon stem cells.The third and fourth passages of cells will be used in cytological studies of tendon stem cells.These cells were photographed and counted.The clonogenicity and multi-lineage differentiation potential of these cells were confirmed before being used for the experiments in this study using standard assays as described previously.A statistically significant increase was found in cell proliferation of IL-6-treated cells as compared to controls.The effects were statistically significant after 5 days in the groups treated with 1,10 or 100 ng/ml IL-6.IL-6 altered the cell cycle of TDSCs.After TDSCs were incubated with 10 ng/ml IL-6 for 3 days,flow cytometry showed activated G1 phase and an increased number of cells in G2/M phase in IL-6-treated cells.The percentages of TDSCs in S phase with and without IL-6 treatment were nearly identical.After the cells were treated with 0,0.1,1,10 or 100 ng/ml IL-6 for 3 days,a significant reduction was shown in the mRNA expression levels of Scx and Coll.At the protein level,the western-blotting consistently showed a significant reduction in the expression of Scx and Coll in the cells incubated with 10 ng/ml IL-6.Quantitative analyses of the protein bands certainly indicated a significant difference in the protein level expression of Scx and Coll between IL-6-treated and control groups(p<0.05).IL-6 also down-regulated the expression of other pivotal genes,Tenomodulin(Tnmd)and Collagen 3(Col3)while it up-regulated Mohawk(Mkx)expression.The western-blotting also showed a significant reduction in the protein expression of Tnmd and Col3 in the cells incubated with 10ng/ml IL-6.The quantitative analyses of the protein bands also indicated a significant difference in the expression of Tnmd and Col3 between IL-6-treated and control groups(p<0.05).Examination of gene expression of other molecules including Lumican(Lum),Decorin(Dcn),early growth response gene 1(Egrl),Fibromoduline(Fmod)and Biglycan(Bgn)in the IL-6-treated cells found a significant reduction in all of them.The western-blotting consistently showed a significant increase in the expression of Phospho-Stat3 in the cells incubated with 10 ng/ml IL-6 for 3 days and there is no significant difference in the expression of Stat3.Quantitative analyses of the protein bands certainly indicated a significant difference in the protein level expression of Phospho-Stat3 between IL-6-treated and control groups(p<0.05).We cultured cells with 10 ng/ml IL-6,with or without Stat3 inhibitor WP1066 at the concentration of 5 ?M for 3 days.A statistically significant reduction was found in cell proliferation of WP1066-treated group as compared to controls(p<0.05).Meanwhile a significant increase was shown in the mRNA expression levels of Coll,Co13,Sex and Tnmd between WP1066-treated and control groups(p<0.05).At the protein level,the western-blotting consistently showed a significant increase in the expression of Coll,Co13,Scx and Tnmd in the WP1066-treated group(p<0.05).Summary:IL-6 exerts dual effects on tendon-derived stem cells in vitro:strongly enhancing their proliferation but inhibiting their tenogenic differentiation via the JAK/Stat3 pathway. |
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