摘要
Background： Mesenchymal stem cells （MSCs） are bone marrow stem cells which play an important role in tissue repair. The treatment with MSCs will be likely to aggravate the degree of fibrosis. The Wnt/β-catenin signaling pathway is involved in developmental and physiological processes, such as fibrosis. Dickkopfs （DKKs） are considered as an antagonist to block Wnt/β-catenin signaling pathway by binding the receptor of receptor-related protein （LRP5/6）. DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/β-catenin signaling pathway. Methods： Stable MSCs were randomly divided into four groups： MSCs control, MSCs ＋ transforming growth factor-β （TGF-β）, MSCs ＋ DKK1, and MSCs ＋ TGF-β ＋ DKK1. Flow cytometry was used to identify MSCs. Cell viability was evaluated by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide test. lmmunofluorescence was used to detect protein expression in the Wnt/β-catenin signaling pathways. Western blotting analysis was employed to test expression of fibroblast surface markers and, finally, real-time reverse transcription polymerase chain reaction was employed to test mRNA expression offibroblast surface markers and Wnt/β-catenin signaling proteins. Results： Cultivated MSCs were found to conform to the characteristics of standard MSCs： expression of cluster of differentiation （CD） 73, 90, and 105, not expression of 34, 45, and 79. We found that DKK1 could maintain the normal cell morphology of MSCs. Western blotting analysis showed that fibroblast surface markers were expressed in high quantities in the group MSCs ＋ TGF-β. However, the expression was lower in the MSCs ＋ TGF-β ＋ DKK1. Immunofluorescence showed high expression of all Wnt/β-catnin molecules in the MSCs ＋ TGF-β group but expressed in lower quantities in MSCs ＋ TGF-β ＋ DKK1 group. Finally, mRNA expression of fibroblast markers vimentin, α-smooth muscle actin and Wnt/β-catenin signaling proteins β-catenin, T-cell factor, and glycogen synthase kinase-3β was significantly increased in MSCs ＋ TGF-β group compared to control （P 〈 0.05）. Expression of the same fibroblast markers and Wnt/β-catenin was decreased to regular quantities in the MSCs ＋ TGF-β ＋ DKK 1 group. Conclusions： DKK1, Wnt/β-catenin inhibitors, blocks the Wnt/β-catenin signaling pathway to inhibit the process of MSCs fibrosis. It might provide some new ways for clinical treatment of certain diseases.
Background： Mesenchymal stem cells （MSCs） are bone marrow stem cells which play an important role in tissue repair. The treatment with MSCs will be likely to aggravate the degree of fibrosis. The Wnt/β-catenin signaling pathway is involved in developmental and physiological processes, such as fibrosis. Dickkopfs （DKKs） are considered as an antagonist to block Wnt/β-catenin signaling pathway by binding the receptor of receptor-related protein （LRP5/6）. DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/β-catenin signaling pathway. Methods： Stable MSCs were randomly divided into four groups： MSCs control, MSCs ＋ transforming growth factor-β （TGF-β）, MSCs ＋ DKK1, and MSCs ＋ TGF-β ＋ DKK1. Flow cytometry was used to identify MSCs. Cell viability was evaluated by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide test. lmmunofluorescence was used to detect protein expression in the Wnt/β-catenin signaling pathways. Western blotting analysis was employed to test expression of fibroblast surface markers and, finally, real-time reverse transcription polymerase chain reaction was employed to test mRNA expression offibroblast surface markers and Wnt/β-catenin signaling proteins. Results： Cultivated MSCs were found to conform to the characteristics of standard MSCs： expression of cluster of differentiation （CD） 73, 90, and 105, not expression of 34, 45, and 79. We found that DKK1 could maintain the normal cell morphology of MSCs. Western blotting analysis showed that fibroblast surface markers were expressed in high quantities in the group MSCs ＋ TGF-β. However, the expression was lower in the MSCs ＋ TGF-β ＋ DKK1. Immunofluorescence showed high expression of all Wnt/β-catnin molecules in the MSCs ＋ TGF-β group but expressed in lower quantities in MSCs ＋ TGF-β ＋ DKK1 group. Finally, mRNA expression of fibroblast markers vimentin, α-smooth muscle actin and Wnt/β-catenin signaling proteins β-catenin, T-cell factor, and glycogen synthase kinase-3β was significantly increased in MSCs ＋ TGF-β group compared to control （P 〈 0.05）. Expression of the same fibroblast markers and Wnt/β-catenin was decreased to regular quantities in the MSCs ＋ TGF-β ＋ DKK 1 group. Conclusions： DKK1, Wnt/β-catenin inhibitors, blocks the Wnt/β-catenin signaling pathway to inhibit the process of MSCs fibrosis. It might provide some new ways for clinical treatment of certain diseases.
基金
Financial support and sponsorship This work was supported by the National Natural Science Foundation of China （No. 81301448）, and the Key Medical Talent Foundation of Jiangsu Provincial Center for Disease Prevention and Control （No. JKRC20110029）.
