Zhao Yanhong, Teng Binhong, Sun Xun, Dong Yunsheng, Wang Shufang, Hu Yongcheng, Wang Zheng, Ma Xinlong, Yang Qiang
Stomatological Hospital of Tianjin Medical University, Tianjin, China.
Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, Beijing, China.
Orthop Surg. 2020 Jun;12(3):938-945. doi: 10.1111/os.12691. Epub 2020 May 28.
To explore the effect of kartogenin (KGN) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSC) in vitro, and the synergistic effects of KGN and transforming growth factor (TGF)-β3 on hUCMSC.
Human umbilical cord mesenchymal stem cells were isolated and cultured. Then the differentiation properties were identified by flow cytometry analysis. HUCMSC were divided into four groups: control group, KGN group, TGF-β3 group, and TK group (with TGF-β3 and KGN added into the medium simultaneously). Cells in all groups were induced for 21 days using the suspension ball culture method. Hematoxylin and eosin, immunofluorescence, and Alcian blue staining were used to analyze chondrogenic differentiation. Real-time reverse transcriptase polymerase chain reaction was performed to investigate genes associated with chondrogenic differentiation.
Hematoxylin and eosin staining showed that cells in the TGF-β3 group and the TK group had formed cartilage-like tissue after 21 days of culture. The results of immunofluorescence and Alcian blue staining showed that compared with the control group, cells in the KGN and TGF-β3 groups demonstrated increased secretion of aggrecan after 21 days of culture. In addition, cells in the group combining KGN with TGF-β3 (5.587 ± 0.27, P < 0.01) had more collagen II secretion than cells in the TGF-β3 alone group (2.86 ± 0.141, P < 0.01) or the KGN group (1.203 ± 0.215, P < 0.01). The expression of aggrecan (2.468 ± 0.097, P < 0.05) and SRY-Box 9 (4.08 ± 0.13, P < 0.05) in cells in the group combining KGN with TGF-β3 was significantly higher than those in the TGF-β3 group (2.216 ± 0.09, 3.02 ± 0.132, P < 0.05).'
The combination of KGN and TGF-β3 had synergistic effects and induced hUCMSC chondrogenesis. This could represent a new approach for clinical application and studies on cartilage repair and regeneration.
探讨软骨生成素（KGN）对人脐带间充质干细胞（hUCMSC）体外增殖及成软骨分化的影响，以及KGN与转化生长因子（TGF）-β3对hUCMSC的协同作用。
分离培养人脐带间充质干细胞，然后通过流式细胞术分析鉴定其分化特性。将hUCMSC分为四组：对照组、KGN组、TGF-β3组和TK组（培养基中同时添加TGF-β3和KGN）。采用悬浮球培养法对所有组的细胞诱导21天。使用苏木精-伊红染色、免疫荧光染色和阿尔辛蓝染色分析成软骨分化情况。进行实时逆转录聚合酶链反应以研究与成软骨分化相关的基因。
苏木精-伊红染色显示，培养21天后，TGF-β3组和TK组的细胞形成了软骨样组织。免疫荧光染色和阿尔辛蓝染色结果显示，与对照组相比，培养21天后，KGN组和TGF-β3组的细胞蛋白聚糖分泌增加。此外，KGN与TGF-β3联合组的细胞（5.587±0.27，P<0.01）比单独TGF-β3组（2.86±0.141，P<0.01）或KGN组（1.203±0.215，P<0.01）分泌更多的Ⅱ型胶原蛋白。KGN与TGF-β3联合组细胞中蛋白聚糖（2.468±0.097，P<0.05）和性别决定区Y框蛋白9（4.08±0.13，P<0.05）的表达明显高于TGF-β3组（2.216±0.09，3.02±0.132，P<0.05）。
KGN与TGF-β3联合具有协同作用，可诱导hUCMSC成软骨分化。这可能为软骨修复和再生的临床应用及研究提供一种新方法。
