Wang Jian-Yang, Yin Chang-Chang, Wu Cui-Cui, Geng Shu-Guo, Yin Ming
Jiujiang Key Laboratory of Translational Medicine, Jiujiang University, Jiujiang 332000, China.
Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, China.
Zhongguo Zhong Yao Za Zhi. 2016 Feb;41(4):694-699. doi: 10.4268/cjcmm20160425.
To investigate the effect of icaritin (ICT) combined with GDF-5 on chondrogenic differentiation of bone marrow stromal cells (BMSCs), and discuss the action of Wnt signaling pathway, full bone marrow adherent method was used to isolate and culture SD rats BMSCs, and the cells at P3 generation were taken and divided into 6 groups： BMSCs group, ICT group, GDF-5 group, GDF-5+ICT group, GDF-5+ICT+SB216763 group, and GDF-5+ICT+ XAV-939 group. The cells were induced and cultured for 14 days. The morphology change was observed by inverted microscope. Alcian blue staining method was used to detect the changes of proteoglycans. RT-PCR was used to detect the mRNA expressions of aggrecan, Col2, Sox9, Dvl1, Gsk3β, and β-catenin. The protein expressions of collagen 2 (COL2) and β-catenin were detected by Western blot. The results indicated that, compared with the BMSCs group, gradual increase was present in proteoglycan Alcian blue staining; mRNA expressions of cartilage differentiation marker genes aggrecan, COL2, Sox9 and the protein expression of COL2, as well as mRNA and protein expressions of Wnt signaling pathway-related gene β-catenin, but with gradual decrease in Gsk3β mRNA expressions in GDF-5 group, GDF-5+ICT group and GDF-5+ICT+SB216763 group. On the contrary, compared with GDF-5+ICT group, there was a decrease in expressions of Dvl1, and β-catenin related to chondrogenic differentiation and Wnt signaling pathway, a increase in Gsk3β mRNA expression, and also a decrease in protein expressions of COL2 and β-catenin in GDF-5+ICT+XAV-939 group, with statistically significant difference between two groups. GDF-5 in combination with icaritin can induce chondrogenic differentiation of BMSCs in rats, and icaritin (ICT) can promote the chondrogenic differentiation. ICT can promote the chondrogenic differentiation of BMSCs in vitro probably by activating the Wnt/β-catenin signaling pathway.
为探讨淫羊藿素（ICT）联合生长分化因子5（GDF-5）对骨髓间充质干细胞（BMSCs）成软骨分化的影响，并探讨Wnt信号通路的作用，采用全骨髓贴壁法分离培养SD大鼠BMSCs，取第3代细胞分为6组：BMSCs组、ICT组、GDF-5组、GDF-5+ICT组、GDF-5+ICT+SB216763组、GDF-5+ICT+XAV-939组。将细胞诱导培养14天。通过倒置显微镜观察形态变化。采用阿尔新蓝染色法检测蛋白聚糖的变化。采用RT-PCR检测聚集蛋白聚糖、Ⅱ型胶原（Col2）、Sox9、Dvl1、糖原合成酶激酶3β（Gsk3β）和β-连环蛋白的mRNA表达。采用蛋白质印迹法检测Ⅱ型胶原（COL2）和β-连环蛋白的蛋白表达。结果表明，与BMSCs组相比，GDF-5组、GDF-5+ICT组和GDF-5+ICT+SB216763组的蛋白聚糖阿尔新蓝染色逐渐增强；软骨分化标志物基因聚集蛋白聚糖、Col2、Sox9的mRNA表达以及COL2的蛋白表达，以及Wnt信号通路相关基因β-连环蛋白的mRNA和蛋白表达均逐渐增加，但Gsk3β的mRNA表达逐渐降低。相反，与GDF-5+ICT组相比，GDF-5+ICT+XAV-939组中与成软骨分化和Wnt信号通路相关的Dvl1和β-连环蛋白表达降低，Gsk3β的mRNA表达增加，COL2和β-连环蛋白的蛋白表达也降低，两组间差异有统计学意义。GDF-5联合淫羊藿素可诱导大鼠BMSCs成软骨分化，淫羊藿素（ICT）可促进成软骨分化。ICT可能通过激活Wnt/β-连环蛋白信号通路促进体外BMSCs的成软骨分化。
