Otic placode cell specification and proliferation are regulated by Notch signaling in avian development

Background: The entire inner ear including the cochlear‐vestibular ganglion arises from a simple epithelium, the otic placode. Precursors for the placode originate from a pool of progenitors located in ectoderm next to the future hindbrain, the pre‐otic field, where they are intermingled with future epibranchial and epidermal cells. While the importance of secreted proteins, such as FGFs and Wnts, in imparting otic identity has been well studied, how precursors for these different fates segregate locally is less well understood. Results: (1) The Notch ligand Delta1 and the Notch target Hes5‐2 are expressed in a part of pre‐otic field before otic commitment, indicative of active Notch signaling, and this is confirmed using a Notch reporter. (2) Loss and gain‐of‐function approaches reveal that Notch signaling regulates both proliferation and specification of pre‐otic progenitors. Conclusions: Our results identify a novel function of Notch signaling in cell fate determination in the pre‐otic field of avian embryos. Developmental Dynamics 244:839–851, 2015. © 2015 Wiley Periodicals, Inc.     

Notch-Hes1 signaling activation in Caroli disease and polycystic liver disease

The Notch signaling pathway plays a key role in the morphogenesis of the biliary tree, but its involvement in cystic biliary diseases, such as Caroli disease (CD) and polycystic liver disease (PLD), has yet to be determined. Immunostaining was performed using liver sections of CD and PLD, and the results were compared with those of congenital hepatic fibrosis (CHF) and von Meyenburg complex (VMC). The expression of Notch receptor 1 (Notch1) was increased in the nuclei of biliary epithelial cells in all cases of CD and PLD, whereas it remained at a low level in CHF and VMC. In addition, Notch2 and Notch3 were preferably expressed in the nuclei of biliary epithelial cells of PLD. Accordingly, the Notch effector Hes1 was highly expressed in biliary epithelial cells of CD and PLD, and the cell proliferative activity was significantly higher in CD and PLD. The expression of the Notch ligand Delta-like 1 was significantly increased in biliary epithelial cells of CD and PLD, which may be causally associated with the nuclear overexpression of Notch1 and Hes1. These results indicate that aberrant activation of the Notch-Hes1 signaling pathway may be responsible for the progression of biliary cystogenesis in CD and PLD.     

腺病毒介导的Hes1基因抑制肝细胞癌细胞的增殖与迁移CSCD

目的探讨Hes1基因对肝细胞癌细胞系Hep1-6细胞增殖、迁移与侵袭的影响。方法通过重组腺病毒载体介导Hes1基因在Hep1-6细胞系中过表达,随后测定细胞克隆形成率、增殖速率及体外迁移与侵袭能力的改变。结果Hep1-6细胞分别感染Ad-Hes1和阴性对照Ad-GFP后,感染Ad-Hes1的细胞系克隆形成数显著少于对照组(P<0.001);感染病毒6天后,CCK-8检测感染Ad-Hes1的细胞系在OD450 nm的吸光度值显著低于对照组(P<0.01);感染Ad-Hes1的细胞系24 h和48 h的划痕愈合率显著低于对照组(P<0.001);感染Ad-Hes1的细胞系在Transwell小室培养48 h后迁移进入Transwell下室的细胞数量显著低于对照组(P<0.001);感染Ad-Hes1的细胞系在铺有Matrigel基质胶的Transwell小室培养48 h后侵袭进入Transwell下室的细胞数量显著低于对照组(P<0.01)。结论Hes1有抑制Hep1-6细胞增殖、迁移与侵袭的作用。     

Notch1信号通路在人脑胶质瘤U87细胞增殖、凋亡中的作用及机制探讨

目的 观察Notch1信号通路在人脑胶质瘤U87细胞增殖和凋亡中的作用,并探讨其机制.方法 将U87细胞随机分为A、B、C组,分别加入Notch信号激活剂rhNF-κB、抑制剂DAPT及PBS共培养48 h.用MTT法检测细胞吸光度值(A值)观察细胞增殖情况,流式细胞术检测细胞凋亡率,RT-PCR、Western blot法分别检测U87细胞中的Notch1、Hes1、Bcl-2 mRNA及其蛋白.结果 培养24、48、72、96 h,A组细胞A值分别为0.185±0.007、0.398±0.012、0.735±0.015、1.083±0.031,B组分别为0.102±0.003、0.130±0.004、0.161±0.006、0.194±0.003,C组分别为0.167±0.005、0.265±0.008、0.496±0.011、0.737±0.016,A、B组与C组比较,P均＜0.05.A、B、C组细胞凋亡率分为0.96％±0.17％、26.51％±3.74％、8.76％±1.40％,A、B组与C组比较,P均＜0.05.与C组比较,A组细胞中Notch1、Hes1、Bcl-2 mRNA及其蛋白表达量均显著增加(P均＜0.05),B组细胞中Notch1、Hes1、Bcl-2 mRNA及其蛋白表达量均显著降低(P均＜0.05).结论 Notch1信号通路在人脑胶质瘤U87细胞增殖、凋亡中发挥了重要的调控作用,其机制可能与调节靶基因Hes1、抗凋亡蛋白Bcl-2表达有关.     

Notch1/Hes1信号调控C/EBPα表达对AECII细胞增殖与分化的影响

目的:探讨Notch1/Hes1信号通路能否通过调控CCAAT/增强子结合蛋白α(C/EBPα)的表达从而影响肺泡Ⅱ型上皮细胞(AECⅡ)的增殖与分化功能。方法:体外培养人AECⅡ,将细胞随机分为对照组、激活剂组(加入Notch通路激活剂Jagged1蛋白500μg/L)和抑制剂组(加入Notch通路抑制剂DAPT 10μmol/L),于干预后24 h收获各组细胞。采用RT-qPCR和Western blot法分别检测Notch1、Hes1及C/EBPα的mRNA与蛋白表达水平;CCK-8法检测细胞活力;细胞计数检测细胞增殖;流式细胞术检测细胞周期及分化。结果:与对照组相比,激活剂组Notch1、Hes1和C/EBPα的mRNA和蛋白表达显著增加(P0.05),促进AECⅡ从S期进入G_2/M期,增殖增加而分化减少(P0.05);抑制剂组Notch1、Hes1和C/EBPαmRNA和蛋白表达水平明显降低(P0.05),AECⅡ被阻滞于G_0/G_1期,增殖减少而分化增加(P0.05)。结论:Notch1/Hes1信号可调控C/EBPα表达并能影响AECⅡ增殖与分化。     

叶酸对体外缺氧新生大鼠神经干细胞Hes5基因表达的影响

目的：探讨叶酸对体外缺氧新生大鼠神经干细胞肌s5mRNA、蛋白表达的影响。方法：分离培养24h新生SD大鼠噙神经干细胞：将细胞分为4组：正常对照组、缺氧模型组、缺氧叶酸添加组、缺氧叶酸缺乏组;于增殖第3天将除正常对照组外的其他3组细胞进行缺氧培养,6h后取出,继续培养至第6天收集细胞;用台盼蓝染色计‘数缺氧损伤后的各组细胞密度;RT—PCR方法检测Hes5mRNA表达;Westernblot方法检测Hes5蛋白表达。结果：缺氧叶酸添加组细胞密度高于其他各组．缺氧叶酸缺乏组最低,差异均有统计学意义（P〈0．05）;RT—PCR结果显示,缺氧叶酸添加组的Hes5mRNA表达量高于其余3组,缺氧叶酸缺乏组表达最少,各组间差异有统计学意义（P〈0．05）;Westernblot检测表明．缺氧叶酸添加组Hes5蛋白表达高于其他各组,缺氧叶酸缺乏组Hes5蛋白表达量最低,差异均有统计学意义（P〈0．05）。结论：体外神经干细胞缺氧损伤造模成功;叶酸对缺氧损伤的神经干细胞增殖有促进作用,可为预防和治疗脑缺血缺氧损伤提供依据。     

New role of Notch-mediated signaling pathway in myocardial ischemic preconditioning

Ischemic preconditioning (IPC) is the strongest endogenous myocardial protective mechanism, but up to now, its specific mechanisms have not been completely understood. The Notch network regulates multiple cellular processes, including cell fate determination, development, differentiation, proliferation, apoptosis, and regeneration. Recent loss-of-function studies have shown that the Notch1 receptor controls the response to injury in the adult heart by limiting myocyte hypertrophy, enhancing myocyte survival, promoting precursor proliferation and reducing interstitial fibrosis. Notch signaling also plays a regulatory role in adult cardiac injury and in protection of myocardial function after ischemia. The Notch pathway cross-talks with the PI3K/Akt and NF-κB signaling pathways, both of which are well-known factors involved in IPC-induced myocardial protection. We therefore hypothesize that Notch signaling may play a regulatory role in myocardial protection during ischemic preconditioning and hope to find new drug targets to attain the same beneficial effects of Notch signaling without ischemic insults.