Wang Yun-Guo, Xie Peng, Wang Yun-Gong, Li Xue-Dong, Zhang Tao-Gen, Liu Zhao-Yong, Hong Quan, Du Shi-Xin
Department of Orthopedics, the Second Hospital of Tianjin Medical University, Tianjin, 300211, China.
Wang Chuanshan College, University of South China, Hengyang, Hunan, 421001, China.
Environ Toxicol Pharmacol. 2014 Sep;38(2):460-8. doi: 10.1016/j.etap.2014.07.008. Epub 2014 Jul 21.
P63 null mice have no or truncated limbs and mutations in human p63 cause several skeletal syndromes that also show limb and digit abnormalities, suggesting its essential role in bone development. In the current study, we investigated the effect of ATRA on chondrogenesis using mesenchymal cells from rat hind limb bud and further examined the mRNA and protein expression of Sox9 and Col2a1 and p63 in rat hind limb bud cells. Limb buds were isolated from embryos from euthanized female rats. Growth of hind limb bud mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. Formation of cartilage nodules was examined by Alcian blue-nuclear fast red staining. The expression of Sox9, Col2al and p63 was determined by Real-time RT-PCR and immunoblotting assays, respectively. Our MTT assays revealed that ATRA at 1 and 10μM significantly suppressed the growth of mesenchymal cells from rat hind limb bud at 24 and 48h (P<0.01 vs. controls). Alcian blue staining further showed that ATRA caused a significant dose-dependent reduction in the area of cartilage nodules (P<0.05 in all vs. controls). At 1μM ATRA, the area of cartilage nodules from hind limb bud cells was reduced to 0.05±0.03mm from 0.15±0.01mm in controls. Real-time RT-PCR assays further indicated that 1 and 10μM ATRA markedly reduced the mRNA expression of Sox9, Col2al and p63 in hind limb bud cells (P<0.05 in all vs. controls). In addition, ATRA time-dependently inhibits the mRNA expression of p63, Sox9 and Col2al. Western blotting assays additionally showed that ATRA dose-dependently reduced the expression of Sox9, Col2al and p63 (P<0.05 in all vs. controls). Together, our results suggest that ATRA suppresses chondrogenesis by modulating the expression of Sox9, Col2al and p63 in primary hind limb bud mesenchymal cells.
p63基因敲除小鼠没有四肢或四肢发育不全，人类p63基因的突变会导致多种骨骼综合征，这些综合征也表现出肢体和指（趾）异常，这表明p63在骨骼发育中起着至关重要的作用。在本研究中，我们使用大鼠后肢芽间充质细胞研究了全反式维甲酸（ATRA）对软骨形成的影响，并进一步检测了大鼠后肢芽细胞中Sox9、Ⅱ型胶原α1（Col2a1）和p63的mRNA及蛋白表达。从安乐死的雌性大鼠胚胎中分离出肢体芽。通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐（MTT）法测定后肢芽间充质细胞的生长情况。通过阿尔辛蓝-核固红染色检测软骨结节的形成。分别通过实时逆转录聚合酶链反应（Real-time RT-PCR）和免疫印迹法检测Sox9、Col2a1和p63的表达。我们的MTT分析显示，1μM和10μM的ATRA在24小时和48小时时显著抑制了大鼠后肢芽间充质细胞的生长（与对照组相比，P<0.01）。阿尔辛蓝染色进一步表明，ATRA导致软骨结节面积显著的剂量依赖性减小（与对照组相比，所有剂量组P<0.05）。在1μM ATRA作用下，后肢芽细胞的软骨结节面积从对照组的0.15±0.01mm²降至0.05±0.03mm²。实时RT-PCR分析进一步表明，1μM和10μM的ATRA显著降低了后肢芽细胞中Sox9、Col2a1和p63的mRNA表达（与对照组相比，所有剂量组P<0.05）。此外，ATRA对p63、Sox9和Col2a1的mRNA表达具有时间依赖性抑制作用。蛋白质免疫印迹分析还显示，ATRA剂量依赖性地降低了Sox9、Col2a1和p63的表达（与对照组相比，所有剂量组P<0.05）。总之，我们的结果表明，ATRA通过调节原代后肢芽间充质细胞中Sox9、Col2a1和p63的表达来抑制软骨形成。
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