Fibronectin modified expression of Sonic hedgehog in ATRA-mediated neuronal differentiation

In this study, the effect of fibronectin on the neurite outgrowth from embryoid bodies (EBs) in neurodifferentiated embryonal carcinoma P19 cells was examined. The neurite outgrowth on fibronectin was maintained for a longer time in comparison with those on collagen or laminin. Quantitative RT-PCR revealed that mRNA level corresponding to sonic hedgehog (Shh) in neurodifferentiated P19 cells was upregulated on fibronectin, whereas collagen or laminin did not affect. Further knockdown of integrin αv subunit in P19 cells demonstrated that expression of Shh was mediated through interaction between fibronectin and integrin. Additionally, exogenous Shh agonist accelerated neurite outgrowth from embryonic stem cell-derived EBs without large change of neuronal phenotype expression. Taken together, fibronectin could maintain neurite outgrowth via increased Shh expression.     

Strontium renelate promotes osteogenesis via regulation of Sonic hedgehog signaling molecules in rat bone marrow mesenchymal stem cells

AIM：To study the role of Sonic Hedgehog (Shh) in strontium ranelate (Sr)-induced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：BMSCs was isolated from 4-week-old rats by adherent culture. The cells in the 3rd～5th generations were induced to differentiate into obteoblasts, and then were treated with different concentrations of Sr and cyclopamine (Cy). The activity of alkaline phosphatase (ALP) was mea-sured by colorimetry. Mineralized nodules were observed by alizarin red staining. The cellular Shh and Runx2 expression was detected by Western blotting. RESULTS：Sr at concentration of 3 mmol/L increased the activity of ALP and induced the formation of mineralized nodules. Sr at concentrations ranging from 0.1 to 5 mmol/L increased the expression of Shh and Runx2 in the BMSCs at 7 d. Furthermore, the peak expression of Shh occurred following the exposure of Sr (1 mmol/L) or Runx2 (3 mmol/L). On the other hand, Sr at concentration of 1 mmol/L showed a time-dependent increase in the expression of Shh and Runx2 from 1 d to 7 d. Cy at concentration of 10 μmmol/L not only obviously inhibited Sr-induced expression of Shh and Runx2, but also antagonized the increase in the ALP activity and mineralization induced by Sr in the BMSCs. CONCLUSION：Sr promotes osteogenic differentiation of BMSCs by increasing the expression of Shh and Runx2.     

牦牛角的形态发生及Shh和β-catenin的时空表达

研究对象为Ed(Embryonic days)60～240,0.5～5岁公牦牛角(原)基.采用石蜡切片、HE染色和免疫组化的方法研究牦牛角的形态发生过程,检测Shh(Sonic hedgehog)和β-catenin的时空表达与互作规律.结果表明,牦牛角的形态发生按照角(原)基毛囊和角质外鞘发生与否分为5个阶段:角原基前期(Ed≤60)表皮增厚,无毛囊发生;角原基中期(Ed60～140)表皮持续增厚,有少量毛囊发生;角原基后期(Ed140～250)表皮变薄,大量毛囊发生;肉角期(≤0.5 a)真皮乳头发生;成角期(≥0.5 a)形成角质外鞘.角原基期的顶部和角边缘处基底层Shh与β-catenin表达趋势相同,均为强-弱-强过程;肉角期的角边缘处基底层Shh与β-catenin同时表达,顶部仪β-catenin表达;成角期的角边缘处基底层Shh与β-catenin同时表达,顶部均无表达.结果提示:Shh在牦牛角的形态发生中负责表皮基底层形态分化,β-catenin则起着维持细胞分化状态并促进细胞增殖的作用.     

Effect of VDUP-1 on apoptosis of renal tubular epithelial cells induced by high glucose

AIM: To investigate the effect of vitamin D3 up-regulated protein 1 (VDUP-1) on apoptosis of renal tubular epithelial cells induced by high glucose and its mechanism. METHODS: Human renal proximal tubular epithelial cell line HK-2 was treated with high glucose. The mRNA and protein levels of VDUP-1 in HK-2 cells were detected by real-time PCR and Western blot. HK-2 cells were transfected with VDUP-1 small interfering RNA (siRNA). Real-time PCR and Western blot were used to detect the inhibitory effect. The HK-2 cells were treated with high glucose, and the change of VDUP-1 expression was detected. The apoptosis was analyzed by flow cytometry. The activities of caspase-3 and caspase-9 in the cells were measured. The tumor necrosis factor-α (TNF-α) content in the culture supernatant was examined by ELISA. The key proteins of Sonic hedgehog (Shh) signaling pathway, Patched 1 (Ptch1), Smoothened (Smo), zinc finger protein Gli2 and Shh, were determined by Western blot. The HK-2 cells were treated with exogenous Shh, and the levels of Ptch1, Smo and Gli2 were detected by Western blot. After the HK-2 cells with VDUP-1 silencing were treated with exogenous Shh and high glucose, the apoptosis was analyzed by flow cytometry, the activities of caspase-3 and caspase-9 in the cells were examined, and the TNF-α content in culture supernatant was measured by ELISA. RESULTS: High levels of VDUP-1 mRNA and protein were observed in the HK-2 cells treated with high glucose. The mRNA and protein levels of VDUP-1 were decreased in the HK-2 cells transfected with VDUP-1 siRNA(P<0.05). Compared with the normally cultured cells, the apoptotic rate of HK-2 cells was increased after high glucose treatment, and the activities of caspase-3 and caspase-9 and the content of TNF-α were also significantly increased (P<0.05). After down-regulation of VDUP-1 expression by siRNA transfection, the apoptotic rate of HK-2 cells decreased after high glucose treatment, and the activities of caspase-3 and caspase-9, and the content of TNF-α were also significantly decreased (P<0.05). The protein levels of Ptch1, Smo, Gli2 and Shh were decreased after high glucose culture, while down-regulation of VDUP-1 partly antagonized the effect of high glucose on the expression of Ptch1, Smo, Gli2 and Shh in the HK-2 cells. Exogenous Shh promoted the expression of Ptch1, Smo and Gli2, and inhibited the apoptosis of the HK-2 cells induced by high glucose. Exogenous Shh and down-regulation of VDUP-1 synergistically inhibited high glucose-induced apoptosis of the HK-2 cells. CONCLUSION: Down-regulation of VDUP-1 expression inhibits high glucose-induced apoptosis and release of TNF-α in renal tubular epithelial cells by activating Shh signaling pathway.     

毛囊发育过程中信号转导调控网络的初步研究

毛囊发育依赖于一系列的信号分子和细胞分子,这些分子包括FGF、EGF、PDGF、Wnt、BMP、Shh和Notch等,它们在毛囊的发育中被反复利用,并相互作用、交织成网.     

Function of Shh gene in the development of embryos

Mammals have three genes with homology to the hh gene. These comprise Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh has been shown to play a crucial role in embryogenesis and the development of ectoderm. Shh has the great relationship with the forming all the system. Shh-Patched-Smoothened signal pathway will lead the embryogenesis in the normal way. Otherwise, the abnormal embryogenesis, malformation, and tumor will be happened.     

半滑舌鳎Shh基因的克隆与表达及甲基化分析

通过RACE方法获得半滑舌鳎(Cynoglossus semilaevis)Hh基因家族的Shh基因,该基因全长1 922 bp,其中5′UTR 266 bp,3′UTR 360 bp,ORF1 296 bp,编码431个氨基酸,经预测该多肽的相对分子质量为47.28 k D,理论等电点6.95,具有Hh基因家族特有的保守结构域。氨基酸序列分析表明,半滑舌鳎Shh与牙鲆的同源性最高,为82%,与其他鱼类同源性为74%~81%,与非洲爪蟾的同源性为61%,与人和鼠的同源性为63%~64%。甲基化结果显示,Shh基因在1龄卵巢中的甲基化程度普遍低于雄鱼与伪雄鱼精巢。基因表达分析显示,Shh基因在胚胎期的囊胚期表达显著高于其它时期(P0.05),在雌雄成鱼8个组织器官均表达,在雌性性腺分化的关键期孵化后50 d,雌性性腺中表达量较前期升高,且显著高于同时期雄性性腺中的表达(P0.05),在雄性性腺分化的关键期80~95 d,Shh基因在雄性中的表达水平显著升高(P0.05),在8月龄、1龄及2龄雌鱼性腺中表达显著高于雄鱼与伪雄鱼。本研究表明,半滑舌鳎Shh基因在进化中高度保守,与胚胎分化、组织器官形成、雌雄性腺分化及性腺发育密切相关。